Novel_Multiplex_PCR_Assay_for_Characterization_and_Concomitant_Subtyping_of_Staphylococcal_Cassette

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分子生物学2-7章作业及答案全

可编辑修改精选全文完整版第二章一、名词解释1、DNA的一级结构：四种脱氧核苷酸按照一定的排列顺序以3’，5’磷酸二酯键相连形成的直线或环状多聚体，即四种脱氧核苷酸的连接及排列顺序。

2、DNA的二级结构：DNA两条多核苷酸链反向平行盘绕而成的双螺旋结构.3、DNA的三级结构：DNA双螺旋进一步扭曲盘绕所形成的特定空间结构。

4、DNA超螺旋:DNA双螺旋进一步扭曲盘绕所形成的特定空间结构，是DNA结构的主要形式，可分为正超螺旋与负超螺旋两大类。

按DNA双螺旋的相反方向缠绕而成的超螺旋成为负超螺旋，反之，则称为正超螺旋。

所有天然的超螺旋DNA均为负超螺旋。

5、DNA拓扑异构体：核苷酸数目相同，但连接数不同的核酸，称拓扑异构体6、DNA的变性与复性:变性（双链→单链）在某些理化因素作用下，氢键断裂，DNA双链解开成两条单链的过程。

复性(单链→双链）变性DNA在适当条件下，分开的两条单链分子按照碱基互补配对原则重新恢复天然的双螺旋构想的现象。

7、DNA的熔链温度（Tm值）：DNA加热变性时，紫外吸收达到最大值的一半时的温度，即DNA分子内50%的双链结构被解开成单链。

Tm值计算公式：Tm＝69.3+0.41（G+C）%；＜18bp的寡核苷酸的Tm计算：Tm＝4（G+C）+2（A+T）。

8、DNA退火：热变性的DNA经缓慢冷却后即可复性，称为退火9、基因：编码一种功能蛋白或RNA分子所必需的全部DNA序列。

10、基因组：生物的单倍体细胞中的所有DNA，包括核DNA和线粒体、叶绿体等细胞器DNA11、C值：生物单倍体基因组中的全部DNA量称为C值12、C值矛盾：C值的大小与生物的复杂度和进化的地位并不一致，称为C值矛盾或C值悖论13、基因家族：一组功能相似、且核苷酸序列具有同源性的基因。

可能由某一共同祖先基因经重复和突变产生。

14、假基因：假基因是原始的、有活性的基因经突变而形成的、稳定的无活性的拷贝。

表示方法：Ψα1表示与α1相似的假基因15、转座：遗传可移动因子介导的物质的重排现象。

多重置换扩增文献解读

多重置换扩增文献解读多重置换扩增（Multiplex PCR）是一种在单个反应中同时扩增多个目标序列的PCR技术。

它在分子生物学和遗传学研究中得到广泛应用，可以高效地检测多个基因、位点或突变，具有高通量、高灵敏度和高效率的特点。

下面我将从多个角度对多重置换扩增文献进行解读。

1. 技术原理：多重置换扩增利用多个引物对多个目标序列进行同时扩增。

引物的设计需要考虑目标序列的特异性和互补性，以确保扩增的准确性和特异性。

在PCR反应中，通过调节引物浓度和扩增条件，使得不同目标序列可以在同一反应中被扩增出来。

2. 应用领域：多重置换扩增在医学诊断、遗传病筛查、基因分型、疾病相关基因检测等领域具有广泛的应用。

例如，在遗传病筛查中，可以同时检测多个与遗传病相关的基因，提高检测效率和准确性。

在基因分型研究中，可以同时检测多个位点，用于人群遗传学研究和个体基因分型。

3. 优势和挑战：多重置换扩增相比传统的单目标PCR具有明显的优势。

首先，它可以节省时间和成本，因为多个目标序列可以在同一反应中扩增。

其次，它可以减少样本消耗，特别适用于样本量有限的情况。

然而，多重置换扩增也存在一些挑战，如引物设计的复杂性和优化反应条件的难度，需要充分考虑引物间的特异性和互补性，以及反应条件的优化，以避免非特异性扩增和引物间的竞争。

4. 实验设计和结果解读：在进行多重置换扩增实验时，需要精确设计引物和优化反应条件。

引物的选择应基于目标序列的特异性和互补性，可以通过生物信息学工具进行预测和评估。

反应条件的优化包括引物浓度、Mg2+浓度、循环数和温度等参数的调节。

在实验结果的解读中，需要根据目标序列的预期扩增大小、特异性和目标序列的存在与否进行判断。

综上所述，多重置换扩增是一种高效、高通量的PCR技术，广泛应用于分子生物学和遗传学研究。

它的应用领域广泛，具有许多优势和挑战。

在实验设计和结果解读中，需要仔细考虑引物设计和反应条件的优化，以确保实验的准确性和可靠性。

多重不对称扩增介绍

前期引物设计时减少最佳退火温度的差异，最佳退火温度的差异不超过3~5°为宜。
引物二聚体
包括引物间的二聚体以及引物自身所形成的发卡结构，还有一类是第三方DNA介导的二聚体，这些二聚体和非特异引物一样都会干扰引物与目标结合位点的竞争，影响扩增效率。
针对不同对引物之间二聚体，目前可以采用Visual OMP6软件（7天试用版）进行验证。
不对称扩增对于低拷贝样本的扩增较难，对于病原体检测需要更详尽的PCR设计和优化。
可以获得大量SSDNA，快速进行下游操作。比如直接进行测序，或者下游杂交检测无需经过变性这一步骤。
03
05
缺点
不对称PCR设计
设计不对称PCR引物时，限制性引物的Tm值较非限制性引物Tm值高4~6°，扩增效果更佳。
不对称PCR设计在于控制限制性引物（低浓度引物）的绝对量，限制性引物过多或过少，均不利于SSDNA的制备。限制性引物量可以考虑设置在之间。
设置限制性引物量后，再通过实验验证限制性引物浓度和非限制性引物浓度的比例，目前常用比例1:3、1:5、1:10、1:15、1:20进行优化，一般情况下，1:10、1:20比例情况下效果可能更佳。
02
导致不平衡的原因： 1. 引物特异性； 2、最佳退火温度不一致； 2. 引物二聚体；4、模板量不同；5、引物扩增效率
引物特异性
如果引物与体系中其他非目的基因片段结合能力更强，那么目的基因结合引物的能力就会受到竞争，从而导致扩增效率下降。严格blast验证
最佳退火温度不一致
将多对引物放置入一个体系中扩增，由于进行PCR反应的退火温度相同，所以要求每一对引物的最佳退火温度接近。
Mpprimer （开放、在线）——最多一次可以设计 6对引物、基于PP3内核。

分子标记

DNA分子标记分子标记
是指能反映基因组某种变异特征的DNA片段。片段。是指能反映基因组某种变异特征的片段
因为生物各种性状的差异主要是遗传物质DNA的差异造成，的差异造成，因为生物各种性状的差异主要是遗传物质的差异造成因而通过DNA分子标记可以直接检测基因组的遗传变异，它分子标记可以直接检测基因组的遗传变异，因而通过分子标记可以直接检测基因组的遗传变异更能准确揭示同一物种内不同种、变种、品种、品系间个体更能准确揭示同一物种内不同种、变种、品种、的差异。
分子标记是指能反映生物个体多态性的生物大分子。大分子。
广义的分子标记包括两类，广义的分子标记包括两类，即：同工酶标记和 DNA标记，狭义的分子标记仅指标记，标记。标记狭义的分子标记仅指DNA标记。标记
同工酶标记
编也有其内在的局限性。同工酶标记也有其内在的局限性。由于翻译后的修饰作用、组织特异性和发育阶段性，修饰作用、组织特异性和发育阶段性，特别是相对较少的多态性位点，对较少的多态性位点，使其在应用上受到一定的限制。限制。
微卫星DNA（Microsatellite）又称简单（微卫星）重复序列（重复序列（Simplified Sequence Repeats））
SSR广泛分布于真核生物基因组中，是一种广泛分布于真核生物基因组中，是一种1~6 广泛分布于真核生物基因组中个碱基组成的长度为几十个核苷酸的简单重复序列，在重复序列的两端，序列，在重复序列的两端，往往是相对保守的限制性内切酶位点。限制性内切酶位点。可根据其两端特异序列设计引物通过PCR反应，反应，可根据其两端特异序列设计引物通过反应检测简单重复序列重复单位书不同的DNA区域检测简单重复序列重复单位书不同的区域的多肽性。的多肽性。

不对称PCR

板配对并进行反应。电泳结果(图3) 显示: 温度低时, 特异性很差。65 ℃退火效果较理想, PCR 产物特异性高。
• 3 反应体系的优化 • 优化反应体系扩增产物的琼脂糖电泳分析表明(图4) , 不对
称PCR 反应最佳体系为: 第1 轮PCR反应时5′末端引物与突变引物配比为50∶1和100∶1 , 第2 轮PCR 反应的退火温度为65～70 ℃。PCR 突变产物1 (290 bp) 和突变产物4 (267 bp) 经上海生工生物公司测序分析, 结果验证引入的突变位点与原来设计的突变位点完全一致。
• Wangh还开发了一系列的Elixirs试剂这些试剂以一 Wangh还开发了一系列的Elixirs试剂这些试剂以一
种独特的方式与反应中的分子相互作用，以避免引物错配，他们是高性能DNA聚合酶HoT Start的引物错配，他们是高性能DNA聚合酶HoT Start的替代物，但与HoT Start不同，替代物，但与HoT Start不同， HoT Start只在零 Start只在零度及第一次热反应后存在。 Elixirs试剂与其他成 Elixirs试剂与其他成分混合在一起，可以避免引物错配的情况发生，但是也会将分离开的基因组链清除掉，他们制止了引物二聚体的出现，改善了有若干成分的多路反应结构。
• 采用分子信标（beacons),或者是Applied 采用分子信标（beacons),或者是Applied
Biosystems公司的TaqMan探针检测退火， Biosystems公司的TaqMan探针检测退火，因为探针温度必须高于引物温度，因此， Wangh认为可以引入一种较低的温度检测 Wangh认为可以引入一种较低的温度检测步骤。方案一是让热循环与退火步骤一样，但是要在限制性引物用尽之后或者是延伸步骤延伸之后引入，另一个方法是将退火温度与检测温度分开。

经典多重PCR参考文献

Research ReportsO. Henegariu, N.A. Heerema, S.R. Dlouhy, G.H. Vance and P.H. Vogt1Indiana University, Indianapo-lis, IN, USA and 1Heidelberg University, Heidelberg, GermanyABSTRACTBy simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous pa-pers and manuals discuss in detail condi-tions influencing the quality of PCR in gen-eral, relatively little has been published about the important experimental factors and the common difficulties frequently en-countered with multiplex PCR. We have ex-amined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multi-plex PCR assay are the relative concentra-tions of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a mul-tiplex PCR assay and suggest ways to over-come commonly encountered problems. INTRODUCTIONMultiplex polymerase chain reac-tion (PCR) is a variant of PCR in whichtwo or more loci are simultaneouslyamplified in the same reaction. Sinceits first description in 1988 (6), thismethod has been successfully appliedin many areas of DNA testing, includ-ing analyses of deletions (2,8), muta-tions (14) and polymorphisms (11), orquantitative assays (10) and reverse-transcription PCR (7).The role of various reagents in PCRhas been discussed (3,9,12,13), andprotocols for multiplex PCR have beendescribed by a number of groups. How-ever, few studies (5,15) have presentedan extensive discussion of some of thefactors (e.g., primer concentration, cy-cling profile) that can influence the re-sults of multiplex analysis. In thisstudy, over 50 loci were amplified invarious combinations in multiplexPCRs using a common, KCl-containingPCR buffer. Because of specific prob-lems associated with multiplex PCR,including uneven or lack of amplifica-tion of some loci and difficulties in re-producing some results, a study of theparameters influencing the amplifica-tion was initiated. Based on this experi-ence, a step-by-step multiplex PCRprotocol was designed (Figure 1), withpractical solutions to many of the prob-lems encountered. This protocol shouldbe useful to those using PCR technolo-gy in both the research and the clinicallaboratories.MATERIALS AND METHODSStandard Solutions and Reagents forthe PCRNucleotides (dNTP) (PharmaciaBiotech [Piscataway, NJ, USA] orBoehringer Mannheim [Indianapolis,IN, USA]) were stored as a 100 mMstock solution (25 mM each dA TP,dCTP, dGTP and dTTP). The standard10×PCR buffer was made as described(Perkin-Elmer, Norwalk, CT, USA) andcontained: 500 mM KCl, 100 mM Tris-HCl, pH 8.3 (at 24°C) and 15 mMMgCl2. Taq DNA Polymerase was pur-chased from Life Technologies(Gaithersburg, MD, USA) or fromPerkin-Elmer. Dimethyl sulfoxide(DMSO), bovine serum albumin (BSA)and glycerol were purchased from Sig-ma Chemical (St. Louis, MO, USA).Primers were either commercially ob-tained (Genosys [The Woodlands, TX,USA] or Research Genetics [Hunts-ville, AL, USA]) or synthesized locallyand were used in a final concentrationof 10–25 pmol/µL each. One set ofprimer pairs (sY) was used to map dele-tions on the human Y chromosome(8,16). Another 10–15 primer pairswere for the Duchenne muscular dys-trophy (DMD) gene on human chromo-some X (4). Other primers representvarious polymorphic loci (microsatel-lites) on human chromosome 12 (Re-search Genetics). Primers were com-bined in multiplex mixtures asdescribed in Table 1 and Figures 2b, 3bMultiplex PCR: Critical Parameters and Step-by-Step ProtocolBioTechniques 23:504-511 (September 1997)and 5e. Genomic DNA was preparedusing a standard sodium dodecyl sul-fate (SDS)/proteinase K protocol(Boehringer Mannheim).Basic PCR ProtocolThe basic PCR (25 µL vol) includ-ed: autoclaved ultra-filtered water; PCR buffer (1×); dNTP mixture (200µM each); primer(s) (0.04–0.6 µM each); DMSO, glycerol or BSA (5% - if used); Taq DNA polymerase (1–2 U/25µL) and genomic DNA template (150 ng/25 µL). The components of the re-action can be added in any order, pro-vided that water is added first. Pipetting was done on ice, and the vials were placed from ice directly into the pre-heated metal block or water bath (94°C) of the thermal cycler. For ra-dioactive labeling, 1 µCi [32P]dCTP (Amersham, Arlington Heights, IL,USA) was added to a 100 µL mastermixture immediately before setting upthe reaction. Results of PCR were thesame when 100- or 25- or 6.2-µL reac-tion volumes were used. With smallervolumes, pipetting is critical, especial-ly for dNTP. Various thermal cyclerswere used during these studies and,with minor cycling adjustments, allperformed well.Gel Analysis of PCR ProductsThe PCR products of non-polymor-phic loci (chromosomes X and Y) wereseparated by electrophoresis on 3%SeaKem®LE or NuSieve®(3:1)Agarose Gels (FMC BioProducts,Rockland, ME, USA) in 1×TAE [0.04M Tris-acetate; 0.001 M EDTA (pH8.0)] or 1×TBE [0.09 M Tris-borate;0.002 M EDTA (pH 8.0)] buffer, re-spectively, at room temperature usingvoltage gradients of 7–10 V/cm. Forany given gel analysis, the same vol-ume of PCR products was loaded ineach gel slot. Results were visualizedafter staining the gels in 0.5–1 µg/mLethidium bromide. Sequencing gels(6% polyacrylamide [PAA]/7 M urea)were used for separation of the PCRproducts when the loci tested werepolymorphic or a higher resolution wasrequired. The equivalent of about 0.2µL radioactively labeled PCR productwas loaded in each gel lane, after mix-ing it in loading buffer. These gels wererun in 0.6×TBE at 1800–2000 V (60A) for about 2 h. Autoradiographs wereobtained after overnight exposure.RESULTS AND DISCUSSIONBased on many experiments, a pro-tocol for establishing a multiplex PCRhas been designed (Figure 1), includinga number of practical solutions to someof the most commonly encounteredproblems. For convenience and ease ofuse, the words in italic characters linkthe scheme with various points present-ed in Materials and Methods and thefollowing subsections.Basic Principles of the MultiplexPCRDNA primers (Steps 1 and 2).Primer selection followed simple rules:primer length of 18–24 bp or higherand a GC content of 35%–60%, thushaving an annealing temperature of55°-58°C or higher. Longer primers(DMD primers, 28-30 bp) allowed thereaction to be performed at a higher an-nealing temperature and yielded lessunspecific products. To calculate themelting point and test for possibleprimer-primer interactions, “Primers1.2” (a freeware that can be down-loaded from ) wasused. To test for possible repetitive se-quences, many of the primers usedwere aligned with the sequence data-bases at the National Center forBiotechnology Information (NCBI) us-ing the Basic Local Alignment SearchTool (BLAST) family of programs.Single locus PCR (Step 3).A PCRFigure 1. Step-by-step protocol for the multiplex PCR.Research Reports program to amplify all loci individuallywas designed. Reaction mixture includ-ed 1×PCR buffer, 0.4 µM each primer,5% DMSO and 1 U Taq DNA poly-merase/25 µL reaction volume. Resultsof PCR were compared when the reac-tions were done consecutively in thesame thermal cycler, or in parallel, inmachines of the same model and in ma-chines of different models or manufac-turers. Results were very reproduciblewhen the same machine or same ma-chine model was used but couldmarkedly differ when the same exactPCR program was used on thermal cy-clers from different manufacturers.However, with adjustments in only thecycling conditions, results became re-producible even in different types ofmachines. We have observed that forthe loci tested (100–300-bp long), yieldof some products was increased by de-creasing the extension temperature. Forindividually amplified loci, the anneal-ing time (from 30–120 s) and the exten-sion time (from 30–150 s) did not visi-bly influence the results, but thespecificity and yield of PCR productwere increased or decreased bychanges in annealing temperature. Toamplify the 22 Y-specific loci (Figure2a), PCR program A gave best results(Table 2).Multiplex PCR: equimolar primermixture (Step 4).Combining the pri-mers in various mixtures and amplify-ing many loci simultaneously (Table 1and Figure 2b), required alteration/opti-mization of some of the parameters ofthe reaction. When the multiplex reac-tion is performed for the first time, it isuseful to add the primers in equimolaramounts. The results will suggest howthe individual primer concentration andother parameters need to be changed.Examples of some useful changes areillustrated and discussed below; howev-er, these examples do not necessarilyfollow the exact order as listed in theprotocol (Figure 1) since a number of parameters (e.g., extension tempera-ture) are referred to more than once.Optimization of Multiplex PCR Cycling ConditionsExtension temperature (Step 5, A–C).Figure 2c illustrates the results obtained when four different amplifica-tion mixtures containing equal amounts(0.4 µM each) of different Y-chromo-some primers were subjected to multi-plex PCR with program A and programB (Table 2); the latter program had ahigher extension temperature (72°C)and longer annealing and extensiontimes. In general, there was a visiblyhigher yield of PCR products for mix-tures Y-1, Y-3* and Y-4 with programA. In addition, with program B, someproducts are missing (in Y-1 and Y-2)and some unspecific products appear(in Y-1 and Y-3*). The results withprogram B were considered less desir-able overall and suggested that thehigher extension temperature in pro-gram B decreased the amplification of Name Size Name Size Name Size Name Size (locus)(bp)(locus)(bp)(locus)(bp)(locus)(bp) Y-1Y-2Y-3Y-4sY84 326sY143311sY86320sY14472 DYS273DYS231DYS148SRYsY134 301sY157285sY105301sY95303 DYS224DYS240DYS201DYS280sY117262sY81209sY82264sY127274DS209DYS271DYS272DYS218sY102 218sY182125Y6HP35226sY109233 DYS198KAL Y DYS274DYF43S1sY151 183sY147100Y6PHc54166sY149132 KAL Y DYS232n.a.DYS1sY94 150sY153139DYS279DYS237sY88 123sY97104DYS276DYS281DMD exon Size DMD exon Size Name Size No.(bp)No. (bp)(locus)(bp) X-1X-312-1 No. 45547PM535AFM263zd1 317-341D12S332PM535No. 3410AFM205ve5 271-291D12S93No. 19459No. 50271AFM205xg3 243-253D12S310No. 17416No. 6202AFM211wb6 228-238D12S98No. 51388No. 60139AFM206ze5 183-201D12S94No. 8360AFM299zd5 165-181D12S349No. 12331AFM135xe3 142-168D12S87No. 44268AFM122xf6 105-125D12S85No. 4196n.a. = locus not assigned.PM = promoter regionTable 1. List of Primers Used in the Multiplex Mixturessome loci, even though we tried to compensate using a longer annealing time and slightly longer extension time.Extension time (Step 5, A, B and D).In multiplex PCR, as more loci are simultaneously amplified, the pool of enzyme and nucleotides becomes a limiting factor and more time is neces-sary for the polymerase molecules to complete synthesis of all the products.Two experiments illustrated the influ-ence of the extension time. In one ex-periment, a Y -chromosome primer pair (Y6BaH34pr, 910bp) was added to a X-chromosome primer mixture (X-3).The results (Figure 3b) showed that in-creasing the extension time in the mul-tiplex PCR (program A vs. program D)increased the amount of longer prod-ucts. In another experiment, four Y multiplex mixtures were amplified us-ing PCR programs C and A (Figure 3a and Table 2). Visibly higher yields of PCR products were obtained for all Y mixtures when a longer extension time was used.Annealing time and temperature (Step 5, A–D; Figure 1).Modification of the annealing time from 20 s to 2min did not alter the amplification effi-ciency (not shown), but the annealing temperature was one of the most im-portant parameters. Although many in-dividual loci could be specifically am-plified at 56°–60°C, our experience showed that lowering the annealing temperature by 4°–6°C was required for the same loci to be co-amplified in multiplex mixtures. This is demonstrat-ed in Figure 3, d–f, which depict an op-timal multiplex annealing temperature of 54°C for primers individually usable at 60°C. At 54°C, although unspecific amplification probably occurs (e.g.,Figure 3c), it is overcome by the con-current amplification of an increased number of specific loci in the multiplex reaction and thus remains invisible.Similarly, when many specific loci are simultaneously amplified, the more ef-ficiently amplified loci will negatively influence the yield of product from the less efficient loci. This is due to the fact that PCR has a limited supply of en-zyme and nucleotides, and all products compete for the same pool of supplies. Number of PCR cycles. Primer mixture Y -3* was used to amplify two different genomic DNA samples, stop-Figure 2.(a) Single-locus PCR. Amplification of the sY loci using 1×PCR buffer and program A. On the gel, the products are arranged in increasing order of sY number (1=sY14, 2=sY81, 3=sY82, 4=sY84,5=sY86, 6=sY88, 7=sY94, 8=sY95, 9=sY97, 10=sY102, 11=sY105, 12=sY109, 13=sY117, 14=sY127,15=sY134, 16=sY143, 17=sY147, 18=sY149, 19=sY151, 20=sY153, 21=sY157 and 22=sY182). All products had the expected length, and there was no visible unspecific amplification. In all gels, lanes without a label show the size marker (1-kb ladder; Life Technologies). (b) Optimized multiplex reactions.Multiplex PCR with primer mixtures Y-1 (sY84, sY134, sY117, sY102, sY151, sY94 and sY88), Y-2(sY143, sY157, sY81, sY182 and sY147), Y-3 (sY86, sY105, sY82, Y6HP35, Y6Phc54, sY153 and sY97) and Y-4 (sY14, sY95, sY127, sY109 and sY149) in 1.6×PCR buffer (PCR program E). Mix Y-3*is mixture Y-3 without primers Y6HP35 and Y6Phc54. Arrows indicate the expected amplification prod-ucts. (c) Extension temperature. Multiplex PCR with mixtures Y-1 to Y-4 with PCR programs A and B (Table 2). All amplification products are visible in the first four lanes (extension at 65°C). In the last four lanes (extension at 72°C), bands are missing in Y-1 and Y-2, and unspecific products appear in Y-1 and Y-3*. Length marker in all figures = 1-kb ladder. In all images, electrophoresis was conducted from top to bottom. Program AProgram B Program C First Denaturing 94°C, 4 min 94°C, 4 min 94°C, 4 min Denature 94°C, 30 s 94°C, 30 s 94°C, 30 s Anneal 54°-56°C, 30 s*54°C, 1 min54°C, 45 sExtend 65°C, 1 min72°C, 1 min, 20 s 65°C, 2 min 32 cycles 32 cycles 32 cycles Final Extension65°C, 3 min 72°C, 3 min 65°C, 3 min Program DProgram E Program F First Denaturing 94°C, 4 min 94°C, 4 min none Denature 94°C, 30 s 94°C, 30 s 94°C, 30-45 s Anneal 55°C, 30 s 54°C, 45 s 56°-58°C, 45 s Extend 65°C, 4 min 65°C, 2 min 68°C, 2 min, 30 s 32 cycles 45 cycles 35 cycles Final Extension65°C, 3 min65°C, 5 minnoneBold characters show most important modifications when programs are com-pared.*Program A was used with two different annealing temperatures, according to the type of PCR amplification (see Results and Discussion).Table 2. Cycling Conditions/PCR ProgramsResearch Reportsping the reaction after increasing num-bers of cycles (Figure 4a). One of the two genomic DNAs was a better tem-plate, possibly due to the higher quality and/or amount of DNA. Both of them,however, show a gradual increase in the yield of all bands with the number of cycles. The most obvious variation in the amount of products was around 25cycles (for ethidium bromide-stained gels). Twenty-eight to thirty cycles are usually sufficient for a reaction; little is gained by increasing cycle number up to 60.Optimization of Multiplex Reaction ComponentsInitially, there was some variation from test to test when the same PCR program was used (e.g., Figures 2c and 3a). Solving this reproducibility prob-lem required adjustments of PCR com-ponents.Amount of primer (Step 5, B and C). Initially, equimolar primer concen-trations of 0.2–0.4µM each were used in the multiplex PCR (Figure 3c), but there was uneven amplification, with some of the products barely visible even after the reaction was optimized for the cycling conditions. Overcoming this problem required changing the pro-portions of various primers in the reac-tion, with an increase in the amount of primers for the “weak” loci and a de-crease in the amount for the “strong”loci. The final concentration of theprimers (0.04–0.6µM) varied consider-ably among the loci and was estab-lished empirically.dNTP and MgCl 2concentrations (Step 5D).dNTP .The significance of the dNTP concentration was tested in a multiplex PCR test with primer mixture Y-4.Magnesium chloride concentration was kept constant (3 mM), while the dNTP concentration was increased stepwisefrom 50–1200µM each (Figure 4b).The best results were at 200 and 400µM each dNTP, values above which the amplification was rapidly inhibited.Lower dNTP concentration (50µM) al-lowed PCR amplification but with visi-bly lower amounts of products. dNTP stocks are sensitive to thawing/freezing cycles. After 3–5 such cycles, multi-plex PCRs often did not work well;products became almost completely in-visible. To avoid such problems, small aliquots (2–4 µL, 10–20 reactions) of dNTP (25 mM each) can be made and kept frozen at -20°C and centrifuged before use. This “low stability” of dNTP is not so obvious when single loci are amplified.MgCl 2. A recommended magne-sium chloride concentration in a stan-dard PCR is 1.5 mM at dNTP con-centrations of around 200µM each. To test the influence of magnesium chlo-ride, a multiplex PCR (mixture Y -3)was performed, keeping dNTP concen-tration at 200 µM and gradually in-creasing magnesium chloride from 1.8–10.8 mM (Figure 4c). Amplifica-tion became more specific (unspecific bands disappeared), and the products acquired comparable intensities (at 10.8 mM). In PCRs with up to 20 mM MgCl 2, products became barely visible,as if the reactions were inhibited (not shown).dNTP/MgCl 2balance.To work properly, Taq DNA polymerase re-quires free magnesium (besides theFigure 3.(a) Extension time. Multiplex PCR of mixtures Y-1 to Y-4, comparing PCR programs C (2-minextension time) and A (1-min extension time, 54°C annealing temperature). Comparison of equivalent lanes shows an improvement in yield when extension time is 2 min. Some faint unspecific bands appear,possibly due to the low buffer concentration (1×). (b) Extension time. Multiplex PCR with mixture X-3(primers for DMD gene exons Nos. PM, 3, 50, 6, 60) and primer pair Y6BaH34 (910-bp product, upper arrow). Primers giving shorter amplification products are preferentially amplified with short extension times (1-min, program A). (c) Equimolar primer mixture. PCR with individual primer pairs of mixture 12–1 (separate and multiplex), using program F. Products are arranged on the gel according to their de-creasing length. Individual products have comparable intensities. When equimolar amounts of primers were mixed for the multiplex reaction (first lane), some products were not efficiently amplified but un-specific products disappeared. (d–f) Annealing temperature, buffer concentration and number of primers.Multiplex amplification of mixture Y-3* (first three lanes in each gel), primer pair sY 153 (lanes 4–6)and mixture Y-3 (lanes 7–12 in 1×or 2×PCR buffer) on three different template DNAs using three PCR programs differing in annealing temperature (48°, 54°or 59°C). Lanes 1–9 on each gel show reactions in 1×PCR buffer. Lanes 10–12 on each gel show reactions in 2×PCR buffer. Lanes 7–12 on each gel (under 1×PCR and 2×PCR) were with primer set Y-3. The very last lane in Figure 3, d and f is the marker (1-kb ladder). Small horizontal arrows indicate the expected products of mixture Y-3* (five products) including the longest specific product on the gel. Oblique arrow (3e) indicates a strong unspecific product. Solid arrowheads indicate the two extra products expected in mixture Y-3 (total of seven products) compared with Y-3*. Arrowhead outlines show positions of some missing products (e.g., 3e, first lane). With mul-tiplex amplification at 48°C, many unspecific bands appear. In 1×PCR buffer, the sY153 product is stronger when amplified in mixture Y-3* (5 primer pairs) than in mixture Y-3 (7 primer pairs), which shows that at least for some products, an increased number of simultaneously amplified loci can influ-ence the yield at some specific loci. Raising the PCR buffer concentration from 1×to 2×allows a more even amplification of all specific products and helps to decrease the intensity of many longer unspecific products (compare lanes 7–9 vs. 10–12). The strong 470–480-bp unspecific band (oblique arrow) seen with 2×buffer was eliminated by varying the proportion of different primers in the reaction (compare with Y-3, Figure 2b). At 59°C the sY153 product can be seen only when 2×buffer is used or when the lo-cus is amplified alone.magnesium bound by the dNTP and theDNA) (9). This is probably why in-creases in the dNTP concentrations(Figure 4b) can rapidly inhibit the PCR,whereas increases in magnesium con-centration often have positive effects (Figure 4c). By combining various amounts of dNTP and MgCl2, it was found that 200 µM each dNTP work well in 1.5–2 mM MgCl2, whereas 800µM dNTP require at least 6–7 mM MgCl2. The threshold for the reaction was roughly 1 mM MgCl2when 200µM dNTP was used, with reduced PCR amplification below this MgCl2con-centration.PCR buffer (KCl) concentration.Comparison of PCR buffers (Step 5, B–D).KCl or PCR buffer concentration.Raising the buffer concentration to 2×(or only the KCl concentration to 100mM) improved the efficiency of the multiplex reaction (Figure 4d and alsoFigure 3, d–f), this effect being moreimportant than using any of the adju-vants tested (DMSO, glycerol or BSA). Generally, primer pairs with longer am-plification products worked better at lower salt concentrations, whereas primer pairs with short amplification products worked better at higher salt concentrations, where longer products become harder to denature (compare 0.4×with 2.8×in Figure 4d). For exam-ple, pair sY94 (melting point ca. 58°C) is favored over both sY88 (melting point ca. 58°C) and sY151 (melting point ca. 52°C) at 0.8×buffer but not at higher salt concentrations. The proper buffer concentration may help over-come other factors (product size, GCFigure 4.(a) Number of cycles. Amplification with two different DNA templates using primer mixture Y-3* in 1.4×PCR buffer, with increasing numbers of cycles by units of three. (b) dNTP concentration. PCR amplification using mixture Y-4 in 2×PCR buffer (3 mM MgCl2) and increasing concentrations of dNTP (50, 100, 200, 400, 600 and 1200 µM). Most efficient amplification is seen at concentrations of 200–400 µM dNTP. Further increase in the dNTP concentration inhibits the reaction when MgCl2 concentration is kept constant. (c) MgCl2concentration. Multiplex PCR was performed with mixture Y-3 in 1.4×PCR buffer, using PCR program E and gradually raising the concentration of MgCl2. (d) PCR buffer concentration. Amplification products of mixture X-1 (DMD gene exons Nos. 45, PM, 19, 17, 51, 8, 12, 44 and 4) using increasing concentrations of PCR buffer and program E. As the stringency in the reaction mixture decreases, shorter products are amplified more efficiently, whereas the intensity of longer products gradually decreases. For this particular primer mixture, the optimal buffer concentration was 1.2×–1.6×. (e) Comparison of PCR buffers. Comparison of multiplex PCR of mixture X-1 in the DMD buffer and the 1.6×KCl-based PCR buffer, using the same proportion of ingredients (DNA, Taq DNA polymerase, primer amount) and PCR program E. For every DNA sample tested, the amounts of products were increased when 1.6×PCR buffer was used. Only four lanes are shown, although the gel had more samples loaded, and identical results were observed. (f) Amount of template DNA. Various amounts of template DNA were amplified with primer sY153 and mixture Y-3* in 2×PCR buffer with program E. Reaction volumes were 25 µL. There were no major differences using 500 or 30 ng DNA; however, some bands became weaker as the DNA amount was further decreased to 0.5 ng/25 µL reac-tion. No major differences due to the DNA template concentration were seen when primer pair sY153 was used alone.Research Reportscontent, etc).Comparison of PCR buffers.We have compared a previously described multiplex PCR buffer (6), called “DMD” for the purpose of this paper,with the less complex, KCl-based buffer in the multiplex reaction. The 5×“DMD” buffer contains 83 mM (NH 4)2SO 4, 335 mM Tris-HCl (pH8.8), 33.5 mM MgCl 2, 50 mMβ-mer-captoethanol, 850µg/mL BSA, and it is used at 1×final concentration together with 10% DMSO and 1.5 mM each dNTP (1,2,4). When tested with the DMD gene primers (mixture X-1) the regular KCl-based PCR buffer at 1.6×worked better than the “DMD” buffer (visibly higher yield of products) (Fig-ure 4e). Results were reproducible indozens of patient DNA samples tested.The KCl-based buffer is less complex and easier to adjust and optimize. Also,since the fidelity of the Taq DNA poly-merase is higher at lower dNTP con-centrations (9), using the KCl-based buffer (which requires much less dNTP) can be beneficial when the PCR products need to be further analyzed for mutations.Amount of template DNA and Taq DNA polymerase (Step 5, A and D).At DNA template quantities between 30 and 500 ng/25 µL reaction, mixture Y-3* showed no significant differences (Figure 4f); however, below 30 ng the amount of some of the products de-creased. When the amount of template DNA is very low (pg of DNA), efficientand specific amplification can be ob-tained by further lowering the anneal-ing temperature, sometimes by as much as 10°–12°C (data not shown).Different concentrations of Taq DNA Polymerase (Perkin-Elmer) were tested using primer mixture Y -3 (Figure 5a). The most efficient enzyme concen-tration seemed to be around 0.4µL or 2U/25µL reaction volume. Too much enzyme, possibly because of the high glycerol concentration in the stock so-lution, resulted in an unbalanced ampli-fication of various loci and a slight in-crease in the background. Five native Taq DNA polymerases, from five dif-ferent sources, performed similarly on mixture Y -4 in 1.6×PCR buffer using 2U/25µL (Figure 5b).Use of adjuvants: DMSO, glyc-erol, BSA (Step 5E). Various authors recommend DMSO and glycerol to im-prove amplification efficiency (higher amount of product) and specificity (no unspecific products) of PCR, when used in concentrations varying between 5%–10% (vol/vol) (9). However, in the multiplex reaction, these adjuvants gave conflicting results. For example,5% DMSO improved the amplification of some products and decreased the amount of others, whereas some loci were not influenced at all (Figure 5c).Similar results were obtained with 5%glycerol (data not shown). Therefore,the usefulness of these adjuvants needs to be tested in each case. BSA, in con-centrations up to 0.8µg/µL (higher than previously described) increased the efficiency of the PCR much more than either DMSO or glycerol. BSA did not have an inhibitory effect on any of the loci amplified (data not shown).Agarose vs. Polyacrylamide Gels Agarose.Multiplex PCR products,differing from each other by 30–40 bp in length could be conveniently sepa-rated on 3% gels of commonly used agaroses, such as SeaKem or NuSieve (FMC BioProducts). Overnight separa-tion of products at lower voltage gradi-ents notably decreased the sharpness of individual PCR bands, especially when the products were smaller than 400–500 bp.Polyacrylamide (PAA) gels.To separate PCR products differing in onlyFigure 5.(a) Amount of enzyme. Amplification products of mixture Y-3, after using 0.5, 1, 2, 4 and 8U/25µL reaction volume are shown. Arrows indicate the expected positions of the amplification prod-ucts. The most appropriate enzyme concentration was between 1–2 U/25µL. (b) Source of enzyme. Mul-tiplex PCR of mixture Y-4 in 1.6×PCR buffer usesTaq DNA polymerases from five sources. Lane 4*shows the products obtained when the enzyme from lane 4 was used in the buffer provided by the vendor.An unspecific product appeared. (c) Use of adjuvants. Comparative multiplex PCR using the Y-specific mixtures with 5% DMSO (superscript D) and without DMSO, in 1×buffer. Loci sY151 and sY88 from mixture Y-1D (oblique arrows) are stronger when no DMSO is used. However, DMSO helps amplify (vertical arrows) locus sY81 in mixture Y-2 and locus sY95 in mixture Y-4. (d) Nondenaturing PAA gel separation. Simultaneous PCR amplification of loci D12S93 and D12S349 performed on genomic DNA from two human-rodent cell lines, GM 10868 (A) and GM 12072 (B), each containing a different copy of human chromosome 12, and their combination (A+B). Although in lanes A and B each locus should have yielded only one allele (i.e., one band), on a nondenaturing polyacrylamide gel, each of the two expected products (arrows) was accompanied by another one running slower on the gel (oblique lines). A similar aspect persisted in lane A+B. Lanes labeled 1 and 2 show separation of amplification products of mixture 12-1 (including eight D12S polymorphic loci, the numbers of which are indicated to the left side of Pan-el e) on two different genomic template DNAs. (e) Denaturing PAA gels. Sequencing gel separation of the same multiplex products as in Figure 4e, after “hot” PCR. Lanes A and B show mono-allelic amplifi-cation of the respective polymorphic loci (D12S93 and D12S349). Lane A+B shows simultaneous am-plification of both alleles at each locus. Lanes 1 and 2 show results using primer mixture 12-1 on two dif-ferent human genomic DNAs, with polymorphisms detected at some loci. Lane 3 shows results after multiplex PCR with mixture 12-1 on DNA from hybridoma cell line GM 10868 yielding homozygous amplification of all loci tested. Numbers to the left of the figure indicate the D12S loci tested.。

新技术在食品微生物检验检测中的应用

分析检测新技术在食品微生物检验检测中的应用　王青青浙江省杭州市萧山区疾病预防控制中心食品是人类生存必不可少的能量来源，一直以来都是人类关注的重点问题，我国每年的食品安全问题层出不穷，需要尽快建立完善的食品安全监控技术，这依赖于食品生物检验检测技术的革新，因此有必要对目前已经出现的几种新技术在食品微生物检验检测中的应用进行分析。

生理生化技术生理生化技术也被称为代谢学技术，其原理是通过研究菌体代谢过程中产生的特异物质和分子的变化特性，检验食品中的菌种。

具体有ＡＴＰ生物发光法和微量生化法。

ATP生物发光法。

ＡＴＰ是一种活跃于所有活体生物体内的物质，ＡＴＰ生物发光法通过注入荧光素酶，观察荧光素酶和ＡＴＰ发生反应所产生的荧光强度，来判断活菌数。

其优点是方便、快速，现多被用于检测乳制品中乳酸菌、啤酒中菌落以及脱水蔬菜中细菌的数量。

微量生化法。

微量生化法同样包括多种具体方法。

菌在生长过程中会产生一定的热量，通过热量的多少可以有效鉴别菌种，这是微热量技法；而放射测量法是将放射性标记物标记在碳水化合物中，通过分析菌种生长过程中产生的具有放射性的碳水化合物来测定菌量，具有快速高效的优点；另外一种电子阻抗法敏感性高、可重复、反应快，目前被广泛运用在大肠菌群、乳酸菌、酵母菌的定量检测上。

分子生物学技术分子生物学技术是在在基因水平上对难以培养或抗原结构复杂的微生物进行检测的技术。

包括常规ＰＣＲ技术和由ＰＣＲ技术衍生出来的ｑＲＴ－ＰＣＲ、ＰＣＲ－ＤＧＧＥ、Ｍｕｌｔｉｐｌｅｘ　ＰＣＲ等技术。

PCR技术。

ＰＣＲ技术通过将聚合酶加入到ＤＮＡ模板和相应引物的混合物中，放大和扩增目标ＤＮＡ片段，以此判断菌种。

凭借着较高的灵敏度、简便的操作和快速性，这种方法早在二十世纪末就被广泛运用，现在常用来检测乳酸菌、大肠杆菌和双歧杆菌等。

qＲT-PCＲ技术。

ｑＲＴ－ＰＣＲ技术即实时荧光ＰＣＲ技术，它比常规的ＰＣＲ技术的特异性更强，而且产生的污染更小，被广泛用于致病菌、霉菌和酵母和乳酸菌的种类和数量检测。

重复引物PCR技术在超大片段动态突变疾病基因检测中的应用_陈晟

遗传Hereditas (Beijing) 2014年11月, 36(11): 1145―1151 综述收稿日期: 2014-07-19; 修回日期: 2014-10-14作者简介: 陈晟，硕士研究生，专业方向：神经遗传病。

E-mail: chensheng0039@通讯作者:吴志英，博士，主任医师，研究方向：神经遗传和变性病。

E-mail: zhiyingwu@ DOI: 10.3724/SP.J.1005.2014.1145 网络出版时间: 2014-10-16 11:39:42URL: /kcms/detail/11.1913.R.20141016.1139.003.html重复引物PCR 技术在超大片段动态突变疾病基因检测中的应用陈晟1，吴志英21. 福建医科大学附属第一医院神经内科，福州 350005；2. 复旦大学附属华山医院神经内科，上海 200040摘要: 动态突变疾病是指基因编码区或非编码区发生核苷酸重复序列异常扩增所导致的一类遗传性疾病。

发生于非翻译区的动态突变常常伴有超大片段重复序列，应用普通PCR 法无法对该片段进行扩增，而传统的Southern blot 等技术费时费力，无法应用于临床基因诊断。

在此背景下，重复引物PCR 技术应运而生，并随着应用范围的扩大而逐渐改进，适用于强直性肌营养不良症、Friedreich 共济失调、脊髓小脑性共济失调10型及C9orf72基因突变引起的额颞叶痴呆或肌萎缩侧索硬化等遗传性动态突变疾病的临床基因检测。

文章简要介绍了重复引物PCR 技术的原理，着重阐述了重复引物PCR 技术在相关超大片段动态突变疾病临床基因检测中的应用进展。

关键词: 重复引物PCR 技术；超大片段重复序列；基因诊断；动态突变疾病Advances on repeat-primed PCR assay for the genetic diagnosis of dy-namic mutation diseases with large pathogenic expansionsSheng Chen 1, Zhiying Wu 21. Department of Neurology , First Affiliated Hospital , Fujian Medical University , Fuzhou 350005, China ;2. Department of Neurology , Huashan Hospital , Fudan University , Shanghai 200040, ChinaAbstract: Dynamic mutation diseases are genetic diseases caused by unstable repeat expansions in coding ornoncoding regions. The unstable repeat expansions located in the noncoding region usually accompany large expan-sions which are difficult to amplify using the standard PCR assay. Traditional detection methods, including South-ern blot, are usually time-consuming and labor-wasting. A new method called fluorescent repeat-primed PCR assay was brought into clinical applications. With the development of genetic diagnoses for dynamic mutation diseases, such as myotonic dystrophy, Friedreich’s ataxia, SCA10, and amyotrophic lateral sclerosis or frontotemporal demen-tia caused by C9orf72 mutations, the clinical use of repeat-primed PCR assay has been broadened. Here, we review the principle of repeat-primed PCR assay and its advances on the genetic diagnoses of related dynamic mutation dis-eases with large pathogenic expansions.1146 遗传Hereditas (Beijing) 2014第36卷Keywords:repeat-primed PCR assay;large pathogenic expansions; genetic diagnosis; dynamic mutation disease动态突变疾病是指基因编码区或非编码区发生核苷酸重复序列异常扩增导致的一类遗传性疾病，根据其发生扩增的分子生物学机制不同，大致可分为三类：第一类是异常扩增引起转录过程受阻，如脆性X综合征(Fragile X syndrome, FXS)、Friedreich 共济失调(Friedreich ataxia, FRDA)等；第二类是异常扩增导致病理性RNA毒性蓄积，如强直性肌营养不良症症1型(Myotonic dystrophy 1, DM1)、脊髓小脑性共济失调10型(Spinocerebellar ataxia 10, SCA10)等；以上两类均为非编码区重复序列异常扩增。

鉴别H5亚型两种分支禽流感病毒双重实时荧光RT-PCR方法的建立及应用

100鉴别H5亚型两种分支禽流感病毒双重实时荧光RT-PCR方法的建立及应用彭　程1，刘　朔1，张　琳1，2，李金平1，于晓慧1，侯广宇1，王静静1，李　阳1，蒋文明1，刘华雷1（1.中国动物卫生与流行病学中心，山东青岛　266032；2.山东农业大学动物科技学院，山东泰安　271000）摘　要：为准确区分H5亚型禽流感病毒2.3.2.1分支和2.3.4.4分支的毒株，通过对GenBank和GISAID数据库中发表的以及本实验室保存的H5亚型禽流感病毒HA基因序列进行比对，设计1对特异性引物和2条探针，建立了区分H5亚型禽流感病毒不同分支毒株的实时荧光RT-PCR方法，并对该方法的反应体系和反应参数进行了优化，同时开展了特异性和敏感性试验，以及临床应用检测。

结果显示：该方法具有良好的特异性，与其他亚型禽流感病毒及常见禽病病毒无交叉反应；对H5亚型禽流感病毒2.3.2.1分支和2.3.4.4分支毒株的检测灵敏度分别为495.0 copies/μL和22.3 copies/μL；100份临床家禽拭子禽流感病毒检测结果与常规RT-PCR和病毒分离检测结果一致，符合率为100%。

结果表明，该方法特异、敏感、准确、耗时少，同时还可区分不同分支，可应用于H5亚型禽流感病毒的准确、快速检测。

关键词：H5亚型禽流感病毒；分支；双重实时荧光RT-PCR；敏感性；特异性中图分类号：S852.65 文献标识码：B 文章编号：1005-944X（2021）01-0100-06DOI：10.3969/j.issn.1005-944X.2021.01.020 开放科学（资源服务）标识码（OSID）：Establishment and Application of a Duplex Real-time RT-PCR Assay for Identifying Two Branches of H5 Subtype Avian Influenza VirusPeng Cheng1，Liu Shuo1，Zhang Lin1，2，Li Jinping1，Yu Xiaohui1，Hou Guangyu1，Wang Jingjing1，Li Yang1，Jiang Wenming1，Liu Hualei1（1. China Animal Health and Epidemiology Center，Qingdao，Shandong　266032，China；2. College of Animal Science，Shandong Agricultural University，Tai'an，Shandong　271000，China）Abstract：In order to accurately distinguish the strains of two evolutionary clades including 2.3.2.1 and 2.3.4.4 of H5 subtype avian inﬂuenza virus（AIV），a pair of speciﬁc primers and two Taq Man probes were designed by comparing HA gene sequences of H5 subtype AIV registered in Genbank and GISAID with the ones reserved in our laboratory，a duplex real-time RT-PCR assay was established to distinguish different clades of the virus，and its reaction system and parameters were optimized. Meanwhile，the speciﬁcity，sensitivity and its clinical application were tested. The results showed that，the speciﬁcity of the method was good with no any cross-reaction with other subtypes AIV or common avian viruses；the detection limits of clades 2.3.2.1 and 2.3.4.4 were 495.0 and 22.3 copies/μL，respectively；100收稿日期：2020-10-10　修回日期：2020-11-09基金项目：国家重点研发计划项目（2017YFC1200503）同等贡献作者：彭程，刘朔通信作者：蒋文明。

采用重叠PCR构建高灵敏度酵母细胞传感器评估遗传毒性化合物_NormalPdf

1材料
1. 1 质粒与菌株酵母重组质粒 pRNR2-yEGFP 由扬州大学医学
院预防医学系李湘鸣教授赠送；大肠埃希菌 DH5α 感受态细胞（上海生工生物工程有限公司）；酿酒酵母 BY4741、pESC-Leu 质粒、pESC-His 质粒（上海柯雷生物科技有限公司）。 1. 2 试剂
SD/-Ura 、SD/-Leu 、SD/-His 、SD/-His-Leu 、SD/ -Ura-Leu、SD/-Ura-His、SD/-Ura-His-Leu 培养基（上海艾礼生物科技有限公司）；PrimeSTAR Max DNA 聚合酶（日本 TaKaRa 生物股份有限公司）；PCR 产物纯化试剂盒、无毒核酸染料、TE 缓冲液、TAE 缓
学报
236
Journal of China Pharmaceutical University 2021，52（2）：236 - 244
采用重叠 PCR 构建高灵敏度酵母细胞传感器评估遗传毒性化合物
何颖 1，2，夏星雅 1，2，魏嘉利 1，郑枫 1，2*
（1中国药科大学药物分析学教研室; 2教育部药品安全与预警重点实验室，南京 210009）
通过采用细胞壁合成与药物转运突变体改善酵母细胞渗透性，已经实现酵母细胞传感器的检测灵敏度与特异性的提高，敲除细胞壁合成基因 erg6、cwp1 和 cwp2 与膜转运蛋白基因 pdr5、snq2 和 yor1 是提高酵母细胞传感器检测遗传毒性灵敏度的有效方法。［18-19］酵母细胞具有较高的同源重组频率和较短的同源片段长度需求，因此采用 40 bp 的同源臂的一步 PCR 产物转化能够进行目标基因的敲除［20］。为了提高酵母细胞基因敲除的效率，需要设计更长的同源臂，而一步 PCR 产物转化法难以满足，通常需要采取酶切、连接的方法构建基因敲除组件［19］。本研究采用重叠 PCR（overlap PCR）构建 pdr5 与 snq2 基因敲除组件，设计的同源臂长度为 600~700 bp，相较于传统方法具有成本更低、操作简便的优点。构建了 pRNR2 调控的基因突变型酵母细胞传感器对遗传毒性化合物进行定量评估，并比较了不同基因突变对酵母细胞传感器检测准确度与灵敏度的影响。

SOX17_过表达慢病毒载体和稳定转染细胞系的构建

第 49 卷第 6 期2023年 11 月吉林大学学报（医学版）Journal of Jilin University（Medicine Edition）Vol.49 No.6Nov.2023DOI：10.13481/j.1671‑587X.20230603SOX17过表达慢病毒载体和稳定转染细胞系的构建黄少婷1,2, 李友1,2, 吴钊淳2, 何嘉文2, 廖科棋2, 李胜男1,2（1. 广东医科大学广东省衰老相关心脑疾病重点实验室，广东湛江524002；2. 广东医科大学附属医院神经病学研究所，广东湛江524002）［摘要］目的目的：构建性别决定区Y盒17（SOX17）过表达慢病毒载体，使用SOX17过表达慢病毒感染PC12细胞并建立稳定过表达SOX17的细胞系。

方法：在NCBI数据库中查找、设计并合成SOX17过表达序列，将其与经Bam HⅠ和AgeⅠ双酶切的慢病毒GV492载体连接，构建GV492-SOX17过表达重组质粒。

琼脂糖凝胶电泳鉴定PCR产物，筛选携带GV492-SOX17过表达重组质粒的阳性菌，克隆后测序。

将GV492空载质粒和GV492-SOX17过表达重组质粒分别转染至人胚肾HEK 293T细胞中，转染48 h后收集GV492对照慢病毒和GV492-SOX17过表达慢病毒进行包装并测定病毒滴度。

将PC12细胞分为空白组、GV492对照组和GV492-SOX17组，空白组不作处理，GV492对照组和GV492-SOX17组分别采用相应慢病毒感染细胞（感染复数＝100），10 mg·L－1嘌呤霉素筛选成功感染慢病毒的PC12细胞，荧光显微镜观察各组PC12细胞生长状态及绿色荧光表达情况。

采用实时荧光定量PCR（RT-qPCR）法检测各组PC12细胞中SOX17 mRNA表达水平，Western blotting 法检测各组PC12细胞中SOX17蛋白表达水平。

结果结果：GV492-SOX17过表达重组质粒的基因片段长度约为744 bp， GV492-SOX17过表达重组质粒基因序列与设计合成的SOX17过表达序列一致。

gexp多重rt-pcr技术在儿童呼吸道病毒感染病原体检测中...

GeXP多重RT-PCR技术在儿童呼吸系统病毒感染病原体检测中的应用于天晓石仲仁李贵霞郭巍巍杨硕王乐【摘要】目的利用GeXP多重基因表达遗传分析系统联合多重逆转录-聚合酶链反应(mRT-PCR)方法同时检测20种呼吸道病毒并探讨其在儿童呼吸系统病毒感染病原体检测中的价值。

方法收集2013年3月-2014年11月在河北省儿童医院呼吸科住院的1305例，年龄0-6岁呼吸系统病毒感染的患儿咽拭子和痰液样本，提取病毒DNA/RNA，利用mRT-PCR扩增技术，通过GeXP分析平台进行毛细管电泳，同时检测20种呼吸道病毒并评价其检测效能。

结果1305例呼吸系统病毒感染患儿病毒检出阳性率为58.77%。

（1）病毒种类：呼吸道合胞病毒阳性率最高（17.32%）；其次，副流感病毒3型，阳性率为16.02%；鼻病毒、腺病毒、博卡病毒和副流感病毒1型的阳性率分别为11.95%、5.06%、3.14%和2.07%。

此外，同时感染两种、以及两种以上病毒的混合感染患儿为92例，阳性率7.05%。

（2）患儿年龄：1-2岁患儿病毒检出率最高（83.61%）；5-6岁患儿病毒检出率最低（39.13%）。

结论利用GeXP分析平台联合多重RT-PCR技术同时检测20种呼吸道病毒，具有高通量、高灵敏度、特异性强且检验速度快等优点，其高效性能够充分满足临床对呼吸道病毒检测的要求，并为儿童急性呼吸道感染的防治提供数据资料，从而指导临床诊断和治疗，避免抗生素的滥用。

【关键词】GeXP分析平台；多重RT-PCR；儿童呼吸系统感染The application of GeXP based multiplex RT-PCR assay for the detection of pathogens in children with viral infection of the respiratory system Yu Tianxiao*,Shizhongren, Li Guixia, Guo Weiwei, Yang Shuo, Wang Le. *Pediatric Research Institute, Children’s Hospital of Hebei Province，Shijiazhuang 050031，chinaCorresponding author：Li Guixia，Email：138****************【Abstract】Objective Combined with the mR T-PCR (multiplex Reverse Transcription-Polymerase Chain Reaction), we used the GeXP (genomelab genetic analysis system) assay to simultaneously detect twenty kinds of virus from respiratory tract; the value of conducting this method in the children respiratory system was also discussed. Methods From 2013-3 to 2013-11, 1305 inpatient children (0-6 years olds) of Children’s Hospital of Hebei Province were recruited. The throat swab and sputum sample s were collected to extract the viral DNA or RNA, mRT-PCR amplification and GeXP capillary electrophoresis were used to test 20 viruses and evaluate the testing efficiency. Results Among 1305 children, infected by respiratory tract virus, the positive relevance rate was 58.77%. (1) Kinds of virus: the highest positive relevance rate was observed on the respiratory syncytial virus (RSV, 17.32%); the parainfluenza virus 3 (PIV-3, 16.02%), human rhinovirus (HRV, 11.95%), adenovirus (ADV, 5.06%), human bocavirus (HBOV, 3.14%) and the parainfluenza virus 1 (PIV-1, 2.07%) were figured out separately; additionally, the positive mixed infection rate was 7.05% (92 children). (2) Ages of children: the highest positive relevance rate was observed in 1-2 years old children (83.61%); the lowest rate was detected in 5-6 years old children (39.13%). Conclusion the combination assay (GeXP with mRT-PCR) we used to examine twenty respiratory tract viruses has the advantages including high throughput, sensitivity, specificity and speed, etc. Its high efficiency could satisfy the clinical examining demand, provide the prevention and curing data on acute respiratory tract infection, there by, conduct the clinical diagnosis and treatment and avoid the antibiotic abusing.【Key words】GeXP; mRT-PCR; Respiratory system infection in children作者单位：050000 石家庄，河北省儿童医院儿研所（于天晓石仲仁李贵霞郭巍巍杨硕王乐）通讯作者：李贵霞，电子邮箱：138****************.急性呼吸道感染（ARTI）是世界范围内婴幼儿发病和死亡的重要病因，ARTI的病原学十分复杂，病毒在儿童急性呼吸道感染中占绝大多数(高于90％)，其中约有30％的病例虽然被认为是由病毒感染引起，但是并未阐明其确切病原[1]。

核苷酸多态性6

C LINICAL M ICROBIOLOGY R EVIEWS, 0893-8512/00/$04.00ϩ0Oct.2000,p.559–570Vol.13,No.4Copyright©2000,American Society for Microbiology.All Rights Reserved.Multiplex PCR:Optimization and Applicationin Diagnostic VirologyELFATH M.ELNIFRO,1AHMED M.ASHSHI,1ROBERT J.COOPER,1AND PAUL E.KLAPPER1,2*School of Medicine,The University of Manchester,1and Clinical Virology,Central Manchester Healthcare Trust,2Manchester,United KingdomINTRODUCTION (559)PRINCIPLE AND DEVELOPMENT OF MULTIPLEX PCR (559)Primers and Multiplex PCR Efﬁciency (559)Other PCR Components (560)Variations in Methodology To Improve Sensitivity and Speciﬁcity (561)General Considerations for Multiplex PCR Development (561)APPLICATION OF MULTIPLEX PCR IN DIAGNOSTIC VIROLOGY (561)Neurotropic Viruses (561)Respiratory Viruses (563)Genito-Urinary Infections (564)Ocular Infections (565)Immunocompromised Patients (565)Other Applications (566)CONCLUSION AND PERSPECTIVES (567)REFERENCES (568)INTRODUCTIONDuring the past decade,advances in PCR technology and other DNA signal and target ampliﬁcation techniques have resulted in these molecular diagnostics becoming key proce-dures(4,107,117).Such techniques are conceptually simple, highly speciﬁc,sensitive,and amenable to full automation(54, 115).The most mature of these technologies,PCR,is in one variant or another now common in research laboratories and is used increasingly in routine diagnostic laboratory settings and undergraduate and high-school teaching(32,38,40,101).In diagnostic laboratories the use of PCR is limited by cost and sometimes the availability of adequate test sample volume.To overcome these shortcomings and also to increase the diagnos-tic capacity of PCR,a variant termed multiplex PCR has been described.In multiplex PCR more than one target sequence can be ampliﬁed by including more than one pair of primers in the reaction.Multiplex PCR has the potential to produce con-siderable savings of time and effort within the laboratory with-out compromising test utility.Since its introduction,multiplex PCR has been successfully applied in many areas of nucleic acid diagnostics,including gene deletion analysis(19,20),mu-tation and polymorphism analysis(86,96),quantitative analy-sis(94,124),and RNA detection(51,126).In theﬁeld of infectious diseases,the technique has been shown to be a valuable method for identiﬁcation of viruses,bacteria,fungi, and/or parasites.A representative list of such agents is shown in Table1.Based upon our own experience with multiplex PCR and those of other authors appearing in the literature during the last10years,we review the theoretical and practical basis of the development and optimization of multiplex PCR systems and discuss the application and potential of this technique in theﬁeld of diagnostic virology.PRINCIPLE AND DEVELOPMENT OF MULTIPLEX PCR A number of review and research articles have provided detailed descriptions of the key parameters that may inﬂuence the performance of standard(uniplex)PCR(17,57,88,91, 112).Fewer publications discuss multiplex PCR(18,28,43).Primers and Multiplex PCR EfﬁciencyTheﬁrst few rounds of thermal cycling have substantial effect on the overall sensitivity and speciﬁcity of PCR(92). Assuming efﬁcient denaturation of the target,overall success of speciﬁc ampliﬁcation depends on the rate at which primers anneal to their target and the rate at which annealed primers are extended along the desired sequence during the early, middle,and late cycles of the ampliﬁcation.Factors preventing optimal annealing rates include poorly designed primers and suboptimal buffer constituents and annealing temperature. The extension rate of speciﬁc primer-target hybrids depends on the activity of the enzyme,availability of essential compo-nents such as deoxyribonucleoside triphosphates(dNTPs),and the nature of the target DNA.Thus,the majority of modiﬁca-tions to improve PCR performance have been directed to-wards the factors affecting annealing and/or extension rates. The optimization of multiplex PCRs can pose several difﬁ-culties,including poor sensitivity or speciﬁcity and/or prefer-ential ampliﬁcation of certain speciﬁc targets(76).The pres-ence of more than one primer pair in the multiplex PCR increases the chance of obtaining spurious ampliﬁcation prod-ucts,primarily because of the formation of primer dimers(9). These nonspeciﬁc products may be ampliﬁed more efﬁciently than the desired target,consuming reaction components and producing impaired rates of annealing and extension.Thus,the optimization of multiplex PCR should aim to minimize or*Corresponding author.Mailing address:Clinical Virology,3rd Floor,Clinical Sciences Building,Manchester Royal Inﬁrmary,Oxford Rd.,Manchester M139WL,United Kingdom.Phone:441612768849. Fax:441612768840.E-mail:pklapper@.559reduce such nonspeciﬁc interactions.Empirical testing and a trial-and-error approach may have to be used when testing several primer pairs,because there are no means to predict the performance characteristics of a selected primer pair even among those that satisfy the general parameters of primer design (43).However,special attention to primer design pa-rameters such as homology of primers with their target nucleic acid sequences,their length,the GC content,and their con-centration have to be considered (26,65,72,88,95,120).Ideally,all the primer pairs in a multiplex PCR should enable similar ampliﬁcation efﬁciencies for their respective target.This may be achieved through the utilization of primers with nearly identical optimum annealing temperatures (primer length of 18to 30bp or more and a GC content of 35to 60%may prove satisfactory)and should not display signiﬁcant ho-mology either internally or to one another (17,26,43).Preferential ampliﬁcation of one target sequence over an-other (bias in template-to-product ratios)is a known phenom-enon in multiplex PCRs that are designed to amplify more than one target simultaneously (68,76,109).Based on both theo-retical modeling and experimental studies,two major classes of processes that induce this bias have been identiﬁed,PCR drift and PCR selection (108).PCR drift is a bias assumed to be due to stochastic ﬂuctuation in the interactions of PCR reagents particularly in the early cycles,which could arise in the pres-ence of very low template concentrations (26,68);variations in the thermal proﬁles of a thermocycler,resulting in unequal ramping temperatures;or simple experimental error.PCR se-lection,on the other hand,is deﬁned as a mechanism which inherently favors the ampliﬁcation of certain templates due to the properties of the target,the target’s ﬂanking sequences,or the entire target genome.These properties include interregion differences in GC content,leading to preferential denatur-ation;higher binding efﬁciency because of GC-rich primers;differential accessibility of targets within genomes due to sec-ondary structures;and the gene copy number within a genome.In addition,the choice of primers has been shown to be crucial to avoid PCR selection.Ampliﬁcation biases that were strongly dependent on the choice of primers and dependent to a lesser extent on the templates have been described (100).Someprimer pairs with high ampliﬁcation efﬁciency resulted in tem-plates being saturated (plateau phase),while other primer pairs produced product independent of starting template con-centrations.Primers with lower ampliﬁcation efﬁciency re-sulted in product concentrations below the saturation concen-trations,and depending on the template,either the expected product ratio or bias was observed.Other PCR ComponentsAlteration of other PCR components such as PCR buffer constituents,dNTPs,and enzyme concentrations in multiplex PCR over those reported for most uniplex PCRs usually results in little,if any,improvement in the sensitivity or speciﬁcity of the test.Increasing the concentration of these factors may increase the likelihood of mis-priming with subsequent pro-duction of spurious nonspeciﬁc ampliﬁcation products.How-ever,optimization of these components in multiplex PCRs that are designed for simultaneous ampliﬁcation of multiple targets may prove beneﬁcial.For example,in the multiplex PCR for the dystrophin gene (nine genomic targets),a Taq DNA poly-merase concentration (with an appropriate increase in MgCl 2concentration)four to ﬁve times greater than that required in uniplex PCR was necessary to achieve optimal nucleic acid ampliﬁcation (19).Variation in concentrations of reaction components above those used in uniplex PCR probably reﬂects the competitive nature of the PCR process.The desired target DNA can be outcompeted by the more efﬁcient ampliﬁcation of other targets (including nonspeciﬁc products),leading to decreases in the efﬁciency of the ampliﬁcation of the desired targets and hence sensitivity of the reaction (79).PCR additives,such as dimethyl sulfoxide,glycerol,bovine serum albumin,or betaine,have been reported to be of beneﬁt in multiplex PCRs (49,62).The components may act to pre-vent the stalling of DNA polymerization,which can occur through the formation of secondary structures within regions of template DNA during the extension process (44).Such cosolvents may also act as destabilizing agents,reducing the melting temperature of GC-rich sequences,or as osmopro-TABLE 1.Representative list of applications of multiplex PCR to the diagnosis of infectious diseasesInfectious agentPathogens targetedClinical manifestation(s)and/or specimenReference(s)Virus HIV-1,HIV-2,HTLV-1,and HTLV-2Blood45HSV-1,HSV-2,VZV,CMV,HHV-6,EBV,and EVs a Meningitis,encephalitis,or meningoencephalitis;CSF 13,14BacteriumHaemophilus inﬂuenzae ,Streptococcus pneumonia ,Mycoplasma catarrhalis ,and Alloiococcus otitidis Upper respiratory tract 42Campylobacter jejuni and Campylobacter coliHuman campylobacteriosis 37Actinomyces actinomycetemcomitans ,Porphyromonas intermedia ,and Porphyromonas gingivalis Periodontal infection 34N.gonorrhoeae and C.trachomatisGenital infections 60,118C.trachomatis ,N.gonorrhoeae ,Ureaplasma urealyticum ,and M.genitalium Genital infections 59Parasite Giardia lamblia and Cryptosporidium parvum Diarrheal disease;water 52,89Leishmania spp.Leishmaniasis5,39CombinationHSV,H.ducreyi ,and T.pallidum Genital ulcer disease 7,73HPVs,HSV,and C.trachomatisGenital swabs64Adenovirus,HSV,and C.trachomatisKeratoconjunctivitisYeo et al.b EV,inﬂuenza viruses A and B,RSV,PIV types 1and 3,adenovirus,M.pneumoniae ,and C.pneumoniaeAcute respiratory tract infections36a EVs,enteroviruses.bAbstr.97th Gen.Meet.Am.Soc.Microbiol.,1997.560ELNIFRO ET AL.C LIN .M ICROBIOL .R EV .tectants,increasing the resistance of the polymerase to dena-turation(44,83).Variations in Methodology To ImproveSensitivity and SpeciﬁcityA straightforward solution to difﬁculties encountered in the development of multiplex PCR has been the use of hot start PCR(21)and/or nested PCR(123).The former often elimi-nates nonspeciﬁc reactions(particularly production of primer dimers)caused by primer annealing at low temperature(4to 25°C)before commencement of thermocycling(21).The pro-cedure has recently been made more practicable through the use of a nonmechanical hot start methodology which involves the use of a form of Taq polymerase,for example,Amplitaq Gold(Roche Diagnostics),which is activated only if the reac-tion mixture is heated at approximately94°C for10min(the ﬁrst denaturation step)(8,53).Nested PCR increases the sensitivity and speciﬁcity of the test through two independent rounds of ampliﬁcation using two discrete primer sets.Al-though this adaptation is undoubtedly effective in most cases,it also considerably complicates the practical application of PCR. The second round of ampliﬁcation delays results,increases the possibility of cross-contamination,and may complicate auto-mation.General Considerations for Multiplex PCR Development Development of multiplex PCRs should follow a rational approach for the inclusion or exclusion of speciﬁc pathogens in the assay.These pathogens can be organ system speciﬁc or symptom speciﬁc with respect to the age of the patient and the epidemiological characteristics of these pathogens.PCR con-ditions,such as compatibility among the primers within the reaction mixture such that there is no interference,is of great technical importance.The primer pairs must be inclusive for as many strains of the target pathogen as possible,and depending on the amplicon detection method,their targets are easily resolvable.The latter may be achieved by using primer pairs that result in PCR products that can be separated and clearly visualized using gel electrophoresis or hybridization probes with maximum speciﬁcity.Prior to application in a clinical setting,multiplex PCRs must be evaluated for their sensitivity as compared with their corresponding uniplex PCRs using both serial dilutions of the target DNA and clinical specimens. Wherever possible,multiplex PCRs should avoid the use of nested primers requiring a second round of ampliﬁcation.The latter is a major contributor to false-positive results due to carryover contamination,although anticontamination proto-cols including PCR controls(reaction and specimens extrac-tion controls)must be implemented in all PCR-based proto-cols(55).Likewise,precautions and methodologies to avoid false-negative results due to reaction failure have to be con-sidered(104).Multiplex PCRs that amplify target sequences along with the presence of external or internal control target nucleic acids to indicate reaction failure have been developed (49,62,69).APPLICATION OF MULTIPLEX PCR INDIAGNOSTIC VIROLOGYDuring the last decade,a number of studies have demon-strated the practicality of identifying viral pathogens in many clinical and epidemiological settings using multiplex PCR(Ta-ble2).The technique has been used to screen for individual or symptom-associated viruses and examine associations of virus infection with disease.In addition,the technique has been shown to be a powerful and cost-effective tool for typing and subtyping virus strains in different epidemiological studies.Neurotropic VirusesPCR has proved to be a powerful tool for investigating meningitis and encephalitis caused by a variety of viruses.In neurological disease the requirement of rapid and reliable diagnosis to provide a rational basis for chemotherapy and limit unnecessary procedures and irrelevant therapy has driven development.The wide range of viruses associated with neu-rological disease includes herpes simplex virus(HSV);cyto-megalovirus(CMV);varicella-zoster virus(VZV);Epstein-Barr virus(EBV);human herpes virus6(HHV-6);the enterovirus group,including echoviruses,polioviruses,and coxsackieviruses;adenoviruses;JC and BK viruses;arenavi-ruses;paramyxoviruses;rabies;and arboviruses.In view of the large number of potentially neuroinvasive viruses and because of the limited volume of the most useful diagnostic specimen—cerebrospinalﬂuid(CSF)—a number of multiplex PCRs have been developed(12–15,82,102).The feasibility of simultaneous screening for viruses,bacte-ria,and parasites in CSF specimens from patients with aseptic meningitis or encephalitis has been described(82).This study by Read et al.(82)utilized three nested multiplex PCRs for detection of HSV and VZV;EBV and HHV-6;and members of the enterovirus group and echovirus type22and23.In addition,two uniplex PCRs were used for detection of CMV and JC virus.In a total of2,233CSF specimens from2,162 patients,the PCR was positive in147specimens from143 patients(6.6%of all patients)including enteroviruses(77pa-tients),HSV-1(20patients),VZV(7patients),HSV type2 (HSV-2)(6patients),CMV(3patients),JC virus(2patients), and HHV-6(1patient).All PCR assays remained negative with28control CSF specimens.The clinical sensitivity and speciﬁcity of this PCR were not determined because full clin-ical information was not available for all of the patients.A nested multiplex PCR for detection and differentiation of HSV-1and-2on the basis of PCR product size has also been described(14).In a prospective analysis,a total of417CSF specimens obtained from395consecutive patients with clinical suspicion of HSV encephalitis,meningitis,or meningoenceph-alitis were tested by multiplex PCR.The test was positive for HSV-1in11specimens(2.6%)from10patients and for HSV-2 in4specimens(1.0%)from3patients;no coinfection with both types was reported.The same multiplex PCR was used to test a total of178CSF samples obtained from171patients with clinical suspicion of herpes virus infection(15).The assay was positive for HSV-1in three samples(1.7%)from two patients (1.2%)and for HSV-2in one sample,and one patient tested positive in a nested uniplex CMV PCR.A similar procedure to detect the DNA of both viruses(HSV-1and-2)was applied to CSF samples from918human immunodeﬁciency virus(HIV)-infected patients with neurological symptoms(22).In patients for whom a diagnosis was conﬁrmed at autopsy,the test was positive for HSV-1or-2for19patients(2%),producing a sensitivity and speciﬁcity of100and99.6%,respectively. Theﬁrst nested multiplex PCR for detection and typing of herpesviruses(HSV-1and-2,VZV,CMV,HHV-6,and EBV) was applied to CSF from patients with meningitis,encephalitis, and other clinical syndromes(102).By utilizing equimolar con-centrations of primers aligning the3Јends with one of two consensus regions within the herpesvirus DNA polymerase gene and the5Јends with the related or nonrelated sequences of each agent to be ampliﬁed,theﬁrst round of ampliﬁcation yielded a194-bp fragment indicating the presence of herpes-V OL.13,2000MULTIPLEX PCR IN DIAGNOSTIC VIROLOGY561virus.The second round of ampliﬁcation utilizing primer mix-tures contained nonhomologous and type-speciﬁc primers se-lected from different regions of the aligned DNA polymerase genes of human herpesviruses produce a product with a dif-ferent size for each related virus.The method ampliﬁed the corresponding virus in infected cells and inﬁve clinical samples (HSV-1PCR-positive CSF from a patient with encephalitis, HSV-2PCR-positive CSF from a patient with meningitis,VZV culture-positive vesicularﬂuid from a patient with shingles, CMV culture-positive urine from a congenitally infected pa-tient,and EBV PCR-positive peripheral blood from a patient with a lymphoproliferative syndrome).In addition,the use of primers targeting consensus regions may allow recognition of new,undescribed human herpesviruses.The detection of a 194-bp fragment after theﬁrst reaction with no positive signal in the second round of ampliﬁcation could reﬂect the detection of a new human herpesvirus.The test was further modiﬁed to include a reverse transcription step and primer pairs to detect enterovirus cDNA(12).This PCR was then evaluated in21 patients with etiologically well-characterized aseptic meningitis and encephalitis.HSV DNA was detected in nine patients, VZV DNA was detected in6patients,and enterovirus RNA was detected in6patients.The test was further evaluated for detection of these same viruses in CSF samples by a prospec-tive study of200neurological-disease patients suspected to have viral infections.Enterovirus was detected in49patients, HSV was detected in3patients,VZV was detected in6pa-tients,CMV was detected in12patients,EBV was detected in 2patients,CMV and HSV were detected in one AIDS patient with encephalitis,and CMV and EBV were detected in an-other AIDS patient with polyradiculomyelitis.For detection of echovirus30in50patients with aseptic meningitis,the multi-plex reverse transcription(RT)-PCR was more sensitive(90% sensitivity)than cell culture(26%sensitivity)and the Amplicor EV test(86%sensitivity).These studies demonstrate the utility of this multiplex RT-PCR for detection of enteroviruses and herpesviruses in CSF samples from patients with various neu-rological manifestations and the usefulness of the technique in patient management and design of antiviral therapy.In the United Kingdom,a nested multiplex PCR for the detection of HSV-1and-2,VZV,and enteroviruses,the four most common causes of viral meningitis and encephalitis,was developed and evaluated using a total of1,683consecutive CSF samples(81).The test was positive in138(8.2%)of the specimens(enteroviruses in51samples,HSV-2in33samples, VZV in28samples,and HSV-1in25samples).Of the51 patients positive for enterovirus RNA,17were babies less than 6months old in whom the CNS infection was detected as partTABLE2.Application of multiplex PCR for diagnosis of viral infectionsClinical manifestation(s)Specimen(s)Viruses and/or other agent(s)targeted Reference(s)Meningitis,encephalitis, and/or meningo-encephalitis CSF HSV-1,HSV-2,and CMV14HSV and VZV;EBV and HHV-681HSV-1,HSV-2,VZV,CMV,HHV-6,and EBV102HSV-1and HSV-215HSV-1,HSV-2,VZV,CMV,HHV-6,EBV,andEVs a12,13CMV,EBV,HHV-6,HHV-7,and HHV-877EBV and T.gondii87Upper and lower respiratory infections Throat,nose,and nasopharyngealswabs;nasopharyngeal andendotracheal aspirates;bronchoalveolar lavageInﬂuenza viruses A and B30PIV types1,2,and327Inﬂuenza virus and RSV98RSVs A and B,inﬂuenza viruses A and B,PIVtypes1,2,and333RSV,PIVs,adenovirus74EV,inﬂuenza viruses A and B,RSV,PIV types1and3,adenovirus,M.pneumoniae,C.pneumoniae36Conjunctivitis,keratitis, keratoconjunctivitis Conjunctival and corneal swabs HSV-1and HSV-215Adenovirus and HSV49Adenovirus,HSV,and C.trachomatis Yeo et al.6Genital ulcer disease Genital ulcer swabs HSV,H.ducreyi,and T.pallidum7,66,73 Genital lesions Lesion and endocervical swabs HPVs,HSV,and C.trachomatis64HPV-associated genital disease Cervical scrapings,smears,andbiopsies;vaginal and vulvalswabsHPVs23,35,56,75,97,123Vesicular rashes Vesicleﬂuids HSV and VZV3 Hepatitis Serum and plasma samples HBV genotypes84HCV,HGV,and GB viruses16Immunocompromised status Plasma HHV-6and HHV-767 Blood HIV-1,HIV-2,HTLV-1,HTLV-245a EVs,enteroviruses.b Abstr.97th Gen.Meet.Am.Soc.Microbiol.,1997.562ELNIFRO ET AL.C LIN.M ICROBIOL.R EV.of a general infection screen and34patients were older chil-dren and adults who had encephalitis and meningitis.In the group positive for VZV(28patients),16patients had menin-gitis and10had encephalitis but clinical details were not avail-able for2patients.HSV-1was detected in two babies less than 6months old and in23adults(22had encephalitis and1had a benign lymphocytic meningitis).The HSV-2-positive patients (33patients)includedﬁve babies less than6months old,two adults with meningoencephalitis,and26patients more than6 months old with benign lymphocytic meningitis.These tests proved suitable for routine use in a diagnostic laboratory and highlighted the importance of screening for more than one virus in patients with meningitis and encephalitis. Although the studies described above(13–15,81,82,102) produced satisfactory results in terms of simultaneous screen-ing for neurological manifestation-associated viruses(for ex-ample herpesviruses),the multiplex PCRs developed utilized a nested strategy.The latter as described earlier may increase the chance of false-positive results due to contamination and may also complicate automation.A recent study(63)utilized a PCR assay which precludes the use of nested primers for si-multaneous ampliﬁcation of herpesviruses DNAs.This assay, termed consensus PCR,uses a pair of“stair”primers,which are based on consensus sequences selected from within the DNA polymerase gene and were76to86%identical to the genomic sequences of the six herpesviruses(HSV-1,HSV-2, CMV,EBV,VZV,and HHV-6)that may infect the CNS.Each stair primer used comprised an equimolar mixture of11oli-gonucleotides corresponding to a consensus sequence:all primers had the same5Јend but extended for20to30nucle-otides in the3Јdirection.The PCR products were analyzed by hybridization in microtiter plates using virus-speciﬁc,biotinyl-ated oligonucleotide probes.The consensus PCR was evalu-ated using142CSF samples previously tested by standard uniplex PCRs.Eighteen samples(12.7%)tested positive by the uniplex PCRs,and37(26%)tested positive by the consensus PCR,including3samples that had coinfections(CMV,VZV, and HSV-2;VZV and HSV-2;and CMV and HHV-6).Of the 142CSF samples,103were classiﬁed as negative by both the uniplex PCRs and the consensus PCR.In addition,the test showed high diagnostic utility in that several cases were found to be positive for viruses for which tests were not requested by the clinician.The problem in evaluating all of the aforementioned multi-plex PCR studies is that most have not included complete patient detail.Thus,while many positive results have been related to compatible clinical illness,positive results are not reported in all patients with similar conditions.Nevertheless, the multiplex PCR detects more positive specimens and is more rapid than conventional techniques such as culture or serology.However,the latter procedures are either insensitive or slow and make an unsatisfactory yardstick(“gold standard”) against which to measure the accuracy of multiplex PCR.Be-cause uniplex PCR has more data available and thus is better substantiated as to its clinical value,results of multiplex PCR should be compared with those of uniplex PCR to ensure that multiplex PCR has equivalent sensitivity,speciﬁcity,and clin-ical relevance.Respiratory VirusesViruses that commonly cause respiratory infection include respiratory syncytial virus(RSV),inﬂuenza viruses and para-inﬂuenza viruses(PIV),and adenovirus,especially in infants and young children.Infection with these viruses may result in severe lower or upper respiratory tract disease requiring hos-pitalization.Thus,sensitive and rapid testing for these viruses is crucial to reduce the potential of nosocomial transmission to high-risk patients,limit unnecessary antibiotic use,and direct appropriate therapy following a speciﬁc diagnosis(119).For this reason,a number of studies have aimed to develop and evaluate multiplex PCR for detection of these viruses and provided substantial evidence of the utility of this technique as an important tool for management of patients presenting with respiratory infections.A number of studies have utilized mul-tiplex PCR to both detect and type or subtype inﬂuenza vi-ruses,PIVs,and RSV in clinical specimens and are summa-rized below.A nested multiplex RT-PCR which included three primer pairs in each round of ampliﬁcation was utilized for the simul-taneous detection,typing,and subtyping of inﬂuenza type A (H3N2and H1N1)and typeB viruses in a prospective surveil-lance of inﬂuenza in England in the1995–1996winter season (30).A total of619combined nose and throat swabs from patients with an inﬂuenza-like illness were analyzed by culture and multiplex PCR.The multiplex RT-PCR detected inﬂuenza viruses in246(39.7%)samples compared to the200(32.3%) which yielded inﬂuenza viruses in culture.In addition,there was excellent correlation between the multiplex RT-PCR and culture for typing and subtyping of inﬂuenza viruses(100%) and for temporal detection of inﬂuenza A H3N2and H1N1 viruses.It was concluded that whereas the multiplex RT-PCR demonstrated its utility in detection of inﬂuenza viruses in patients with inﬂuenza-like illness,patients with inﬂuenza-like illness who are negative for inﬂuenza viruses may harbor a pathogen(s)producing a syndrome difﬁcult to distinguish clin-ically from true inﬂuenza(for example,RSV).Indeed,when this multiplex RT-PCR was modiﬁed so that it was capable of detecting and subtyping inﬂuenza A(H1N1and H3N2)and B viruses as well as RSV subtypes A and B in respiratory clinical samples(98),the assay again demonstrated excellent(100%) correlation with the results of culture and serology.The ability of the test to detect viral coinfection in both simulated speci-mens and clinical samples was also demonstrated.A nested multiplex RT-PCR using three primer pairs was developed to detect PIV types1,2,and3in throat and naso-pharyngeal swabs(27).In theﬁrst round of ampliﬁcation, similar-size fragments are produced.In the second round of ampliﬁcation a series of three internal primer pairs are intro-duced,producing type-speciﬁc amplicons that were easily dif-ferentiated based on size upon gel electrophoresis.The test detected and correctly typed PIV in15isolates and26of30 (87%)previously positive nasopharyngeal specimens but re-mained negative in naso-or oropharyngeal specimens and/or culture isolates of33unrelated respiratory tract pathogens.In a modiﬁed version,the test was also used to detect RSV and adenovirus utilizingﬁve primer sets to amplify cDNA of RSV subtypes A and B;PIV types1,2,and3;and DNA of adeno-virus types1to7(74).The test was sensitive and speciﬁc for all 12tissue culture-grown prototype viruses and when applied to respiratory specimens was more sensitive(41of112)than direct immunoﬂuorescence or antigen detection following cul-ture(34of112).Among positive samples,multiple respiratory viruses were found in four specimens,further illustrating the potential utility of this multiplex PCR assay.A multiplex quantitative RT-PCR enzyme hybridization as-say(Hexaplex;Prodesse,Inc.,Milwaukee,Wis.)which com-bines primers originating from highly conserved regions of7 respiratory viruses(RSV subtypes A and B;PIV types1,2,and 3;and inﬂuenza viruses A and B)with probes for the detection of PCR products using enzyme hybridization assay has also been described(33).The assay provides rapid simultaneousV OL.13,2000MULTIPLEX PCR IN DIAGNOSTIC VIROLOGY563。

Brilliant Multiplex QPCR Master Mix, Part Number 6

Brilliant Multiplex QPCR Master Mix, Part Number 600553*************(24小时)化学品安全技术说明书GHS product identifier 应急咨询电话（带值班时间）::供应商/ 制造商:安捷伦科技贸易（上海）有限公司中国（上海）外高桥自由贸易试验区英伦路412号（邮编:200131)电话号码： 800-820-3278传真号码: 0086 (21) 5048 2818Brilliant Multiplex QPCR Master Mix, Part Number 600553化学品的推荐用途和限制用途2X Brilliant Multiplex QPCR Master Mix600553-51Reference Dye 600530-53部件号:物质用途:分析试剂。

2X Brilliant Multiplex QPCR Master Mix2.5 ml（毫升）Reference Dye0.1 ml（毫升） (100 µl 1 mM)部件号（化学品试剂盒）:600553安全技术说明书根据 GB/ T 16483-2008 和 GB/ T 17519-2013GHS化学品标识:Brilliant 多重 QPCR反应预混液，部件号600553危险性类别信号词:警告Reference Dye无信号词。

GHS标签要素物质或混合物的分类根据 GB13690-2009 和 GB30000-2013H316皮肤腐蚀/刺激 - 类别 3H320严重眼损伤/眼刺激 - 类别 2B紧急情况概述2X Brilliant Multiplex QPCR Master Mix 液体。

Reference Dye 液体。

2X Brilliant Multiplex QPCR Master Mix 无资料。

Reference Dye 无资料。

2X Brilliant Multiplex QPCR Master Mix 无资料。

pcr荧光探针法英文

pcr荧光探针法英文Polymerase Chain Reaction (PCR) is a powerful molecular technique widely used in laboratories for amplifying specific DNA sequences. It has revolutionized many fields of research, including genetics, forensic science, and molecular diagnostics. One commonly used method for detecting PCR amplification is the PCR Fluorescence Probe Technique.The PCR Fluorescence Probe Technique utilizes a fluorescent probe that binds to the amplified DNA during PCR amplification. This probe contains a specific fluorophore molecule and a quencher molecule. When the probe is intact, the fluorescence of the fluorophore is quenched due to the proximity of the quencher. However, during PCR amplification, the probe specifically binds to the target DNA sequence and gets cleaved by the DNA polymerase. This results in the separation of the fluorophore and the quencher, leading to fluorescence emission.There are various types of PCR fluorescence probes, including TaqMan probes, Molecular Beacons, and Scorpion primers. TaqMan probes are most commonly used and consist of a fluorescent dye attached to the 5' end of the probe and a quencher molecule at the 3' end. During PCR amplification, the DNA polymerase extends the probe and cleaves it, causing the fluorophore to separate from the quencher, resulting in a fluorescence signal.Molecular Beacons are hairpin-shaped probes that form a stem-loop structure when in solution. The fluorophore is attached to one end of the hairpin, and the quencher is at the other end. When theMolecular Beacon comes into contact with the specific target DNA sequence, it binds and forms a double-stranded DNA molecule, causing the fluorescence signal to be emitted.Scorpion primers are designed as a part of the PCR primer itself. They contain a specific DNA probe sequence with the fluorophore and quencher attached to opposite ends. During PCR amplification, the Scorpion primer anneals to the template DNA and upon extension, forms a hairpin structure. This separation of the fluorophore and the quencher results in the detection of fluorescence.The advantages of PCR Fluorescence Probe Technique include high specificity, sensitivity, and quantitative capabilities. It allows for the detection of specific DNA sequences in a complex mixture, with minimal risk of false-positive results. Moreover, the real-time detection capability of the fluorescence signal allows for the monitoring of the amplification reaction during each round of PCR.PCR Fluorescence Probe Technique finds applications in various fields. In genetics, it is used for genotyping and identifying genetic variations associated with diseases. In forensic science, it aids in DNA profiling and identifying individuals based on their unique DNA sequences. In medical diagnostics, it is employed for the detection of pathogens, genetic disorders, and monitoring disease progression.In conclusion, PCR Fluorescence Probe Technique is a valuable tool in molecular biology, allowing for sensitive and specific detection of DNA sequences. The use of fluorescent probesenables real-time monitoring of PCR amplification, providing researchers with valuable insights into various genetic phenomena. Advancements in this technique continue to drive advancements in genetics, diagnostics, and other scientific disciplines.。

多重pcr nc 算法

多重pcr nc 算法
多重PCR（Multiplex PCR）是一种在同一个PCR反应体系中加入多对引物，同时扩增出多个核酸片段的PCR反应。

其主要用于多种病原微生物的同时检测或鉴定某些病原微生物、某些遗传病及癌基因的分型鉴定。

多重PCR的生信算法主要包括：
1. MultiPLX：用于计算现有PCR引物的相容性评分，并对多重PCR引物进行分组。

2. Oli2go：将多个序列作为输入，为所有序列设计多重引物和探针，其特异性检查不仅限于单个物种，而且可以在多种物种上进行。

3. MPprimer和PrimerStation：设计多重引物组，其扩增子的大小不同，以便通过电泳分离。

4. MCMC-ODPR：采用Markov chain Monte Carlo优化方法，围绕单核苷酸多态性设计多重简并引物，注重引物的可重复使用性，以降低成本。

5. SADDLE：一种二聚体似然估计的模拟退火设计算法。

在大型扩增子法测序panel中实现了超低的引物二聚体水平。

请注意，对于生信算法的选择和使用，应依据实际研究需求和目标来选择最合适的方法。

如果需要更具体的信息或对某一算法有进一步的了解，建议查阅相关的专业文献或咨询专业人士。

A组轮状病毒原位捕获RT-qPCR检测体系的建立及评估

•轮状病毒胃肠炎的预防与控制•A组轮状病毒原位捕获RT-qPCR检测体系的建立及评估鲁飞凤吕陈昂石镇涛王大鹏上海交通大学农业与生物学院食品科学与工程系200240通信作者：王大鹏，Email:norovirus@，【摘要】目的建立基于原位捕获反转录实时定量PCR方法（/« ■s/M capture real timequantitative reverse transcription PCR，ISC-RT-qPCR)检测感染性A组轮状病毒（group A rotavirus,RVA)的体系，并对其进行评估。

方法以自行构建的NSP3重组质粒为对象，绘制标准曲线；通过优化ISC-RT-qPCR体系中猪胃粘膜提取物（porcine gastric mucin，PGM)包被浓度、孵育时间、孵育缓冲液pH和封闭时间的参数，建立、完善RVA的ISC-RT-qPCR检测体系，并评估该体系的特异性、检测限及稳定性。

结果ISC-RT-qPCR最佳试验参数分别为：PGM包被浓度为1.0 mg/mL、畔育时间为60 min、孵育pH为9.0、封闭时间为60 min;体系的检测限约为5拷贝/mL,检测特异性强、稳定性好。

结论本研究所建立的ISC-RT-qPCR是一种可用于检测感染性RVA的方法，具有操作简单、绿色环保、可高通量检测等优点，适合于临床、尤其是低病毒载量的食品和环境样品中RVA的快速检测。

【关键词】A组轮状病毒；原位捕获；反转录实时定量PCR基金项目：国家重点研发计划项目（2017YFF0210200)D0I：10.3760/cma.j.issn.1673-4092.2021.02.003Development and evaluation of in situ capture RT-qPCR for detection of group A rotavirusLu Feifeng, Lyu Chen 'ang, Shi Zhentao, Wang DapengDepartment o f Food Science and Technology, School o f Agriculture and Biology, Shanghai Jiao TongUniversity, Shanghai 200240, ChinaCorresponding author: WangDapeng,Email:*****************,Tel: 0086-21-34206918【Abstract 】Objective To develop and evaluate the in situ capture real time quantitative reversetranscription PCR (ISC-RT-qPCR)method for the detection of infectious group A rotavirus (RVA).Methods The standard curve for the assay was plotted with a self-constructed NSP3 recombinant plasmid.The ISC-RT-qPCR for the detection of RVA was established and optimized.The optimized parametersincluded coating concentration of porcine gastric mucin (PGM),incubation time,pH of incubation bufferand blocking time.Besides,the specificity,detection limit and stability of the system were evaluated.Results The optimal test parameters of ISC-RT-qPCR were:PGM coating concentration of1.0 mg/mL,incubation of samples for60 min at pH9.0 and blocking time of60 min.Under the optimal test conditions,the detection limit of ISC-RT-qPCR was about 5 copies/mL with high specificity,accuracy and stability.Conclusions The ISC-RT-qPCR established in this study is a simple,environmentally friendly and high-throughput method to detect the infective RVA and is suitable for rapid detection of low-load RVA in foodand environmental samples.【Key words 】Group A rotavirus; //?s z Y w capture;Real-time quantitative reverse transcription PCRFund program: National Key Research and Development Project of China(2017YFF0210200)DOI：10.3760/cma.j.issn.1673-4092.2021.02.003A组轮状病毒（group A rotavirus,RVA)是引之一*，每年导致约四十万患者死亡，其中80%集起全球5岁以下婴幼儿急性肠胃炎的重要病原体中在发展中国家1"。

遗传病基因突变分析

SSCP原理是在不含变性剂的中性聚丙烯酰胺凝胶中，单链DNA 迁移率除与DNA长度有关外，更主要取决于DNA单链所形成的空间构象，相同长度的单链DNA因其顺序不同或单个碱基差异，所形成的构象就会不同。PCR产物经变性后进行单链DNA凝胶电泳时，每条单链处于一定的位置，靶DNA中若发生碱基缺失、插入或单个碱基置换时, 就会出现泳动变位，从而提示该片段有基因变异存在。
DMD mix
22 l
Primer R+F (混合)
1.5 l
DNA 模板
1.5 l
DMD-e8,DMD-e19,DMD-e45,DMD-e48, DMD对照
石蜡油
1滴
放置在PCR循环仪上，循环参数设置为：
Step 1 (1个循环) 94C
Step 2 (30个循环) 94C
56C
72C
Step 3 (1个循环)
72C
5分钟 30秒 30秒
30秒
5 分钟
2. 琼脂糖凝胶电泳:
① 配制2%的琼脂糖凝胶
配制1×TBE电泳缓冲液70ml于三角烧瓶中，称取1.4克的琼脂糖粉放入后，微波炉加热熔化，冷却至60℃，加入溴化乙锭(DURED)，倒入电泳槽中，插入梳子，待凝固。
② 向电泳槽中倒入1×TBE，没过胶面2mm，小心移去梳子。
5、硝酸银价格较贵，可回收使用，适当延长处理时间。
6、PCR产物特异性不高时，要增加退火温度，减少退火时间及延伸时间，降低引物和Taq DNA聚合酶的浓度，改变Mg++浓度，一般情况下是减少浓度，减少循环次数。
(１) 8%的聚丙烯酰胺凝胶28ml，包括：
H2O 蔗糖
14.8 ml 6%
30%丙烯酰胺 7.5 ml

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10.1128/JCM.43.10.5026-5033.2005.2005, 43(10):5026. DOI:J. Clin. Microbiol. Louie and John M. ConlyKunyan Zhang, Jo-Ann McClure, Sameer Elsayed, ThomasaureusStaphylococcus Methicillin-Resistant Types I to V in mec Chromosome Subtyping of Staphylococcal Cassette Characterization and Concomitant Novel Multiplex PCR Assay for /content/43/10/5026Updated information and services can be found at: These include:REFERENCES/content/43/10/5026#ref-list-1at: This article cites 32 articles, 21 of which can be accessed free CONTENT ALERTSmore»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new /site/misc/reprints.xhtml Information about commercial reprint orders: /site/subscriptions/To subscribe to to another ASM Journal go to: on August 4, 2012 by guest/Downloaded fromJ OURNAL OF C LINICAL M ICROBIOLOGY,Oct.2005,p.5026–5033Vol.43,No.10 0095-1137/05/$08.00ϩ0doi:10.1128/JCM.43.10.5026–5033.2005Copyright©2005,American Society for Microbiology.All Rights Reserved.Novel Multiplex PCR Assay for Characterization and Concomitant Subtyping of Staphylococcal Cassette Chromosome mec TypesI to V in Methicillin-Resistant Staphylococcus aureusKunyan Zhang,1,2,3,4,5*Jo-Ann McClure,1Sameer Elsayed,2,3,5Thomas Louie,3,4and John M.Conly1,2,3,4,5Centre for Antimicrobial Resistance,Calgary Health Region/Calgary Laboratory Services/University of Calgary,Calgary,Alberta,Canada1;Departments of Pathology&Laboratory Medicine,2Microbiology and Infectious Diseases,3and Medicine,4University of Calgary,Calgary,Alberta,Canada;and Calgary Laboratory Services,Calgary,Alberta,Canada5Received21April2005/Returned for modiﬁcation15June2005/Accepted15July2005Staphylococcal cassette chromosome mec(SCC mec)typing is essential for understanding the molecularepidemiology of methicillin-resistant Staphylococcus aureus(MRSA).SCC mec elements are currently classiﬁedinto types I to V based on the nature of the mec and ccr gene complexes,and are further classiﬁed into subtypesaccording to their junkyard region DNA segments.Previously described traditional SCC mec PCR typingschemes require multiple primer sets and PCR experiments,while a previously published multiplex PCR assayis limited in its ability to detect recently discovered types and subtypes such as SCC mec type V and subtypesIVa,b,c,and d.We designed new sets of SCC mec type-and subtype-unique and speciﬁc primers and developeda novel multiplex PCR assay allowing for concomitant detection of the methicillin resistance(mecA gene)(alsoserving as an internal control)to facilitate detection and classiﬁcation of all currently described SCC mec typesand subtypes I,II,III,IVa,b,c,d,and V.Our assay demonstrated100%sensitivity and speciﬁcity in accuratelycharacterizing54MRSA strains belonging to the various known SCC mec types and subtypes,when comparedwith previously described typing methods.Further application of our assay in453randomly selected localclinical isolates conﬁrmed its feasibility and practicality.This novel assay offers a rapid,simple,and feasiblemethod for SCC mec typing of MRSA,and may serve as a useful tool for clinicians and epidemiologists in theirefforts to prevent and control infections caused by this organism.Methicillin,theﬁrst semisynthetic penicillin to be devel-oped,was introduced in1959to overcome the problem of penicillin-resistant Staphylococcus aureus due to␤-lactamase (penicillinase)production(17).However,methicillin-resistant S.aureus(MRSA)strains were identiﬁed soon after its intro-duction(2,14).MRSA strains have acquired and integrated into their genome a21-to67-kb mobile genetic element,termed the staphylococcal cassette chromosome mec(SCC mec),which harbors the methicillin resistance(mecA)gene and other an-tibiotic resistance determinants(10,12,19).Since then,strains of MRSA have spread and become established as major nos-ocomial pathogens worldwide(1,5,8,26,32).Recently,these organisms have evolved and emerged as a major cause of community-acquired infections(16,31).These newly emerging community-acquired MRSA strains possess novel,small,mo-bile SCC mec type IV or V genetic elements which contain the mecA gene with or without additional antibiotic resistance genes and are more easily transferred to other strains of S. aureus than larger SCC mec(types I,II,and III)elements(22, 31).The emerging spread of these community-acquired MRSA strains poses a signiﬁcant threat to public health(16,31). SCC mec is a mobile genetic element characterized by the presence of terminal inverted and direct repeats,two essential genetic components(the mec gene complex and the ccr gene complex),and the junkyard(J)regions(10,12,19).The mec gene complex is composed of IS431mec,mecA,and intact or truncated sets of regulatory genes,mecR1and mecI.The ccr gene complex encodes the recombinases(ccr)that mediate the integration of SCC mec into and its excision from the recipient chromosome and are,therefore,responsible for its mobility. The rest of the SCC mec element is comprised of J regions(J1, J2,and J3)that are located between and around the mec and ccr complexes and contain various genes or pseudogenes whose does not appear to be essential or useful for the bacte-rial cell,although notable exceptions include plasmid-or trans-poson-mediated resistance genes for non-␤-lactam antibiotics or heavy metals(13).To date,there are three classes(A,B, and C)of mec complex and four allotypes(types1,2,3,and5) of ccr complex.Different combinations of these complex classes and allotypes generate various SCC mec types(Table 1).SCC mec elements are currently classiﬁed into types I,II, III,IV,and V based on the nature of the mec and ccr gene complexes,and are further classiﬁed into subtypes according to differences in their J region DNA(10,12,19).A thorough understanding of the molecular epidemiology and evolution of MRSA is required to help detect,track,con-trol and prevent human disease due to this organism.Full characterization of MRSA requires deﬁnition of not only the putative bacterial genetic background but also of the complex*Corresponding author.Mailing address:Department of Pathology &Laboratory Medicine,#9-3535Research Road N.W.,Calgary,Al- on August 4, 2012 by guest / Downloaded fromoutbreaks is increasingly being recognized (16,22,31).The new MRSA nomenclature scheme recently set by the Interna-tional Union of Microbiology Societies incorporates SCC mec typing information in conjunction with that provided by mul-tilocus sequence typing (7,28,29).Previously described SCC mec traditional PCR typing schemes have required the use of many (20to 30)primer sets and multiple individual PCR experiments (23).The only previously described multiplex PCR assay for SCC mec typing (24)is more difﬁcult to interpret and is limited in its ability to detect SCC mec subtypes IVa,b,c,and d plus the newly described type V,these groups being implicated in currently emerging community MRSA outbreaks (12,31).Here we report a recently developed novel multiplex PCR assay capable of detecting and classifying all currently de-scribed SCC mec types plus the major subtypes,with simulta-neous discrimination of MRSA from methicillin-susceptible S.aureus (MSSA).MATERIALS AND METHODSBacterial strains and isolates.The SCC mec typing standard MRSA control strains,including type I (NCTC10442),type II (N315),type III (85/2082),type IVa (CA05),type IVb (8/6-3P),type IVc (MR108),type IVd (JCSC4469),and type V (WIS [WBG8318]-JCSC3624)(Table 1),were obtained from K.Hira-matsu and T.Ito at the Juntendo University in Tokyo,Japan (10,12,19,23).Additional SCC mec reference strains,including type I (COL and PER34)and type III (ANS46),were kindly provided by H.de Lencastre,Rockefeller Uni-versity,New York (24).The Canadian epidemic MRSA reference strains,CMRSA-1to 6,and strain N02-590were provided by M.Mulvey,National Microbiology Laboratory,Health Canada,Winnipeg,Canada (30).Our local strains of MRSA belonging to various SCC mec types were obtained from Calgary Laboratory Services (CLS),Calgary,Alberta,Canada,and which had previously underwent complete phenotypic and genotypic analyses at the Centre for Antimicrobial Resistance,Calgary,Alberta,Canada (Table 3).Clin-ical MRSA isolates used for assessing the applicability and utility of our multi-plex PCR (M-PCR)assay were randomly selected from the CLS frozen clinical isolate stock collected over the August 1999to November 2004time period.Additional historical clinical MRSA strains were recovered from ﬁve tertiary acute-care teaching hospitals located in 4cities in 3provinces of the Canadian Prairies (Winnipeg,Manitoba;Saskatoon,Saskatchewan;Calgary,Alberta;and Edmonton,Alberta)during the 1989to 1994period (6).Identiﬁcation and phenotypic susceptibility testing of staphylococcal isolates.The staphylococcal isolates were identiﬁed morphologically and biochemically by standard laboratory procedures (21).The coagulase plasma test (Remel,Lenexa,KS)was performed on organisms exhibiting typical staphylococcal colony mor-done by VITEK (bioMerieux,Inc.Durham,NC)along with the NCCLS oxacillin agar screen,while conﬁrmation of methicillin resistance was achieved using an in-house assay for the mecA gene (9).Sequence alignment and primer design.New sets of SCC mec type-and sub-type-unique and speciﬁc primers,as well as the novel speciﬁc primers for mecA gene,and for typing mec and ccr gene complexes (Table 2)were designed based on the comprehensive analyses and alignments of the S.aureus and MRSA genomes and SCC mec sequences currently available in the GenBank database (National Center for Biotechnology Information;updated as of December,2004).Gene targets,strains and accession numbers for each primer pair are as follows:type I,ORF E008of strain NCTC10442(AB033763);type II,kdpE of strain N315(D86934);type III,ORF CZ049of strain 85/2082(AB37671);type IVa,ORF CQ002of strain CA05(AB063172);type IVb,ORF CM001of strain 8/6-3P (AB063173);type IVc,ORF CR002of strain MR108(AB096217);type IVd,ORF CG001of strain JCSC4469(AB097677);type V,ORF V011of strain JCSC3624(AB12121);mecA ,mecA gene of strain NCTC8325(X52593);mecI ,of strain N315;IS 1272and mecR1,R of strain CA05;ccrC ,of strain JSCS 3624.The ccrAB primers are as previously described (10).The oligonucleotide primers used in this study were synthesized and purchased from QIAGEN Operon (QIAGEN Inc.,Alameda,CA).DNA extraction.Frozen bacteria were subcultured twice onto 5%sheep blood Columbia agar plates (PML Microbiologicals,Wilsonville,OR)prior to DNA extraction.For rapid DNA extraction,one to ﬁve bacterial colonies were sus-pended in 50␮l of sterile distilled water and heated at 99°C for 10min.After centrifugation at 30,000ϫg for 1min,2␮l of the supernatant was used as template in a 25-␮l PCR (33).PCR ampliﬁcation.Our SCC mec M-PCR typing assay contained 9pairs of primers including the unique and speciﬁc primers for SCC mec types and sub-types I,II,III,IVa,IVb,IVc,IVd,and V,and the primers for the mecA gene (Table 2).The M-PCR assay used for characterization of mec gene and ccr gene complexes,respectively,contained 4primers each (mecI-F,mecI-R,IS1272-F and mecR1-R for mec gene M-PCR,and ccrAB-␤2,ccrAB-␣2,ccrAB-␣3,and ccrAB-␣4for ccr gene M-PCR)(Table 2).The single target ampliﬁcation PCR was used to determine type 5ccr using ccrC-F and ccrC-R primers (Table 2).These primers and their respective concentrations used in the PCR are listed in Table 2.All PCR assays were performed directly from bacterial suspensions obtained after the rapid DNA extraction method.An aliquot of 2␮l of this suspension was added to 23␮l of PCR mixture containing 50mM KCl,20mM Tris-HCl (pH 8.4),2.5mM MgCl 2,0.2mM of each deoxynucleoside triphos-phate(dATP,dUTP,dGTP,and dCTP)(Invitrogen Inc.,Carlsbad,CA),various concentrations of the respective primers (Table 2),and 1.0unit of Platinum Taq DNA polymerase (Invitrogen Inc.,Carlsbad,CA).The ampliﬁcation was performed in a GeneAmp PCR system 9700or 9600Thermal Cycler (Applied Biosystems,Foster City,CA)beginning with an initial denaturation step at 94°C for 5min followed by 10cycles of 94°C for 45seconds,65°C for 45seconds,and 72°C for 1.5min and another 25cycles of 94°C for 45seconds,55°C for 45seconds,and 72°C for 1.5min,ending with a ﬁnal extension step at 72°C for 10min and followed by a hold at 4°C.For the single target ampliﬁcation,PCR was run in 23␮l of PCR mixture but containing 0.2␮M of each primer,with cycling parameters beginning with an initial denaturation step at 94°C for 5min followed by 30cycles of 94°C for 1min,50°C for 1min,and 72°C for 2min,ending with a ﬁnal extension step at 72°C for 10min.For comparative purposes,SCC mec typing using Oliveira’s method was performed using primer and PCR conditions described previously (24).All PCR assay runs incorporated a reagent control (without template DNA).The PCR amplicons were visualized using a UV light box after electrophoresis on a 2%agarose gel containing 0.5␮g/ml ethidium bromide.Limiting dilution experiments for estimation of M-PCR sensitivity.The sen-sitivity of ampliﬁcation of various pairs of primers by M-PCR was estimated by limiting dilution experiments.Brieﬂy,bacterial cultures from overnight growth at 37°C on 5%sheep blood agar plates were suspended in sterile saline to a density corresponding to a 1.0McFarland turbidity standard.These suspensions were then used to prepare serial 10-fold dilutions using sterile double distilled water.DNA extraction,using the rapid method described previously,was performed on 50␮l of each dilution.The standard M-PCR assay was performed to determine its sensitivity.The lower limits of detection (or minimal numbers of CFU de-tectable)of the target genes by M-PCR were then calculated based on correla-tion of the 1.0McFarland standard to 3ϫ108CFU/ml.Validation and application of SCC mec typing method.Our M-PCR assay was ﬁrst optimized in the standard control strains and then validated with other control strains,and simultaneously compared with the traditional SCC mec typing TABLE 1.Current SCC mec types and type IV subtypesSCC mec type amec complex bccr complex cOriginal strainGenBank no.(reference)I Class B Type 1NCTC10442AB033763(10)II Class A Type 2N315D86934(10)III Class A Type 385/2082AB37671(10)IVa Class B Type 2CA05AB063172(19)IVb Class B Type 28/6-3P AB063173(19)IVc Class B Type 2MR108AB096217(13)IVd Class B Type 2JCSC4469AB097677VClass C Type 5WIS [WBG8318]-JCSC3624AB12121(12)a Subtypes of SCC mec IV differ based on the junkyard (J)region DNA.bClass A mec:IS 431-mecA-mecR1-mecI ;class B mec:IS 431-mecA-⌬mecR1-IS 1272;class C mec:IS 431-mecA-⌬mecR1-IS 431.cType 1ccr :ccrB1-ccrA1;type 2ccr :ccrB2-ccrA2;type 3ccr :ccrB3-ccrA3;type 5ccr :ccrC.V OL .43,2005PCR FOR MRSA SCC mec TYPING 5027on August 4, 2012 by guest/Downloaded fromapplicability and utility of our SCC mec typing assay,453randomly selected local clinical isolates from our MRSA clinical isolate frozen stock collection for the 1989to 2004time period were tested.To verify our assay’s ability to differentiate MRSA from MSSA,comparison of our assay with standard phenotypic suscep-tibility testing (VITEK)and the conventional mecA gene PCR test (above)was conducted in 150randomly selected local clinical MSSA isolates,in addition to the above 453clinical MRSA isolates.RESULTSIdentiﬁcation and selection of unique and speciﬁc loci and primer design for SCC mec types and subtypes.To design the SCC mec type-and subtype-unique and speciﬁc primers,an extensive BLAST sequence similarity search was conducted and was followed by comprehensive analyses and alignments of the S.aureus and MRSA genomes and SCC mec sequences currently available in the GenBank database.These loci con-sisted of open reading frames (ORFs)or sequence fragments,including ORF E008of strain NCTC10442(AB033763),kdpE of strain N315(D86934),ORF CZ049of strain 85/2082MR108(AB096217),ORF CG001of strain JCSC4469(AB097677),and ORF V011of strain JCSC3624(AB12121),and were found to be unique and speciﬁc for SCC mec types and subtypes I,II,III,IVa,IVb,IVc,IVd,and V,respectively (refer to Materials and Methods for details of gene targets,strains,and accession numbers).The corresponding SCC mec type-and subtype-unique and speciﬁc primers were designed (Table 2)and their uniqueness and speciﬁcity were further conﬁrmed with a GenBank database BLAST search.Utiliza-tion of these primers in our novel M-PCR assay allowed us to speciﬁcally detect the currently described SCC mec types and subtypes of MRSA strains and clinical isolates.New M-PCR for typing and subtyping SCC mec types I to V,and simultaneous detection of methicillin resistance (mecA gene).We developed a new and simple single M-PCR assay to determine (classify)SCC mec types and subtypes I,II,III,IVa,IVb,IVc,IVd,and V,and simultaneously discriminate MRSA from MSSA.The M-PCR assay targeted the unique and spe-TABLE 2.Primers used in this studyPrimer Oligonucleotide sequence (5Ј-3Ј)Concn (␮M)Amplicon size (bp)Speciﬁcity ReferenceType I-F GCTTTAAAGAGTGTCGTTACAGG 0.048613SCC mec I This study Type I-R GTTCTCTCATAGTATGACGTCC Type II-F CGTTGAAGATGATGAAGCG 0.032398SCC mec II This study Type II-R CGAAATCAATGGTTAATGGACC Type III-F CCATATTGTGTACGATGCG0.04280SCC mec III This study Type III-R CCTTAGTTGTCGTAACAGATCG Type IVa-F GCCTTATTCGAAGAAACCG 0.104776SCC mec IVa This study Type IVa-R CTACTCTTCTGAAAAGCGTCG Type IVb-F TCTGGAATTACTTCAGCTGC 0.092493SCC mec IVb This studyType IVb-R AAACAATATTGCTCTCCCTC Type IVc-F ACAATATTTGTATTATCGGAGAGC 0.078200SCC mec IVc This study Type IVc-R TTGGTATGAGGTATTGCTGG Type IVd-F5CTCAAAATACGGACCCCAATACA 0.28881SCC mec IVd This study Type IVd-R6TGCTCCAGTAATTGCTAAAG Type V-F GAACATTGTTACTTAAATGAGCG 0.06325SCC mec V This study Type V-R TGAAAGTTGTACCCTTGACACC MecA147-F GTG AAG ATA TAC CAA GTG ATT 0.046147mecA This study MecA147-R ATG CGC TAT AGA TTG AAA GGA T mecI-F CCCTTTTTATACAATCTCGTT 0.08146Class A mec This study mecI-R ATATCATCTGCAGAATGGG IS1272-F TATTTTTGGGTTTCACTCGG0.081,305Class B mecThis study mecR1-R CTCCACGTTAATTCCATTAATACC ccrAB-␤2ATTGCCTTGATAATAGCCITCT 0.0810ccrAB-␣2AACCTATATCATCAATCAGTACGT 0.08700Type 1ccr 10ccrAB-␣3TAAAGGCATCAATGCACAAACACT 0.081,000Type 2ccr 10ccrAB-␣4AGCTCAAAAGCAAGCAATAGAAT 0.081,600Type 3ccr 10ccrC-F ATGAATTCAAAGAGCATGGC 0.08336Type 5ccrThis studyccrC-RGATTTAGAATTGTCGTGATTGC5028ZHANG ET AL.J.C LIN .M ICROBIOL .on August 4, 2012 by guest/Downloaded fromserving as a determinant of methicillin resistance but also serv-ing as an internal positive control for the assay.To ensure the individual primer pairs were adequate for the ampliﬁcation of all nine loci (gene fragments),the single-target PCR protocol with each individual primer pair was conducted prior to the M-PCR optimization,using eight SCC mec stan-dard control strains:type I (NCTC10442),type II (N315),type III (85/2082),type IVa (CA05),type IVb (8/6-3P),type IVc (MR108),type IVd (JCSC4469)and type V (WIS [WBG8318]-JCSC3624)(Tables 1and 3).Each individual PCR ampliﬁca-tion reaction yielded the fragment of the expected size,i.e.,613,398,280,776,493,200,881,325,and 147bp for the unique and speciﬁc loci of SCC mec types and subtypes I,II,III,IVa,IVb,IVc,IVd,and V,and mecA gene in their corresponding strains,respectively.The optimized M-PCR condition as de-scribed above was obtained through assaying different primer concentrations and other PCR components.Ampliﬁcation in a single M-PCR produced distinct bands corresponding to their respective molecular sizes that were easily recognizable in aga-rose gels stained with ethidium bromide (Fig.1).Sensitivity of M-PCR.The sensitivity of our M-PCR assay was examined in 8SCC mec standard control strains for type I (NCTC10442),type II (N315),type III (85/2082),type IVa (CA05),type IVb (8/6-3P),type IVc (MR108),type IVd (JCSC4469)and type V (WIS [WBG8318]-JCSC3624).This assay was capable of detecting,with reproducibility,a band in ethidium bromide-stained gels at dilutions corresponding to 6ϫ104CFU per PCR for all eight type-and subtype-speciﬁc genes.However,the sensitivity for the internal control mecA gene varied slightly depending on the strains examined,being 6ϫ105CFU per PCR for the strains NCTC10442(type I),JCSC4469(type IVd)and WIS (type V),and 6ϫ104CFU per PCR for all other type or subtype strains [N315(type II),85/2082(type III),CA05(type IVa),8/6-3P (type IVb),and MR108(type IVc)].This sensitivity is quite compatible with the single target PCR assay (1ϫ104to 6ϫ105)(data not shown),suggesting that our M-PCR assay is sufﬁciently robust.Validation of M-PCR assay.To validate the M-PCR assay,we simultaneously compared our assay with the traditional PCR SCC mec typing scheme including mec and ccr gene com-plex typing and a previously described M-PCR assay (24).Validation of our assay was performed by testing a total of 54well-characterized MRSA strains with known SCC mec types including type I (n ϭ3),type II (n ϭ14),type III (n ϭ9),type IVa (n ϭ18),type IVb (n ϭ4),type IVc (n ϭ3),type IVd (n ϭ2),type V (n ϭ1).We found a 100%concordance in typing SCC mec types I-IV between the PCR results of our M-PCR,traditional SCC mec typing method,and Oliveira’s assay (Table 3)except for one type V strain.However,in the WIS strain (type V),both our assay and the traditional SCC-mec typing method correctly identiﬁed this strain as SCC mec type V,but Oliveira’s M-PCR falsely categorized the strain as SCC mec type III (Table 3).In addition,our assay had more discriminatory power and was able to further classify type IV strains into subtypes IVa,b,c,and d (Table 3).To address our assay’s ability in differentiating MRSA from MSSA,we tested 150randomly selected local clinical MSSA isolates,in addition to the above 54MRSA control strains and isolates,hence being 100%concordant with phenotypic sus-ceptibility (VITEK)and conventional mecA gene PCR test results.Applicability and accuracy of M-PCR.To assess the appli-cability and accuracy of the M-PCR assay,we further applied our SCC mec typing assay to test a total of 453local clinical MRSA isolates randomly selected from our clinical stock col-lection for the 16-year period from 1989to 2004.Among them,235(51.88%),122(26.93%),74(16.34%),5(1.1%),and 4(0.88%)isolates belonged to SCC mec types and subtypes II,III,IVa,IVb,and IVc,but no SCC mec types and subtypes I,IVd,or V were found among the isolates tested.However,there were 13(2.87%)isolates that were not typeable using our assay,with ﬁve (1.10%)isolates having multiple bands and eight (1.77%)isolates with ampliﬁcation of only the mecA gene.These not-typeable isolates were further characterized using the traditional PCR SCC mec typing method and Oliveira’s M-PCR assay.In ﬁve multiple-band isolates,one isolate pre-senting two bands of 200bp and 280bp (corresponding to types IVc and III by our new assay)was also not-typeable by the traditional PCR but was found to be type III by Oliveira’s M-PCR,while the other four isolates with bands of 398bp and either 613bp or 200bp (corresponding to types II and either type I or IVc by our new assay)were typed as types II in both other assays (Table 4),and may represent undescribed new variant subtypes of SCC mec type II.However,among the other eight isolates with ampliﬁcation of only the mecA gene,only one isolate (mecA -band 8)was determined to be type IV by both the traditional PCR SCC mec typing method and Olivei-ra’s M-PCR assay,while the remaining (7isolates)had incon-gruent typing results among the two other typing methods (Table 4),potentially representing new types or variant sub-types.DISCUSSIONSCC mec typing is one of the most important molecular tools available for understanding the epidemiology and clonal strain relatedness of MRSA,particularly with the emerging out-breaks of community-acquired MRSA occurring on a world-wide basis.However,due to the very complex and diverse structure of the SCC mec element,SCC mec typing is usually achieved by DNA sequence analysis (21to 67kb)(10,11,25),Southern blot analysis using three or more restriction enzymes and several key probes speciﬁc for each SCC mec type (25),and by PCR.Previously described traditional PCR SCC mec typing schemes target the individual regions of the classes of the mec complex (IS431-mecA ,IS1272-mecA ,and mecI -mecRI ),the al-lotypes of the ccr complex (ccrA1,ccrA2,ccrA3,ccrB1,ccrB2,ccrB3,and ccrC ),and individual subtypes of the J regions,and therefore require the use of many primer sets and PCR exper-iments (12,23).These methods are laborious,time-consuming and expensive,resulting in limited utility for clinical and sur-veillance purposes.Oliveira and de Lencastre developed a multiplex PCR strat-egy for mec element type assignment and deﬁned types of SCC mec based on genes located within the J-regions of V OL .43,2005PCR FOR MRSA SCC mec TYPING 5029on August 4, 2012 by guest/Downloaded fromTABLE parison of our assay with the traditional PCR and an M-PCR SCC mec typing methodStrain aTraditional PCR typing bOliveira’s M-PCR cOur novel assaymec complex typeccr complex typeSCC mec typeNCTC10442B 1I I I COL B 1I I I PER34B 1I I I N315A 2II II II CMRSA-2A 2II II II MRSA-80A 2II II II CLS-5153A 2II II II CLS-5371A 2II II II CLS-440A 2II II II CLS-72251A 2II II II CLS-69500A 2II II II CLS-68961A 2II II II CLS-6146A 2II II II CLS-4021A 2II II II CLS-2516A 2II II II CLS-52692A 2II II II CLS-19095A 2II II II 85/2082A 3III IIIB III ANS46A 3III III III CMRSA-3A 3III IIIA III CMRSA-6A 3III III III CLS-5861A 3III III III CLS-1777A 3III III III H163A 3III III III H478A 3III III III H527A 3III III III CA05B 2IV IV IVa N02-590B 2IV IV IVa CLS-2207B 2IV IV IVa CLS-3860B 2IV IV IVa CLS-2772B 2IV IV IVa CLS-1236B 2IV IV IVa CLS-884B 2IV IV IVa CLS-2772B 2IV IV IVa CLS-4550B 2IV IV IVa CLS-2245B 2IV IV IVa CLS-5897B 2IV IV IVa CLS-847B 2IV IV IVa CLS-846B 2IV IV IVa CLS-2525B 2IV IV IVa CLS-3497B 2IV IV IVa CLS-5401B 2IV IV IVa CLS-5381B 2IV IV IVa CLS-284B 2IV IV IVa 8/6-3P B 2IV IV IVb CLS-4584B 2IV IV IVb CLS-5827B 2IV IV IVb CLS-6572B 2IV IV IVb MR108B 2IV IV IVc CLS-1040B 2IV IV IVc H434B 2IV IV IVc JCSC 4469B 2IV IV IVd CMRSA-5B 2IV IV IVd JCSC 3624WIS [WBG8318]C25VIIIVaThe SCC mec typing standard MRSA control strains were:type 1(NCTC10442),type II (N315),type III (85/2082),type IVa (CA05),type IVb (8/6-3P),type IVc (MR108),type IVd (JCSC4469),and type V (WIS [WBG8318]-JCSC3624);additional SCC mec reference strains were type 1(COL and PER34)and type III (ANS46);The Canadian epidemic MRSA reference strains were CMRSA-1to 6and strain N02-590.Our local SCC mec type control strains are designated CLS and H.5030ZHANG ET AL.J.C LIN .M ICROBIOL .on August 4, 2012 by guest/Downloaded fromto the kdp operon,which is speciﬁc for SCC mec type II;locus C,internal to the mecI gene present in SCC mec types II and III;locus D,internal to the dcs region present in type I,II,and IV;locus E,located in the region between integrated plasmid pI 258and transposon Tn 554,speciﬁc for SCC mec type III;locus F,which is also speciﬁc for SCC mec type-III located in the region between Tn 554and orfX ;locus G,the left junction between IS 431and pUB110;and locus H,the left junction between IS 431and pT181(24).This is the only single-step multiplex PCR assay published to date,but it too has its lim-itations.Since it is much simpler and easier to perform than the traditional (nonmultiplex)PCR assays for SCC mectyping,FIG.1.New multiplex PCR assay identiﬁes SCC mec types and subtypes I,II,III,IVa,IVb,IVc,IVd,and V,and simultaneously detects the methicillin resistance (mecA gene).Type I,lanes 1to 3(strains NCTC10442,COL,and PER34,respectively);type II,lanes 4to 6(strains N315,CLS-5153,and CLS-440,respectively);type III,lanes 7to 9(strains 85/2082,ANS46,and CMRSA-3,respectively);type IVa,lanes 10to 12(strains CA05,N02-590,and CLS-2207,respectively);type IVb,lanes 13to 15(strains 8/6-3P,CLS-4584,and CLS-5827,respectively);type IVc,lanes 16and 17(strains MR108and CLS-1040,respectively);type IVd,lanes 18and 19(strains JCSC4469and CMRSA-5,respectively);type V,lane 20(strain WIS [WBG8318]-JCSC3624);lane 21,negative control;and lanes M,molecular size markers,100-bp DNA ladder (BioLabs).Refer to Table 3for details of each strain.TABLE parison of SCCmec typing results for traditional PCR and Oliviera’s multiplex PCR assays for isolates not typeable by ourmultiplex PCR assayIsolate aOur novel assay bTraditional PCR typing cOliveira’s M-PCR dSpeciﬁc PCR product(s)(bp)Corresponding to:Multiband 1200ϩ280Type IVc ϩIII Not typeable Type III Multiband 2398ϩ613Type II ϩI Type II Type II Multiband 3398ϩ613Type II ϩI Type II Type II Multiband 4398ϩ200Type II ϩIVc Type II Type II Multiband 5398ϩ200Type II ϩIVc Type IIType II mecA band 1147mecA gene Not typeable Type IV mecA band 2147mecA gene Not typeable Not typeable mecA band 3147mecA gene Type IV Type ImecA band 4147mecA gene Type IV Not typeable mecA band 5147mecA gene Type II Type IV mecA band 6147mecA gene Type II Type IV mecA band 7147mecA gene Type I Not typeable mecA band 8147mecA gene Type IV Type IVa Not-typeable isolates (multiple bands or single mecA gene band)using our new assay.bOur new multiplex PCR assay.V OL .43,2005PCR FOR MRSA SCC mec TYPING 5031on August 4, 2012 by guest/Downloaded from。