2013 HBV检测

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HIVID技术检测HBV病毒整合

HIVID:An ef ﬁcient method to detect HBV integration using low coverage sequencing ☆Weiyang Li a ,b ,1,Xi Zeng a ,1,Nikki P.Lee c ,1,Xiao Liu a ,Shengpei Chen a ,Bing Guo a ,Shang Yi a ,Xuehan Zhuang a ,Fang Chen a ,Guan Wang a ,Ronnie T.Poon c ,Sheung Tat Fan c ,Mao Mao d ,Yingrui Li a ,Songgang Li a ,Jun Wang a ,JianWang a ,Xun Xu a ,Hui Jiang a ,Xiuqing Zhang e ,⁎aBGI-Shenzhen,Shenzhen,518083,ChinabSchool of Bioscience and Bioengineering,South China University of Technology,China cDepartment of Surgery,University of Hong Kong,Hong Kong,China dP ﬁzer Oncology,San Diego,CA 92121,USA eThe Guangdong Enterprise Key Laboratory of Human Disease Genomics,BGI-Shenzhen,Shenzhen,Chinaa b s t r a c ta r t i c l e i n f o Article history:Received 18January 2013Accepted 8July 2013Available online xxxx Keywords:Hepatocellular carcinoma Integration CaptureHigh-throughput Cost-effectiveWe reported HIVID (high-throughput Viral Integration Detection),a novel experimental and computational method to detect the location of Hepatitis B Virus (HBV)integration breakpoints in Hepatocellular Carcinoma (HCC)genome.In this method,the fragments with HBV sequence were enriched by a set of HBV probes and then processed to high-throughput sequencing.In order to evaluate the performance of HIVID,we compared the results of HIVID with that of whole genome sequencing method (WGS)in 28HCC tumors.We detected a total of 246HBV integration breakpoints in HCC genome,113out of which were within 400bp upstream or downstream of 125breakpoints identi ﬁed by WGS method,covering 89.3%(125/140)of total breakpoints.The integration was located in the gene TERT,MLL4,and CCNE1.In addition,we discovered 133novel breakpoints missed by WGS method,with 66.7%(10/15)of validation rate.Our study shows HIVID is a cost-effective methodol-ogy with high speci ﬁcity and sensitivity to identify viral integration in human genome.©2013The Authors.Published by Elsevier Inc.All rights reserved.1.IntroductionMore than 350million people are infected by hepatitis B virus (HBV)all over the world [1,2],and approximately 30%–50%of the estimated 320,000annual HBV-related deaths are consequences of hepatocellular carcinoma (HCC)[3].Previous studies have discovered evidence of the involvement of HBV in the tumorigenesis [4,5],indi-cating HBV integration in host genome is suspected to be one of the most important etiological events in HBV-induced HCC [6].The recur-rent HBV integration event was ﬁrst reported to be located at the TERT gene in two HCC tumor samples [7,8],and subsequent studies also identi ﬁed HBV integration breakpoints in FAR2,ITPR1(IP3R1),IRAK2,MAPK1,MLL2and MLL4genes [9,10].Previous reports have suggested that a number of cellular genes such as hTERT and FN1were frequently targeted by HBV in HCC tissue [7,9,10].Although HBV integration into the host genome has been reported in tumors from HBV-infected individuals,the mechanism of viral-host interactionremains elusive.However,previous studies indicated integration of HBV can change the expression level and function of endogenous parative analysis of gene expression levels indicated that samples with HBV integration demonstrated higher expression of TERT,MLL4and CCNE1than those samples not harboring HBV DNA integration [11].It is intriguing that the overall MLL4transcription output is much higher in the affected genome,but the resulting fusion transcript lacks the AT-hook DNA-binding domain of MLL4[12].Moreover,HBV integration was also reported to relate with somatic copy number variations [11].DNA copy number analysis in this region revealed that this viral integra-tion colocalized precisely with the junction of a large DNA copy number loss [12].Traditionally,most of the HBV integration breakpoints are detect-ed by PCR-based methods,such as Alu-HBV PCR.In the process of Alu-HBV PCR,signi ﬁcant bias happens towards Alu regions;in other words,only the integration near Alu region can be ef ﬁciently detected [9,13].It is still a challenge to study integrations at genome-wide scale using PCR-based methods.With the rapid development of mas-sive parallel sequencing technology,whole genome sequencing (WGS)brings new insight to detect HBV integration in HCC genome [11,12,14].However,this approach is also limited by the high cost to perform population-scale studies.Thus,a sensitive low cost method is urgently needed to study the relationship between virus integration and tumorigenesis.Genomics xxx (2013)xxx –xxx☆This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-No Derivative Works License,which permits non-commercial use,distribution,and reproduction in any medium,provided the original author and source are credited.⁎Corresponding author.Fax:+8675525273884.E-mail address:zhangxq@ (X.Zhang).1These authors contributed equally to this work.YGENO-08536;No.of pages:7;4C:0888-7543/$–see front matter ©2013The Authors.Published by Elsevier Inc.All rights reserved./10.1016/j.ygeno.2013.07.002Contents lists available at ScienceDirectGenomicsj o u r n a l h o m e p a ge :w ww.e l s e v i e r.c o m/l o c a t e /y g e n oHere,we present a novel experimental and computational meth-od,HIVID (high-throughput viral integration detection),to effectively detect hbv integration at a single base pair resolution using HBV cap-ture sequencing (Fig.1).Probes were prepared to capture the HBV inserted fragments in host genome and these fragments were further sequenced on Illumina HiSeq 2000platform.Based on the sequence data of enriched DNA,we recruited a pair-read assemble strategy to locate the integration breakpoints.To examine the performance of HIVID,this method was tested in 28HCC samples that were previously sequenced at the whole-genome level for identifying HBV integrations [11].The integration hotspots of human genome were located in the genes TERT,MLL4and CCNE1.Most of the breakpoints were signi ﬁcantly enriched in a 500bp region on HBV genome from 1500bp to 2000bp.We compared the results of HIVID and WGS method,and validated the novel HBV integration using PCR and Sanger sequencing.Our study provided an accurate and cost-effective method for HBV integration re-search,which can also be adapted to other virus integration studies.2.Materials and methodsBased on our previous study [11],28Chinese HCC samples from Queen Mary Hospital,Hong Kong were selected.All patients had been diagnosed with HCC with concurrent HBV rmed written consents were obtained from each patient.Approvals were acquired from the BGI Ethics Committee and the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster.All samples had been previously used to discover HBV integration breakpoints.The results from 30×WGS data were used to evaluate sensitivity and speci ﬁcity of HIVID.2.1.HBV probes preparationFull-length HBV genome of type B and type C were ampli ﬁed by PCR using biotin-labeled dNTPs.PCR mix was prepared as follows:5μl of HBV DNA (1ng/μl);5μl of 10*LA Taq Buffer;26.5μl of H 2O;4μl of 2.5mM dNTPs (1/4dNTPs biotin-labeled);2.5μl of 10pmol P1(TTT TTC ACC TCT GCC TAA TCA);2.5μl of 10pmol P2(AAA AAGTTG CAT GGT GCT GG);and 0.5μl of LA Taq enzyme (Takara Bio,Inc.).PCR was subjected to the following cycling condition:initial denaturation for 3min at 94°C;32cycles of denaturation for 30s at 94°C,annealing for 50s at 56°C,and extension for 2.5min at 68°C;ﬁnal extension for 10min at 72°C.Amplicons were puri ﬁed by Ampure beads (Beckman Coulter,Inc.)and were then fragmented by Covaris E-210(Covaris,Inc.,Woburn,MA)to 250bp –500bp through the 2%agarose electrophoresis.Single-stranded HBV probes were generated by high temperature denaturation at 94°C for 5min.2.2.HBV-inserted fragments enrichment and sequencingSequencing libraries of 170bp insert size were constructed for 28samples following the instruction of Illumina.Genomic DNA was sheared to around 150bp –200bp DNA fragments by Covaris E-210(Covaris,Inc.,Woburn,MA).These fragments were puri ﬁed,end blunted,“A ”tailed,and adaptor ligated.10cycles of PCR were performed after size selection in 2%agarose gel.The concentration of libraries was quanti ﬁed by Bioanalyzer 2100(Agilent Technologies,Santa Clara,CA).Libraries were hybridized with HBV probes at 47°C for 24hours and then washed to remove un-captured fragments.The eluted fragments were ampli ﬁed by 16cycles of PCR to generate libraries for sequencing (Fig.2).Libraries were quanti ﬁed and proceeded to 101cycles paired-end index sequencing in the Illumina HiSeq 2000sequencer according to manufacturer's instructions (Illumina Inc.,San Diego,CA).2.3.HBV integration breakpoints detectionThe whole pipeline for data analysis in HIVID consists of six steps (Fig.1).After we isolated the paired-end reads which may contain the signals of HBV integration,paired-end read assembly was conducted to obtain reconstructed inserted fragment and detected HBV integra-tionbreakpoints.Fig.1.Overall work ﬂow of HIVID.The pipeline includes the work ﬂow of experiment and bioinformatics process.In the process,we do Raw data ﬁlter ﬁrst,then perform Raw-mapping with ﬁltered raw data.After that we pick out the chimeric paired-end reads to do Paired-end read assembly.PE-assembled reads are conducted to go through Re-mapping to locate the HBV integrations sites.Finally,Signal merging and Signal ﬁlter are performed to obtain the ﬁnal results.To do the step of Paired-end reads assembly,we used a program developed by ourselves.2W.Li et al./Genomics xxx (2013)xxx –xxx2.3.1.Raw data ﬁlterLow quality reads and duplication reads,as well as adaptor contami-nation reads,was ﬁrst removed to obtain clean reads for subsequent analysis.If a read whose bases with quality value (b =5)occupy 50%of the read length,the read is considered as a low quality read.We then had the clean reads for detecting the HBV integration after the low qual-ity reads and duplication reads removal.2.3.2.Raw-mappingClean reads were mapped to human (NCBI build 37,HG19)and HBV genome using SOAP2(-l 40-v 5-r 1)[15].We removed reads that perfectly paired-end aligned to human or HBV genome and reserved chimeric paired-end reads (partial read sequence aligned to human genome and partial aligned to HBV genome)for assembly.This might help identify HBV integration breakpoint.2.3.3.Paired-end reads assemblyPaired-end reads assembly was used to reconstruct around 170bp of fragment sequences,assembly which might increase the potential to locate the exact position of breakpoints.The paired-end reads were ﬁrst changed to the same strand.In case that the tail of an upstream end (read1)and the head of a downstream end (read2)overlap by more than 5bp where the mismatch rate was less than 0.2,the two ends would be spliced into one continuous sequence,called PE-assembled read,which was reconstructed inserted fragment (Fig.3A).2.3.4.Re-mappingTo determine the exact location of HBV integration breakpoint,the PE-assembled reads were re-mapped to human and HBV genome using BWA 0.5.9-r16(-a 1,-b 2,-q,5,-r,2)[16].If the match length on both HBV genome reference and human genome reference for one PE-assembled read was larger than 30bp,the PE-assembled read alignment result was reported to detect a precise breakpoint.The joint position of human and HBV sequence was the breakpoints for HBV inte-gration (Fig.3B).2.3.5.Signal mergingWe also calculated the total number of support PE-assembled reads (NSS)of each HBV integration breakpoint to examine its reliability.Considering errors in the procedure of experiment and bioinformatics analysis and highly heterogeneous nature of tumors,we merged breakpoints within 20bp to select the breakpoint with largest NSS as the representative,and reset its NSS as sum of NSS of all breakpoints in this region.2.3.6.Signal ﬁlterTo minimize the impact of total sequencing data for each breakpoint frequency and remove the noise signals,we normalized NSS to Normal-ized number of support PE-assembled reads (NNSS)using its number of effective paired-reads (EFR)as following:NNSS ¼NSS Â106=EFREffective paired-reads are de ﬁned as reads used to search breakpoints except contaminated data.NNSS could be considered as the number of supporting reads of each HBV integration breakpoint out of every million read-pairs and represents the reliability and data utilization with no bias.In this study,HBV breakpoints with NNSS N =1was regarded as true signal.2.4.PCR and Sanger sequencing validationPCR and Sanger sequencing was used to verify the selected HBV integration breakpoints from HIVID.PCR primers were designed based on the PE-assembled fragment,in which one primer located in human genome and the other in HBV genome.PCR were performed by GeneAmp®PCR System 9700thermal cycler and then preceded to Sanger sequencing on Applied Biosystems 3730x DNA analyzer (Life Technologies,Inc).3.Results3.1.HBV integration breakpoints detectionTo get the overall evaluation of the accuracy of breakpoint detection using HIVID approach,we compared our results to that of previously re-ported ~30×WGS approach [11].Sung et al.reported a total of 399HBV integration breakpoints in 81HBV-positive and 7HBV-negative HCC and adjacent non-tumor samples,with a validation rate of 82.0%[11].We randomly selected 28samples for HIVID to conduct HBV capture sequencing on Illumina HiSeq 2000,generating ~1.43Gb on average (Table 1).The effect of paired-end reads for the following analysis were about 6millions,among them around 83.72%and 0.08%can be aligned to human genome and HBV genome,respectively.After paired-end assembly and re-mapping,we ﬁnally obtained an average of 1654reads supporting HBV integration breakpoints for each sample.A total of 246HBV integration breakpoints were detected within these 28sam-ples,in average 8.8breakpoints per sample.The number of integration breakpoints varied from 1to 27among samples (Table S1).3.2.HBV integration breakpoint detection comparisonWe ﬁrstly compared HBV integration breakpoints between HIVID and WGS approach in 28selected samples.To minimize the impact of different work ﬂows we de ﬁned a shared breakpoint in HIVID and WGS method when the distance of two breakpoints from the same sam-ple was less than 400bp.There were 140HBV integration breakpoints from Sung's study with at least two paired-end reads supporting and 89.3%of them (125/140)was shared with HIVID test (Tables S2and S3),suggesting that the current approach has high speci ﬁcity when com-pared with WGS approach.In contrast,only 45.9%of HBV integration breakpoints (113/246)from HIVID approach were shared with WGS approach (Fig.4).Fourteen of 113HBV integration sites that were select-ed for validation were all validated in previous WGS study,showing 100%validation rate [11].Further analysis for the average NNSSvalue,Fig.2.HBV capture work ﬂow.The different colored images marked represented differ-ent meaning.The dark yellow color represented the HBV fragments,while the glaucous color represented the human genome fragments.DNA libraries were hybridized with HBV probes at 47°C for 24hours and then washed to remove un-captured fragments.The eluted fragments were ampli ﬁed by 16cycles of PCR to generate libraries for se-quencing.(For interpretation of the references to color in this ﬁgure legend,the reader is referred to the web version of this article).3W.Li et al./Genomics xxx (2013)xxx –xxxwhich was 13.6for 113WGS-shared HBV integration breakpoints and 8.5for all breakpoints,indicated WGS approach might tend to identify high frequency HBV integration breakpoints due to a relatively limited sequencing depth.The comparative analysis indicated HIVID test could detect almost the same HBV integration breakpoints as WGS approach with much less sequencing data.In the 133novel breakpoints we randomly picked 15novel HBV integration breakpoints from six samples for PCR and Sanger sequencing validation test.66.7%(10/15)of novel HBV integration sites were suc-cessfully con ﬁrmed (Table S4),which is signi ﬁcantly lower than the validation rate for WGS-shared breakpoints.We then investigated the average NNSS value of the 133novel breakpoints,which were only 4.2,compared to 13.6for WGS-shared breakpoints and 8.5for the total ones.It was obvious that HIVID showed a notable advantage on breakpoints enrichment ef ﬁciency.On average,the HIVID approach generated 6mil-lion paired-end reads,compared to 600million read pairs for WGS meth-od.Moreover,the average NNSS values of two methods were 8.5and 0.0142,representing that HIVID approach could enrich HBV integration fragments close to 600times than WGS approach.This high ef ﬁciency for HBV integration sites enrichment would be bene ﬁt for large-scale virus-related tumor development studies.3.3.Characteristics of HBV integration breakpointsWe also investigated the characteristics of HBV integration breakpoints,which might be related with HCC development.Integra-tion events were detected in HBV subtype C (AB014381.1).Among them,33.5%(68/203;P =2.7×10−5)were signi ﬁcantly enriched in a 500bp region on HBV genome from 1500bp to 2000bp (Supplemen-tary Fig.S1),which was consistent with the results of the WGS study [11,12].Additionally,we also surveyed the frequency of integration breakpoints around HBV genes,showing 38.9%(79/203)breakpoints were located in the region of pre-S1(Table S5).The analysis for recurrent genes with HBV integration breakpoints in those 28samples was also consistent with previous studies (Fig.5)[10,17–19].Using HIVID approach,HBV integration breakpoints were detected in TERT ,MLL4and CCNE1genes,affecting 25%,17.9%,and 7.1%of HCC samples (Table 2).Notably,we identi ﬁed HBV integration breakpoint in TERT gene for sample 90T and 41T,which was missed in previous WGS approach.Besides the frequent bias of HBV integration sites in HBV and human genome,we also noticed that HBV integration events with the deletion of human genome.For example,a 49bp human genome sequence in sample 43T was replaced by a 41bp HBV sequence on ChrX:20,156,148(Table S4).In sample 49T the human genome fragment of 728,975bp in chromosome 2were replaced by 127bp HBV sequence (Fig.6).The abundant supporting reads for breakpoints provided by HIVID may be helpful to explore the complex relation.4.DiscussionIn this study,we developed a novel approach for HBV integration breakpoint detection and employed 28HCC samples to estimate the performance on integration sites with high frequency and low frequen-cy.With only 1.5Gb HBV capture sequencing data,HIVID approach can identify about 90%of HBV integration breakpoints identi ﬁed from ~30×WGS data,and over 50%of novel low frequent integration sites missed by WGS approach.HIVID approach shows its strength on several aspects compared with previously reported approaches.Unlike the widely used HBV-Alu PCR method,HIVID can be uti-lized to detect integration sites at the genome-wide level instead of the regions close to Alu repeats.In fact,83.3%(205/246)of the discov-ered HBV integration breakpoints were more than 10,000bp away from Alu repeats (Table S2),signi ﬁcantly larger than 7.8%of WGS ap-proach [11].Although both HIVID and WGS approach are based on mas-sively parallel sequencing platform,HIVID approach has dramatically higher ef ﬁciency on virus integration detection.First,HIVID approach requires much less sequencing data due to the process of HBVfragmentFig.3.Insight of computational process of HIVID.A,principle of pair-end reads assembly.The ﬁgure shows that the tail of an upstream end (read1)and the head of a downstream end (read2)were spliced into one continuous sequence,called PE-assembled read;B,determination of breakpoint.The ﬁgure shows that one PE-assembled read consists of the part of human sequence and part of HBV sequence.The joint position was the breakpoint for HBV integration.4W.Li et al./Genomics xxx (2013)xxx –xxxcapture,but with an enrichment close to 600times compared with WGS approach.HIVID could also be suitable for detecting the breakpoints of other types of viruses,such as human papillomavirus,human immuno-de ﬁciency virus.Moreover,it also could decrease the cost and shorten the time to generate the results.Secondly,HIVID can detect 89.3%(125/140)integration breakpoints which had been identi ﬁed by WGS method,and those integration sites are also characterized by higher NNSS and validation rate.These results demonstrate the HIVID method has a high speci ﬁcity of detecting viral integration breakpoints.Thirdly,HIVID method is more sensitive for detecting low frequency HBV inte-gration breakpoints.Although the validation rate was 66.7%(10/15),HIVID methods could identify 50%more HBV integration sites.The reduced average NNSS value of the novel sites (4.2)compared with the WGS shared ones (13.6)indicates HBV integration in these sites may be less frequent among the tumor cells.The other cause for the lower veri ﬁcation rate of new breakpoints could be attributed to the primer design dif ﬁculty for the shorter support reads.The validationrate might be improved if we adopt the 454long reads sequencing.The long reads also will help us to get more information about the HBV insertion sequence.The major limitation of this approach com-pared with WGS is that we can only target speci ﬁcally one or more virus that have genome references,and identify their integrations.This is a hypothesis driven approach.Another technical limitation is that human genome reads and free virus reads take up a high propor-tion of the data.The method had a low capture speci ﬁcity,although it had an obvious enrichment.We will get more integration breakpoints and need less sequencing data if the capture speci ﬁcity could get im-proved.The single cell study for virus integration breakpoints could ﬁg-ure out monoclonal evolution and the roles of high and low frequency integration events.Since we used part of samples from our previous study,the similar hotspot regions in human and HBV genome were de-tected with these two methods.However,with more supporting reads we annotated the complicated types of HBV integration events,which were omitted in our previous study.Herein,we found a trend of dele-tion in human genome accompanying with HBV fragments insertion,which might induce chromosomal instability and further carcinogene-sis.Genetic instability triggered by HBV integration has been considered in some reports to be an important contributing factor in the pathogen-esis of HCC [20,21].HBV insertions are commonly associated with large genetic alterations:deletions,duplications and chromosomal transloca-tions,which might re ﬂect the abrogation of control mechanisms that safeguard chromosomal integrity [22].However,the impact of those human structure variations on molecular mechanism of HCC still re-mains unclear.In summary we reported the HIVID approach to detect HBV inte-gration breakpoints with high speci ﬁcity and sensitivity using only 1.5Gb sequencing data,for which this approach can be used to screen virus integration in a large cohort of samples and leads to a systematic study for its relationship with disease etiology and tumorigenesis in a comprehensive and unbiased way.Supplementary data to this article can be found online at /10.1016/j.ygeno.2013.07.002.Table 1Data production of 28tested samples.Sample Total Bases Reads number EFR (M paired-reads)Human alignment (pair end)HBV alignment (pair end)Integration rate 101T 1.50G 7.51M;7.51M 5.8895.58%0.03%738(0.01%)43T 1.32G 6.58M;6.58M 5.6686.30%0.04%2520(0.02%)71T 1.49G 7.45M;7.45M 6.4685.05%0.11%1858(0.01%)58T 1.47G 7.33M;7.33M 6.1684.35%0.05%1230(0.01%)34T 1.39G 6.93M;6.93M 5.9884.35%0.02%480(0.00%)55T 1.53G 7.63M;7.63M 6.4782.91%0.10%602(0.00%)35T 1.64G 8.22M;8.22M 7.0585.13%0.02%188(0.00%)41T 1.15G 5.77M;5.77M 4.9784.23%0.12%1042(0.01%)32T 0.70G 3.52M;3.52M 3.1287.08%0.08%424(0.01%)186T 1.99G 9.94M;9.94M 8.5986.70%0.01%374(0.00%)49T 1.17G 5.83M;5.83M 4.9582.61%0.11%2026(0.02%)26T 1.40G 7.01M;7.01M 5.9481.78%0.03%442(0.00%)90T 1.34G 6.70M;6.70M 5.8188.05%0.20%1212(0.01%)182T 1.55G 7.74M;7.74M 6.6887.93%0.03%474(0.00%)266T 1.36G 6.80M;6.80M 5.7786.27%0.03%860(0.01%)145T 1.41G 7.03M;7.03M 5.7486.09%0.08%1398(0.01%)174T 1.35G 6.75M;6.75M 5.7887.22%0.03%290(0.00%)122T 1.52G 7.59M;7.59M 6.4885.11%0.15%1086(0.01%)114T 1.85G 9.25M;9.25M 7.5986.53%0.01%176(0.00%)17T 1.47G 7.36M;7.36M 6.4386.35%0.23%584(0.00%)200T 1.45G 7.25M;7.25M 5.6274.33%0.18%1546(0.01%)95T 1.24G 6.21M;6.21M 5.1877.77%0.04%1546(0.01%)23T 1.17G 5.83M;5.83M 4.8879.94%0.08%2634(0.03%)64T 1.17G 5.83M;5.83M 4.8777.04%0.01%136(0.00%)30T 1.66G 8.28M;8.28M 6.9380.35%0.05%572(0.00%)65T 1.80G 9.01M;9.01M 7.5677.03%0.05%482(0.00%)70T 1.04G 5.19M;5.19M 4.3479.30%0.30%1752(0.02%)82T1.90G9.51M;9.51M8.0278.64%0.03%288(0.00%)This table shows the data production of 28samples.Including human alignment ratio of pair-end reads,HBV alignment ratio of pair-end reads,raw data quantity and integration rate,etc.Fig.4.Overview of breakpoints detected by HIVID.The ﬁgure shows that HIVID detect-ed 246breakpoints on human genome in total.There were 113novel breakpoints and 133breakpoints shared with WGS method.The shared 133breakpoints covered 89.3%of WGS result.In addition the ﬁgure also provides the information of NNSS value and validated rate.5W.Li et al./Genomics xxx (2013)xxx –xxxFig.5.Distribution of breakpoints on human genome.In this CIRCOS ﬁgure,we showed the human chromosomes,the integration frequency of each breakpoint on the level of supporting reads and on the level of samples.The outer circle represents 24chromosomes and mitochondria with different color-code and number labeled.In the middle circle,the integration frequency of each breakpoint one the level of supporting reads is displayed as height of each blue point.In the inner circle,the height of each red point represents the integration frequency of each breakpoint on the level of samples.To explore the distribution of frequency of all integration breakpoints on the level of supporting reads,we pooled all breakpoints from all samples.We merged those breakpoints which appeared in several different samples but in the same location and reset NNSS of the new merged breakpoint as sum of NNSS of all breakpoints in this location.The new NNSS could be seen as the integration frequency of each merged breakpoint on the level of supporting reads.The middle circle was ﬁgured based on the process above and the NNSS of each merged breakpoint is displayed as the height of each blue point.To explore the distribution of frequency of integration breakpoints on the level of samples,the human genome sequence was divided into about three million windows,each with length of 1000bp.The total number of samples that the breakpoints in each window belonged to,was calculated.Each window was considered as one new breakpoint and the number of samples in each win-dow calculated above was considered as supporting samples that supported the window as a new breakpoint.The number could be seen as the integration frequency of each breakpoint on the level of samples.The inner circle was ﬁgured based on the process above and the height of each red point represents the integration frequency of each breakpoint on the level of samples.(For interpretation of the references to color in this ﬁgure legend,the reader is referred to the web version of this article).Table 2The enriched region of HBV integration.Sample ID PositionNSS NNSS Gene266T chr5:1,295,4639716.82TERT:Promoter 34T chr5:1,295,3759616.05TERT:Promoter 58T chr5:1,295,3398012.98TERT:Promoter 65T chr5:1,295,4429712.82TERT:Promoter 90T chr5:1,295,6397212.38TERT:Promoter41T chr5:1,295,12119 3.82TERT:NM_001193376:5-UTR;TERT:NM_198253:5-UTR;64T chr5:1,295,2959 1.85TERT:Promoter49T chr19:36,212,74121242.83MLL4:NM_014727:Intron;71T chr19:36,213,142467.12MLL4:NM_014727:Intron;186T chr19:36,214,00716619.34MLL4:NM_014727:CDS;70T chr19:36,212,313465107.03MLL4:NM_014727:CDS;95T chr19:36,212,5665711.01MLL4:NM_014727:CDS;145T chr19:30,303,49923140.24CCNE1:NM_001238:Intron;200Tchr19:30,315,3669416.72CCNE1:DownstreamThis table shows the hotspots of HBV integration in the 28samples.The relative genes of these integration positions have been shown in the table.6W.Li et al./Genomics xxx (2013)xxx –xxx。

实时荧光定量PCR检测HBV DNA室内质控分析

实时荧光定量PCR检测HBV DNA室内质控分析目的探讨实时荧光定量PCR在HBV DNA定量检测过程中的室内质控问题。

方法用实时荧光定量PCR法检测2013年1~7月自制的乙肝阳性室内质控血清，与临床标本同步检测，计算每次阳性质控结果的常用对数、标准曲线的斜率、截距和相关系数(r)及相关结果的均值(x)、标准差(s)和变异系数(cv)，利用EXCEL折线散点图画出质控图进行质控。

结果本室2013年1~7月测定自制的阳性室内质控结果测定值均在控，标准差和变异系数均在允许范围内，符合要求。

结论本实验室采用实时荧光定量PCR检测HBV-DNA定量检测过程中，选用的质控方法和自制的质控品稳定性良好，能可靠有效地为临床提供准确的检验结果。

Abstract：ObjectiveTo explore the indoor quality controlproblems in HBV DNA quantitative detection of real-time fluorescence quantitative PCR. Indoor quality control serum HBsAg positive self detection.MethodsFor 1~7 months of 2013 by real-time fluorescence quantitativePCR method and clinical specimens, synchronous detection, curve of standard logarithm, calculated for eachpositive quality control results of the slope, intercept and correlation coefficient (R) value and related results ,standard deviation (s) and the coefficient of variation(CV), scatter picture quality control chart for quality control by using the EXCEL line. ResultsThe 1~7 months of 2013 were positive results of internal quality control of homemade measured values are in control, the standard deviation and variation coefficient were within the allowable range, meet the requirements. ConclusionThis laboratory detection of HBV-DNA quantitative real-time fluorescence quantitative PCR detection process,quality control methods and quality control of homemadegood stability, can provide valid and reliable accurate test results for clinical.Key words：Real time fluorescence quantitative PCR; Polymerase chain reaction; HBV-DNA; Quality controlHBV DNA含量是诊断和监测乙型病毒性肝炎的重要指标[1]。

HBsAg与HBV DNA定量测定的临床信息互补

HBsAg与HBV DNA定量测定的临床信息互补武建国【摘要】2013年是HBV表面抗原(HBsAg)发现五十周年.HBsAg的定性测定一直是判定HBV感染的依据,但近年来其定量检查受到临床高度重视,主要原因是定性检查只能反映HBV感染是否存在,而定量检测才能反映病情,指导治疗,判断预后.HBsAg与反映病毒复制的HBV DNA合成途径不同,二者在血清中的表达水平并不平行,这在核苷酸类似物(NAs)的抗病毒治疗时表现尤为突出.因此,为有效控制慢性乙肝(CHB)的病情发展,优化抗病毒治疗策略,追求患者临床结局的改善,HBsAg 定量与HBV DNA定量的联合检测已成为肝病专家的共识,并被纳入各种诊断、治疗的方案中.【期刊名称】《临床检验杂志》【年(卷),期】2013(031)011【总页数】4页(P801-804)【关键词】乙型肝炎病毒;表面抗原;乙肝病毒脱氧核糖核酸;定量【作者】武建国【作者单位】南京军区南京总医院临床中心实验科,南京210002【正文语种】中文【中图分类】R446.61963年，美国国立卫生研究院的Baruch S. Blumberg偶然发现澳大利亚一原住民的血清与多次输血的血友病患者血清在琼脂双向扩散试验时出现一条沉淀线，对该不明物质取名为“澳大利亚抗原”(Australia antigen，Au抗)。

其后研究发现，Au抗与肝炎有关。

1968年Alfred Prince确认Au抗是HBV的一部分，并正名为HBV表面抗原(HBsAg)。

1970年Dane DS在电镜下观察到完整的HBV颗粒(Dane颗粒)。

研究发现，HBV感染与输注污染血密切相关。

1972年在建立血清中HBsAg的测定法(Abbott公司放射免疫法，AUSRIA-125)后，世界卫生组织(WHO)正式建议对献血员进行筛查，此举使美国因输血感染HBV的概率下降了75%[1]。

上世纪80年代，酵母菌重组乙肝疫苗(HBsAg亚单位疫苗)获美国食品和药物管理局(FDA)批准，广泛用于新生儿和普通人群预防接种，使乙肝的流行得到了很大程度的控制。

荧光定量PCR检测HBV

荧光定量PCR检测HBV[摘要] 目的探讨荧光定量pcr法检测hbv-dna与elisa法乙肝免疫学指标的相关性。

方法采用elisa 法和荧光定量pcr技术分别对353 例乙型肝炎患者血清进行免疫学指标的检测及hbv-dna 的定量检测。

结果 elisa法呈大三阳组患者的hbv-dna阳性率及病毒平均拷贝数均高于小三阳组；hbeag（+）组的阳性率及病毒平均拷贝数高于hbeag（-）组；hbeab（+）组有较低的hbv-dna阳性率及病毒平均拷贝数。

结论 hbv-dna 的含量与乙肝免疫学检测结果具有相关性。

临床上应注意两者联合应用。

[关键词] hbv-dna；elisa法；乙肝；免疫标志物[中图分类号] r512.62 [文献标识码] b [文章编号] 2095-0616（2013）06-110-03乙型肝炎是目前最常见的威胁人类健康的传染病之一。

我国是乙型肝炎的高发区，并且近年来其发病率有上升趋势[1]。

因此做好乙肝的检测对于传染病的防控及临床诊治有重要的意义。

目前临床最常用的乙肝筛查及诊断治疗的方法是检测乙肝免疫学指标，即乙肝五项指标。

而在免疫指标检测方面最常用的是酶联免疫吸附试验（elisa）法，它反映人体对hbv的免疫反应状态，具有快速、价廉、简便的特点，适合乙肝的大批量筛查及快速诊断；但是不能直接反映hbv在患者体内的复制情况。

而荧光定量pcr检测hbv-dna 法可直接监测血清中乙肝病毒核酸的载量，是判断病毒复制活跃程度及是否具有传染性的可靠依据，对临床抗病毒治疗及判断预后均具有指导意义。

1 资料与方法1.1 一般资料收集2011年6月～2012年6月在本院门诊、住院及健康查体的hbsag（+）标本共353例，其中女157例，男196例。

按患者的乙肝病毒血清免疫学标志物组合分为6组：a组（大三阳）hbsag（+）、hbeag（+）、hbcab（+），共112例；b组（小三阳）hbsag（+）、hbeab （+）、hbcab（+），共93例；c组 hbsag（+）、hbeag（+），共45例；d组 hbsag（+）、hbeag（-），共31例；e组 hbsag（+）、hbeab （+），共53例。

检测乙肝患者血清标志物与HBV—DNA含量浅析

检测乙肝患者血清标志物与HBV—DNA含量浅析作者：车梦楠来源：《中国保健营养·中旬刊》2013年第10期【摘要】目的：探讨乙型肝炎患者血清标志物与HBV-DNA定量检测之间的关系及其临床意义；方法：选择258例经我院确诊的乙肝患者，采用酶联免疫吸附试验法检测患者体内血清标志物，采用荧光定量聚合酶链反应检测HBV-DNA含量，比较血清标志物与HBV-DNA检测量之间的关系；结果：HBsAg+/HBeAg+组与HBsAg+/HBeAg+/抗-HBc+组阳性检出率分别为100%（16/16）与95.45%（84/88），HBV-DNA对数值分别为7.85±2.12拷贝数/ml和7.08±2.65拷贝数/ml；27例HBsAg+/HBc +组、85例HBsAg+/抗-HBe+/抗-HBc+组、11例抗-HBs +/抗HBe+/抗-HBc +组、8例抗-HBe +/抗-HBc+组、23例抗-HBs+组阳性检出率分别为62.96%、52.94%、37.50%、27.27%、4.35%；结论：乙肝患者血清HBV-DNA阳性率与血清标志物存在相关性；同时进行血清标志物的检测与HBV-DNA定量检测能较准确直接地反应病毒复制程度及传染强度，对于早期诊断、治疗及预后判断具有指导意义。

【关键词】乙肝患者；血清标志物；HBV-DNA【中图分类号】R440 【文献标识码】A 【文章编号】1004—7484（2013）10—0087—02乙型肝炎是一种以肝脏炎性病变为主累及器官损害的传染性病症疾病，常用的诊断指标有HBV-DNA与血清标志物（乙肝二对半）。

为探讨乙肝患者体内血清标志物与HBV-DNA定量之间的关系，本文选择258例乙肝患者，采用酶联免疫吸附试验（ELISA）检测血清标志物，采用荧光定量聚合酶链反应（FQ-PCR）技术检测其血清HBV-DNA的含量，旨在为临床诊断、治疗及预后判断提供参考数据。

北京市卫生局关于同意卫生部北京医院等44家医疗机构开展临床基因扩增检验技术的通知

北京市卫生局关于同意卫生部北京医院等44家医疗机构开展临床基因扩增检验技术的通知文章属性•【制定机关】北京市卫生局•【公布日期】2013.12.31•【字号】京卫医字[2013]221号•【施行日期】2013.12.31•【效力等级】地方规范性文件•【时效性】现行有效•【主题分类】健康促进正文北京市卫生局关于同意卫生部北京医院等44家医疗机构开展临床基因扩增检验技术的通知（京卫医字［2013］221号）各区县卫生局，各有关医疗机构：根据原卫生部《医疗机构临床基因扩增实验室管理办法》（卫办医政发〔2010〕194号）和《北京市卫生局关于印发北京市医疗机构临床基因扩增实验室技术审核相关文件的通知》（京卫医字〔2012〕160号），我局委托北京市医学检验质量控制与改进中心组织专家对我市申请开展临床基因扩增检验技术的医疗机构进行了技术审核和现场评估。

经审核研究，现就有关工作通知如下：一、同意给予卫生部北京医院等44家医疗机构开展的临床基因扩增检验项目登记备案（机构名单和检验项目见附件）。

二、各医疗机构应按照《北京市卫生局转发国家卫生计生委关于印发医疗机构临床检验项目目录（2013版）的通知》（京卫医字〔2013〕156号，以下简称《目录》）要求认真核查准予开展的检验项目。

涉及因《目录》调整等而导致检验项目超出已登记的诊疗科目专业范围的，应当按照有关规定先行办理相关诊疗科目的变更登记手续。

三、符合条件的医疗机构可携《医疗机构执业许可证副本》到准予登记注册的卫生行政部门办理临床基因扩增检验技术的登记手续。

各卫生行政部门审核后，在医疗机构《医疗机构执业许可证》副本备注栏予以登记“允许开展临床基因扩增检验技术”。

四、完成相关诊疗科目及临床基因扩增检验技术登记后的医疗机构可在备案项目范围内开展临床基因扩增检验工作。

请各区县卫生局将本文转发至辖区有关医疗机构。

北京市卫生局2013年12月31日附件：。

电化学发光定量检测乙肝五项指标分析及临床意义

电化学发光定量检测乙肝五项指标分析及临床意义发表时间：2013-04-16T10:04:54.967Z 来源：《医药前沿》2013年第5期供稿作者：孙学青韩景银刘兆军[导读] HBV感染人体后在组织细胞与血液中会出现各种类型的感染标志物，通过对这些感染标志物的检测分析。

孙学青韩景银刘兆军（山东省东营市人民医院检验科山东东营 257091）【摘要】目的分析电化学发光定量检测乙肝五项指标与定性检测结果的差异及定量检测的临床意义。

方法对292份血标本分别采用ELISA 法定性检测和电化学发光法定量检测乙肝五项指标。

结果 ELISA法和电化学发光法分别检出9种和12种乙肝五项指标模式，电化学发光法HBsAg、HBsAb、HBeAg、HBeAb、HBcAb的阳性检出率均高于ELISA法（P均<0.05）。

结论电化学发光定量检测乙肝五项指标敏感性高于定性法并且具有良好的重复性，适合用于HBV感染的早期诊断以及临床抗病毒药物治疗疗效预测和评估。

【关键词】定量检测乙肝五项指标临床意义【中图分类号】R446 【文献标识码】A 【文章编号】2095-1752（2013）05-0389-03 Electrochemiluminescence analysis and clinical significance of the quantitative detection of hepatitis B five indicators Sun Xue-qing, Han Jing-yin, Liu Zhao-jun (Dongying City, Shandong Province, People's Hospital Laboratory shandong Dongying 257091) 【Abstract】Objective To analyze the the electrochemiluminescence quantitative detection of hepatitis B five indicators and qualitative differences and quantitative detection of the test results of clinical significance. Methods 292 blood samples using ELISA the legal detection and electrochemiluminescence quantitative detection of hepatitis B five indicators. Results The ELISA and ECLIA were detected 9 and 12 hepatitis B five indicators mode, the ECLIA HBsAg , HBsAb, HBeAg, HBeAb, HBcAb positive were higher than the ELISA method (P<0.05). Conclusion Electrochemiluminescence quantitative detection of hepatitis B five indicators more sensitive than the qualitative method with good reproducibility, and suitable for use in the early diagnosis of HBV infection and the clinical antiviral therapy prediction and assessment. 【Key words】Quantitative detection Hepatitis B five indicators Clinical significance 乙肝病毒（HBV）在我国以及整个亚洲地区有很高的感染率，根据世界卫生组织（WHO）的统计，全世界1/3的人曾感染过HBV，其中约2%-5%为慢性感染[1]。

HBV-DNA荧光定量与ELISA测定乙肝五项指标及肝功能相关性分析

HBV-DNA荧光定量与ELISA测定乙肝五项指标及肝功能相关性分析发表时间：2013-08-05T15:45:20.967Z 来源：《中外健康文摘》2013年第26期供稿作者：容文潮苏明茂黄秀华赵一帆[导读] HBV-DNA定量与HBV-M关系密切，与ALT、AST和r-GT有一定的相关性，全面检测各项指标有利于乙肝患者的诊断和治疗。

容文潮苏明茂黄秀华赵一帆（台山市人民医院检验科广东江门 529200）【摘要】目的探讨HBV-DNA定量与不同血清学标志物(HBV-M)及肝功能的关系；方法对352例HBsAg阳性患者血清用RQ-PCR定量法测定HBV-DNA含量，用ELiSA法测定HBV-M，同时进行肝功(ALT、AST、r-GT)测定。

结果 HBV-DNA定量大三阳组显著高于小三阳组和（HBsAg+HBcAb）组（P<0.05）；HBV-DNA定量与ALT、AST、r-GT含量呈一定的正相关性（P<0.05）。

结论 HBV-DNA定量与HBV-M 关系密切，与ALT、AST和r-GT有一定的相关性，全面检测各项指标有利于乙肝患者的诊断和治疗。

【关键词】 HBV-DNA HBV-M 肝功三酶检测乙肝病毒是引起乙型肝炎的病原体，是我国常见的传染病，其传染性和病死率均居高不下，我国每年约有1.2亿人感染乙型肝炎病毒（HBV），每年约有30万人死于慢性乙型肝炎、肝硬化、肝癌等相关性疾病，严重危害人们的健康[1]，目前，乙型肝炎病毒感染诊断检测手段较多，主要是血清学标志物（HBV-M）的检测和HBV-DNA含量的检测。

荧光定量聚合酶链式反应（FQ-PCR）具有较高的特异性和灵敏度，克服了常规PCR假阳性的特点，实现对HBV-DNA的定量检测可直接监测血清中HBV-DNA的复制情况，能为乙肝患者提供准确的病原诊断和抗病毒治疗的正确评估。

酶免疫吸附试验(ELISA)法检测乙肝血清学标志物（HBV-M）是目前诊断乙型肝炎的免疫状态[2]，是一种定性分析。

乙型肝炎病毒大蛋白检测的临床应用

乙型肝炎病毒大蛋白检测的临床应用作者：高云芳等来源：《中国保健营养·下旬刊》2013年第12期【摘要】目的研究乙型肝炎病毒外膜大蛋白在血清中的表达及其临床意义。

方法收集78例乙型肝炎病毒感染者的血清标本，检测血清乙型肝炎病毒外膜大蛋白，同时检测这些标本中的乙肝病毒脱氧核糖核酸载量、乙肝表面抗原含量及血清谷丙转氨酶水平。

结果大蛋白阳性组的病毒脱氧核糖核酸载量、表面抗原含量和谷丙转氨酶水平均明显高于阴性组。

结论血清乙型肝炎病毒大蛋白的表达在乙型肝炎病毒活动及发病机制中可能有重要作用。

【关键词】乙型肝炎病毒大蛋白；乙型肝炎病毒脱氧核糖核酸；乙肝表面抗原文章编号：1004-7484（2013）-12-7104-02我国是乙型肝炎的高发地区，目前大约有1.2亿乙型肝炎病毒感染者，乙型肝炎病毒（hepatitis B virus，HBV）是该病的主要病原体[1]。

乙型肝炎的进展和病毒的复制活动密切相关。

长期以来，临床上主要通过酶联免疫法和聚合酶链反应（polymerase chain reaction，PCR）监测病毒复制情况。

近年来，随着对乙型肝炎病毒机构研究的深入，人们发现乙型肝炎病毒外膜大蛋白（hepatitis B virus large protein，HBV LP）是乙型肝炎病毒颗粒成熟包装的重要成分，可以反映病毒在体内的复制[2]。

本文采用酶联免疫法检测乙型肝炎病毒大蛋白，探讨其与病毒复制及肝功能损害的关系。

1 资料与方法1.1 病例资料 2012年7月至2012年12月来我院就诊的门诊或住院的表面抗原阳性患者78例，其中男性60例，女性18例，年龄20-73岁，平均42.8岁。

1.2 方法患者均为清晨空腹采血5ml，经离心后分离血清备用。

血清ALT检测采用日本日立公司的7600型全自动生化分析仪，试剂购自日本和光纯药工业株式会社。

乙型肝炎病毒表面抗原（HBsAg）检测使用美国PerkinElmer公司的Auto DELFIA1235型全自动时间分辨荧光免疫分析仪，试剂购自中山大学达安基因股份有限公司。

HBVDNA

HBVDNA与乙肝两对半检测结果的相关性探讨作者：唐耀敏来源：《维吾尔医药》2013年第04期摘要：目的：探讨HBVDNA的检出情况与乙肝两对半结果之间的关系。

方法：运用荧光定量PCR（FQPCR）检测307例疑似乙肝患者血清中的HBVDNA，同时运用ELISA方法进行两对半指标的检测，并对结果进行相关性探讨。

结果：HBsAg（+）HBeAg（+）HBcAb（+）组（大三阳组）患者血清HBVDNA检出率为98.8%（84/85），平均拷贝数为1.82E+07/ml；HBsAg（+）HBeAb（+）HBcAb（+）组（小三阳组）患者血清HBVDNA检出率为64.6%（53/82），平均拷贝数为1.82E+04/ml；HBsAg（+）HBcAb（+）组血清HBVDNA检出率为63.6%（7/11），平均拷贝数为7.94E+04/ml。

结论：血清HBVDNA水平与HBV M表现模式有关，HBsAg（+）/HBeAg（+）/HBcAb（+）的标本HBVDHA值显著高于HBsAg（+）/HBeAb（+）/HBcAb（+）的标本和HBsAg（+）/HBcAb（+）的标本，提示HBsAg与HBeAg的存在影响HBVDNA水平。

FQPCR能实现准确定量，可以检测HBV的真实感染和复制情况，对于乙型肝炎的临床诊断、治疗方案的选择和疗效考察有较大的指导意义。

关键词：肝炎病毒；乙型；DNA；定量PCR乙型肝炎病毒（HBV）感染是影响我国人民健康最重要的传染病之一，现我国约有1.2亿人口为HBV携带者[1]，HBV感染所致的慢性乙型肝炎病程迁延，有进一步发展为肝硬化或肝功能衰竭的可能，甚至于发展成肝癌，因此对HBV的检测是较为关注的课题。

我们采用荧光定量聚合酶链反应方法检测了211份临床血清标本，并同时采用酶联免疫吸附测定（ELISA）进行对照检测，报告如下。

1 材料与方法1.1 材料1.1.1 标本来源 211份临床血清标本来自208年1月至2013年1月到我科作乙型肝炎血清标志检测者标本，根据其HBV M表现模式分为9组，所有标本均为随机抽样。

化学发光免疫分析与酶联免疫吸附试验检测乙型肝炎病毒标志物比较分析司志霖

2结果 210 份标本用 CLIA 和 ELISA 检测 HBsAg、抗-HBs、HBeAg、
【摘要】目的比较化学发光免疫分析（CLIA）与酶联免疫吸附试验（ELISA）两种方法检测乙型肝炎病毒标志物
（HBV-M）的临床价值。方法分别用 CLIA 和 ELISA 对 210 份血清标本进行 HBV-M 平行检测，然后对两种方法检测结
果进行比较分析。结果 CLIA 和 ELISA 检测血清 HBV-M 结果比较，差异无统计学意义（P>0.05）。结论两种方法检
【Abstract】 Objective To compare the clinical value of the enzyme-linked immunosorbent assay（ELISA） and the chemiluminescence immunoassay（CLIA）for detecting hepatitis B vivus（HBV）markers. Methods 210 serum samples were parallellydetected by CLIA and ELISA respectively and the detection results were comparatively analyzed. Results No statistical difference between the results of CLIA and ELISA were found （P>0.05）. Conclusion The 2 methods of CLIA and ELISA have the high consistency in the detection results of HBV markers ，but CLIA is more accurate and more sensitive than ELISA.

乙肝免疫标志物检测及其实验结果解读

>100，1年后复检； >960，2-3年后复检。
（二）HBeAg 和HBeAb
<1> e抗原(HBeAg)： ➢是可溶性蛋白质 ➢只存在于血液中 ➢是病毒复制和传染性的标志 <2> e抗体(HBeAb)： ➢对HBV感染有一定的保护作用（与肝细胞表面HBeAg结合，破坏受感染细胞，建立部分免疫力） ➢病毒复制停止标志 ➢其出现标志着病毒复制减少、传染性降低。
HBcAb
临床意义
复查建议临床建议
6
+
+
-
-
+
(（2） HBeAg/HBeAb浓度复查，且定期复查
低(3)亚临床型感染早期
HBcAb不稀
（4）不同亚型HBV二次感释
染(5)抗原抗体转化期
7
+
+
_
+
-
（1)亚临床型或非典型感复查，且定期复查
染（2） HBcAb经生理盐水 HBcAb不稀
稀释低于下限
993 17833
25722
HBsAg>0.2（mIU/ml）检测人数（人）
0
2011年
2012年
2013年
HBV阳性率分别为：4.8%，5.6%，5.6%
4.HB(V)的诊断指标
病毒标记物 HBV DNA HBeAg HBsAg
肝脏损伤标记物 ALT AST Bilirubin
感染与免疫标记物 Anti-HBc, Anti-HBcIgM Anti-HBe Anti-HBs
<2> HBsAg阴性表明排除HBV感染或有S基因突变株存在（隐匿性感染）。
<3> HBsAg定量水平可反映感染细胞数量

800例乙肝两对半监测结果分析论文

800例乙肝两对半监测结果分析【摘要】目的：分析800例乙肝两对半的监测结果，以便为有效防控hbv感染提供建议。

方法：采集检验者的血液标本，对其进行离心处理，进行elisa检验。

结果：hbv呈阳性的共有105例，占比13.125%，其中男52例，占比49.52%，女53例，占比50.48%；21岁至30岁的阳性率较高，为34.75%。

结论：监测乙肝五项以及接种疫苗是预防hbv感染者不断增加的有效措施。

【关键词】监测；乙肝两对半；；hbv【中图分类号】r446.6 【文献标识码】a 【文章编号】1004-7484（2013）05-0656-01在检测人体是否感染hbv时，常将乙肝两对半作为标志物，对乙肝两对半进行监测，还能够初步判断病毒复制情况以及复制能力，将小三阳与大三阳区分开来，为hbv感染情况的分析提供必要依据[1]。

本文分析了156例乙肝两对半的检测结果，报告如下。

1研究资料与方法1.1研究资料本文的研究资料均来源于要求对乙肝五项进行检测的人群，共选取了800例进行研究；在800例中，有398例为男性，402例为女性；年龄最小者为11岁，最大者为58岁，平均年龄为31.8岁。

1.2方法先采集检验者的血液标本：采集静脉血，标本容量为3.5ml；采集好全血标本之后，对其进行离心处理，离心时间为15min，转速为2500r/min，将血清完全分离出来之后，观察是否出现溶血现象，如发生溶血，则不能作为监测标本。

采用标准试剂盒对血清进行elisa检验，检测的项目包括hbv的五项指标，即hbcab（核心抗体）、hbeab（e抗体）、hbeag（e抗原）、表面抗体（hbsab）以及表面抗原（hbsag）。

检测完成之后，对不同性别以及年龄的阳性结果进行对比分析。

2 结果监测结果如下：在800例中，hbv呈阳性的共有105例，占比13.125%，其中男52例，占比49.52%，女53例，占比50.48%；从年龄情况来看，21岁至30岁的阳性率较高，为34.75%，31岁至40岁的阳性率次之，为28.53%；55岁以上的人群hbv的阳性率较低。

HBV—DNA乙肝患者的检测结果观察与分析

HBV—DNA乙肝患者的检测结果观察与分析作者：董天珍董天雄来源：《医学美学美容·中旬刊》2014年第02期【摘要】目的：观察并分析乙肝患者的乙肝病毒脱氧核糖核酸（HBV-DNA）的检测结果。

方法：随机选取我院门诊及住院部在2012年2月-2013年4月收治的1000例慢性乙肝患者为研究对象，应用荧光定量-核酸扩增（PCR）检测法以及酶联免疫吸附试验（ELISA）检测所有患者血清标本的HBV-DNA含量。

结果：所有乙肝病毒血清两对半检测组合（HBVm）模式中所占比例最高的是小三阳模式449例（44.9%）和大三阳模式358例（35.8%），两种检测组合模式中HBV-DNA阳性检出率对比具有明显差异，其中大三阳模式的HBV-DNA阳性检出率（62.3%）明显高于小三阳模式（33.4%），差异对比具有统计学意义（P【关键词】乙肝；HBV-DNA；PCR检测；检测结果；观察分析【中图分类号】R512.6+2 【文献标识码】B【文章编号】1004-4949（2014）02-0095-01乙型肝炎是我国较为流行的一种传染性疾病，分布范围较广，该病主要是由于乙型肝炎病毒（HBV）感染所致，经体液和血液均可传播，具有HBV乙肝病毒慢性携带状态[1]。

乙型肝炎是我国发病率和感染率最高的传染性疾病，据相关数据统计[2]，乙肝病毒脱氧核糖核酸（HBV-DNA）含量可直接反映HBV的传染性以及复制程度，为进一步提高临床诊断水平，为临床治疗提供重要的科学依据，本文对我院1000例HBV-DNA慢性乙肝患者检测结果进行深入分析，具体报道如下。

1资料与方法1.1 研究对象及方法随机选取我院门诊及住院部在2012年2月-2013年4月收治的1000例慢性乙肝患者为研究对象，所有患者均符合全国第6次病毒性肝炎会议关于慢性乙型肝炎的诊断标准[3]。

1000例患者中男700例，女300例；病程均超过6个月；年龄7-65岁，平均（27.2±4.1）岁。

专家解读NICE2013年慢性乙型肝炎诊断和管理指南

庄辉教授英国国立优质卫生和保健研究所（National Institute for Health and Care Excellence，NICE）是英国非政府的公共卫生机构，为英格兰和威尔士医疗体系服务，其主要职责是社会保健，并由特殊的卫生权力机构转变为非政府的公共卫生机构。

NICE曾于2006年制订了关于阿德福韦酯和PEG-IFN-α2a治疗慢性乙型肝炎第96号技术评估指南；2008年分别制订了替比夫定和恩替卡韦治疗慢性乙型肝炎第153号和第154号技术评估指南；2009年制订了替诺福韦酯治疗慢性乙型肝炎第173号技术评估指南。

2013年6月，NICE发布了更新的《儿童、青年和成人慢性乙型肝炎诊断和管理指南》，该指南更新了第96号技术评估指南，并将第153号、第154号和第173号技术评估指南包含其中。

该指南包括前言、以患者为中心的保健、所用术语、实施的关键要点、建议、对今后研究的建议、其他信息、以及指南制订小组、国家协作中心和NICE项目组组成等。

该指南与其他指南的主要不同点是：提出了各类慢性乙型肝炎患者抗病毒治疗的用药顺序。

现简要介绍如下。

■HBeAg阳性慢性乙型肝炎且为代偿期肝病成年患者的治疗顺序1.给予PEG-IFN-α2a一线治疗48周。

2.如PEG-IFN-α2a治疗24周后，HBV DNA下降<2log IU/mL和（或）HBsAg>20000 IU/mL，给予替诺福韦酯二线治疗；对替诺福韦酯不能耐受或禁忌的患者，则选择恩替卡韦二线治疗。

3.如PEG-IFN-α2a治疗24周后，HBV DNA下降>2log IU/mL和（或）HBsAg<20000 IU/mL，则继续用PEG-IFN-α2a治疗至48周，如未达到HBeAg血清学转换或HBeAg血清学转换后又阳转（复发）的患者，则给予替诺福韦酯二线治疗。

对替诺福韦酯不能耐受或禁忌的患者，选择恩替卡韦二线治疗。

慢性乙肝患者血清H B V c c c D N A 检测的临床意义作者

慢性乙肝患者血清H B V c c c D N A 检测的临床意义作者：编辑时间：2013-01-29 10:42 点击：57 慢性乙肝患者血清H B V c c c D N A 检测的临床意义摘要：目的探讨慢性乙肝患者血清HBV cccDNA与现有血清学指标(AST、ALT、TBIL、HBeAg、HBV DNA)的相关性。

方法选取309医院2009年5月-2012年5月就诊的慢性乙肝患者58例，采用Roche C6000电化学发光法检测HBeAg；日立Hi7600全自动生化分析仪检测ALT、AST、TBIL；采用实时荧光定量PCR检测血清HBV DNA、HBV cccDNA载量。

结果血清HBV cccDNA与HBV DNA有很好的相关性(r=0.880，P=0.000)。

HBeAg阳性组HBV cccDNA阳性的比例为67.6%，显著高于HBeAg阴性组的37.5%(χ2=5.170，P=0.023)。

血清HBV cccDNA与肝组织炎症的严重程度呈正相关(χ2=4.819，P=0.028)。

结论血清HBV cccDNA定量检测是评价HBV复制情况和肝细胞损伤程度的较为可靠的指标。

关键词：慢性乙型肝炎；共价闭合环状DNA；HBV DNA；乙肝e抗原；谷丙转氨酶目前慢性乙肝的实验室检查常采用免疫学指标HBeAg、HBV DNA反映病毒复制情况，其特异性好但灵敏度相对较低；采用生化指标ALT、AST、TBIL反映肝细胞损伤程度，其灵敏度好但特异性相对较差。

共价闭合环状DNA(cccDNA)是HBV基因组复制中间体mRNA和前基因组RNA的模板，可长期存在于细胞核中，是HBV持续染的关键，也是乙肝慢性化的主要原因[1-2]。

当肝细胞变性坏死时，肝细胞核内的cccDNA可释放入血。

有研究表明血清cccDNA与肝内cccDNA有非常好的相关性[3]，且血清cccDNA可反映肝细胞损伤的严重程度。

本文通过对58例慢性乙型肝炎患者血清HBV cccDNA与现有血清学指标的相关性分析，探讨其在慢性乙肝患者病毒复制和肝细胞损伤评价中的意义。

ALT低于2倍正常值上限的慢性HBV感染者肝脏病理结果的预测因素分析

ALT低于2倍正常值上限的慢性HBV感染者肝脏病理结果的预测因素分析杨瑞;张亚平;王昕红;朱斌;张光文【期刊名称】《临床肝胆病杂志》【年(卷),期】2014(000)009【摘要】目的：探索在ALT＜2倍正常值上限（2×ULN）的慢性HBV感染人群中，一般临床指标对肝脏病理结果的预测作用。

方法收集2009年1月至2013年6月新乡医学院第一附属医院收治的122例ALT＜2×ULN的慢性HBV感染者，在超声引导下行肝穿刺活组织检查术，判断肝组织炎症活动度及纤维化程度，同期化验肝功能、乙型肝炎血清标志物、HBV DNA等指标，应用Logis-tic回归分析法探索该类患者的一般临床指标对其肝脏病理结果的预测作用。

结果122名患者中有明显炎症或纤维化（G≥2或S≥2）者共94例（77．0％），早期肝硬化者5例（4．1％）。

G＜2组与G≥2组相比，除HBV DNA外，其余各指标间差异均无统计学意义；S＜2组与S≥2组相比，年龄、HBeAg、HBV DNA、AST、血小板差异均有统计学意义；Logistic回归分析提示，年龄、HBeAg和AST是肝脏明显纤维化（S≥2）的独立预测因子。

结论对血清ALT＜2×ULN的慢性HBV感染者，年龄＞40岁、HBeAg阴性、AST＞40 U/L者应积极进行肝穿刺活组织检查术，必要时尽早抗病毒治疗。

【总页数】4页(P895-898)【作者】杨瑞;张亚平;王昕红;朱斌;张光文【作者单位】新乡医学院第一附属医院感染二科，河南新乡453100;新乡医学院第一附属医院感染二科，河南新乡453100;新乡医学院第一附属医院感染二科，河南新乡453100;新乡医学院第一附属医院感染二科，河南新乡453100;新乡医学院第一附属医院感染二科，河南新乡453100【正文语种】中文【中图分类】R512.62【相关文献】1.ALT≤2倍正常值上限的HBeAg+/-慢性HBV感染者肝脏病理及临床特征 [J], 耿晓霞;林健梅;杨兴祥;黄仁刚;江南2.HBeAg阴性高病毒载量谷丙转氨酶≤2倍正常值上限慢性HBV感染者伴明显肝脏组织学改变的独立预测因素 [J], 李强;黄玉仙;陈良3.ALT小于2倍正常上限的HBeAg阴性慢性HBV感染者肝脏病理改变及相关因素分析 [J], 耿晓霞;林健梅;杨兴祥;黄仁刚;江南4.肝硬度值在ALT低于2倍正常值上限慢性HBV感染者肝纤维化中的应用价值[J], 唐伯莹; 杨晴; 王岩; 关欣5.GP模型对ALT小于2倍正常值上限的慢性HBV感染者肝纤维化的诊断价值[J], 张杰灵; 邹桂舟; 郜玉峰; 李家斌因版权原因，仅展示原文概要，查看原文内容请购买。

荧光定量PCR检测HBV DNA室内质控的建立与应用评价

荧光定量PCR检测HBV DNA室内质控的建立与应用评价发表时间：2013-04-08T08:41:03.187Z 来源：《医药前沿》2013年第4期供稿作者：刘兆军[导读] 自制质控血清无需灭活及其他特殊处理，完全能够满足PCR实验室室内质量控制的需要。

刘兆军（山东省东营市人民医院检验科 257091）【摘要】目的自制临床基因扩增实验室HBV DNA检测的室内质控品并对其应用进行评价。

方法将试剂盒自带的弱阳性对照品、自制未灭活质控血清、自制灭活质控血清作为室内质控品的选择对象，每天与样本一起检测，连续30天，通过不精密度、L-J质控图，t检验，单因素方差分析来评价三者哪一个更适合基层医院PCR实验室作为室内质控品。

结果弱阳性对照品检测结果日间不精密度为7.72%,未灭活自制质控血清检测结果日间不精密度为2.92%,灭活自制质控血清检测结果日间不精密度为3.70%；灭活与未灭活自制质控血清配对t检验,t=-0.860，P=0.397，P＞0.05，两组检测结果无显著性差异；对灭活前后两组数据进行单因素方差分析，F=0.901，P=0.346，P＞0.05，两组检测结果均值无显著性差异；SPSS17.0统计学软件绘制弱阳性对照品、未灭活自制血清质控品L-J质控图11月份30天的变化情况。

结论自制质控血清无需灭活及其他特殊处理，完全能够满足PCR实验室室内质量控制的需要。

【关键词】 HBV DNA 自制质控血清 L-J质控图【中图分类号】R446 【文献标识码】A 【文章编号】2095-1752（2013）04-0032-02 临床实验室的分析中质量控制是全过程质量控制的一个重要环节，临床实验室的误差有很大一部分是发生在样本的分析阶段[1]，因此加强室内质控的分析是实验室质量控制的一个重要环节。

目前荧光定量PCR检测HBV DNA室内质控品的缺失是各基层检验科PCR实验室普遍存在的问题，因为市场上无商品化的质控品可以购买，大部分供应商只提供阴、阳性对照及标准品，因此通过实验室自制质控品是解决目前困境的主要途径。

检测试剂技术规格及要求2013

国产核酸检测试剂技术规格及要求一、用途：血液核酸筛查二、数量：2万人份（人份定义为：汇集之前或单人份检测标本数）注：因为要与计划编号为300028201300004，金额为35万元的核酸检测试剂合并采购，因此试剂数量两次合计起来，实际应为3万人份。

三、使用要求1.检测项目：乙肝病毒DNA、丙肝病毒RNA，人免疫缺陷病毒RNA；2.★检测报告：能够提供单项病毒核酸检测结果；3.★单人份检测灵敏度(≥95%可信度):1.1H BV ≤100 IU/ml；1.2H CV ≤100IU/ml；1.3H IV ≤100 IU/ml；4.试剂含有内标质控（Internal Control），全程监控检测过程，包括核酸提取，扩增及检测步骤，以防止假阴性；5.试剂覆盖HIV-1(M),HIV-1(O), HBV, HCV所有基因型，对HBV的亚型检出能力强，能够检测基因型A、B、C、D、E、F、G、H6.适用检测样本种类：血清以及EDTA、ACD抗凝血浆；7.检测技术：实时荧光聚合酶链反应，8.★.试剂数量人份以出报告人数计算。

9.每一人份包含检测（包括拆分和鉴别试验）所需的所有试剂、试验耗材、对照品、质控品和试剂损耗。

必须免费提供鉴别试验用试剂、试验耗材、对照品。

10.★审核批准1.4中标方必须提供国家药品监督管理部门审核批准的血站使用核酸血筛试剂批准文件；四、配套要求:1.无偿提供相关配套设备：1.1.中标方提供所有核酸检测相关检测仪器（加样、核酸提取、纯化、扩增、检测和鉴别设备、信息管理设备）以及配套实验台等，进行自动核酸提取、扩增、检测和鉴别；中标方无偿提供设备运输、安装、调试、培训和维修的服务；1.2.检测设备需求量：配置设备满足常规6小时800个标本检测的需求；并能满足设备故障时应急检测需要。

2.无偿提供配套耗材：2.1.提供实验所需一次性带滤芯的无DNA酶、无RNA酶加样头；3.试剂和设备提供的时间:3.1.中标方30天内提供所有设备与试剂耗材。