FASTDNA SPIN KIT FOR SOIL中文说明书
土壤试剂盒操作手册和常见问题
FastDNA Spin Kit for Soil实验步骤1.Add up to 500 mg of soil sample to a Lysing Matrix E tube.在裂解介质管E中最多加入500mg土壤样品。
注意：推荐最多加入500mg土壤样品，含水量比较多的土壤或者碎屑多的土壤可适量减少样品量。
2.Add 978 μL Solution Phosphate Buffer to sample in Lysing Matrix E tube.在裂解介质管E中加入978μl Sodium Phosphate Buffer3.Add 122 μL MT Buffer.What’s happening: Begin to solubilize membrane proteins with detergents as well asextra-cellular proteins and contaminations in soil.加入122μl MT Buffer发生的反应：用洗涤剂溶解细胞膜蛋白以及细胞外蛋白和土壤中的污染物。
注意：为了得到更好的样品处理效果，加入土壤样品及两个缓冲液后，在裂解介质管中仍能保留有250-500μl空间。
4.Homogenize in the FastPrep Instrument for 40 seconds at a speed setting of 6.0What’s happening: mechanical disruption of cell walls of soil organisms and releasing nucleic acids into the protective buffer.将样品置于FastPrep®仪器上匀浆40s，速度为6.0m/s发生的反应：机械破碎土壤微生物的细胞壁，将核酸释放入保护缓冲液中。
5.Centrifuge at 14,000×g for 5-10 minutes to pellet debris.14,000 x g离心5-10min至沉渣注意：如果把离心时间延长到15min，可以更好地使样品量较大的，或者细胞壁结构较复杂的细胞碎片沉降到管底。
FastDNASPINKitforSoil土壤DNA快速提取试剂盒提取步骤
FastDNASPINKitforSoil土壤DNA快速提取试剂盒提取步骤1.准备样品：选择合适的土壤样品，将其称重至1g。
如果样品中有根、树枝等杂物，需要将其去除。
2. 加入试剂：将1g土壤样品加入50ml试样管中，然后加入4ml试剂A。
用旋转混合器将土壤样品和试剂彻底混合。
3.离心：将混合物离心10分钟，以去除土壤颗粒和杂质。
离心后，样品将分为上清液和沉淀。
4. 转移上清液：使用移液器将上清液转移到新的1.5ml离心管中。
注意不要转移沉淀物。
5. 加入试剂：将2ml试剂B加入离心管中，用手轻轻颠倒离心管，使试剂B和上清液充分混合。
6. 震荡：将离心管放入震荡器中，以6000rpm的速度震荡10分钟，以充分裂解细胞。
7.离心：将离心管离心10分钟，以去除残余的土壤颗粒。
8. 转移上清液：使用移液器将上清液转移到新的1.5ml离心管中。
注意不要转移沉淀物。
9. 加入试剂：将0.5ml试剂C加入离心管中，用手轻轻颠倒离心管，以充分混合。
10.离心：将离心管离心10分钟，以去除残余的杂质。
注意不要转移沉淀物。
12. 加入试剂：将0.8ml试剂D加入离心管中，用手轻轻颠倒离心管，以充分混合。
13.离心：将离心管离心10分钟，以去除残余的杂质。
14. 转移上清液：使用移液器将上清液转移到新的1.5ml离心管中。
注意不要转移沉淀物。
15. 加入试剂：将0.5ml试剂E加入离心管中，用手轻轻颠倒离心管，以充分混合。
16.离心：将离心管离心10分钟，以去除残余的杂质。
17. 转移上清液：使用移液器将上清液转移到新的1.5ml离心管中。
注意不要转移沉淀物。
18. 加入试剂：将0.5ml试剂F加入离心管中，用手轻轻颠倒离心管，以充分混合。
19.离心：将离心管离心10分钟，以去除残余的杂质。
20. 转移上清液：使用移液器将上清液转移到新的1.5ml离心管中。
注意不要转移沉淀物。
21. 加入试剂：将0.5ml试剂G加入离心管中，用手轻轻颠倒离心管，以充分混合。
木醋液的抑菌活性及其对连作番茄根际土壤微环境生态的影响
第43卷 第1期2024年 1月华中农业大学学报Journal of Huazhong Agricultural UniversityVol.43 No.1Jan. 2024，40~51木醋液的抑菌活性及其对连作番茄根际土壤微环境生态的影响肖健1，谭俊杰2，林泽毅3，林强3，杨尚东1，谭宏伟41.广西大学农学院/广西农业环境与农产品安全重点实验室/植物科学国家级实验教学示范中心，南宁 530004；2.广西民族师范学院，崇左 532200；3.广西贺州匠心科技有限公司，贺州 542800；4.广西农业科学院/广西甘蔗遗传改良重点实验室，南宁 530007摘要 为探索木醋液对番茄根际土壤微生态的作用，以番茄为供试材料，连续3 a 常规种植于相同田块，设置木醋液稀释300倍（A ）、稀释600倍（B ）、稀释900倍（C ）处理，以无菌去离子水为对照（CK ），探究不同稀释度木醋液的抑菌活性及其对连作番茄植株根系生长、根际土壤细菌和真菌群落结构的影响。
结果显示：不同稀释度木醋液对青枯病菌和枯萎病菌均具有显著的抑制作用；3种稀释度的木醋液中，稀释600倍的木醋液浇灌不仅有利于连作番茄根系的生长，而且有助于提高番茄连作根际土壤细菌和真菌的多样性；与对照（CK ）相比，不同稀释度木醋液处理均不同程度地降低了放线菌门（Actinobacteriota ）细菌的相对丰度，提高了酸杆菌门（Acido‐bacteriota ）细菌以及子囊菌门（Ascomycota ）、新赤壳属（Neocosmospora ）、曲霉属（Aspergillus ）、青霉属（Penicilli⁃um ）、木霉属（Trichoderm a ）、毛壳菌属（Chaetomium ）和镰刀菌属（Fusarium ）真菌等有益微生物的相对丰度。
结果表明，木醋液具有显著抑制番茄青枯病及枯萎病病原微生物的作用，浇灌稀释木醋液有利于连作番茄根系生长，改善连作番茄根际土壤微环境生态的作用；其中，以稀释600倍（B ）木醋液的改良效果最佳。
Fasttrack磨刀器说明书
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平菇发酵培养料制备过程中黄曲霉毒素降解变化分析
中国瓜菜2023，36（11）：93-99收稿日期：2023-07-29；修回日期：2023-09-07基金项目：河南省现代农业产业技术体系专项（HARS-22-08-S ，HARS-22-08-G1）；河南省重大公益性专项（201300110700）作者简介：崔筱，女，副研究员，主要从事食用菌育种及功能基因研究。
E-mail ：********************通信作者：孔维丽，女，研究员，主要从事食用菌育种及平菇发酵料栽培机制研究。
E-mail ：*********************玉米芯是黄淮海地区栽培平菇的主要原料，秋季收获后的玉米芯存放不当易产生黄曲霉毒素。
黄曲霉毒素（aflatoxins ，AFT ）是一组由曲霉属（As-pergillus ）的一些霉菌产生的具有强毒性和致癌性的次级真菌代谢产物[1-3]。
据统计，曲霉属中有28个种的霉菌可产生黄曲霉毒素，其中最重要及最广为人知是黄曲霉（A.flavus ）、寄生曲霉（A.parasiti-cus ）和红绶曲霉（A.nomius ）[4]。
目前已分离鉴定出20余种黄曲霉毒素异构体，其中最常见的包括B1、B2、G1、G2、M1、M2。
黄曲霉毒素具有稳定的结构，通过物理（臭氧、微波、高压、紫外照射、吸附剂等）或化学方法（碱处理法、氧化法等）仅能降低黄曲霉毒素的含量，很难将其完全去除。
熟料栽培的金针菇菌渣中检测出平菇发酵培养料制备过程中黄曲霉毒素降解变化分析崔筱，胡素娟，刘芹，吴杰，师子文，张玉亭，孔维丽（河南省农业科学院食用菌研究所郑州450002）摘要：为解析平菇发酵培养料制备过程中黄曲霉毒素降解的变化规律，采用非靶向代谢组学技术和宏基因组学技术，对平菇发酵培养料制备过程中5个时期黄曲霉毒素含量及相关微生物基因丰度进行关联分析。
结果表明，发酵初期（T1）培养料含有AFM1、AFG2两种黄曲霉毒素，升温阶段（T2）其含量均升至最高；第2次至第4次翻堆（T3至T5）时期未检测到黄曲霉毒素。
FastDNA SPIN Kit for Soil使用方法
FastDNA SPIN Kit for Soil使用方法1.加入500 mg土壤到Lysing Matrix E管中。
加入978 μl Sodium Phosphate Buffer（磷酸钠缓冲液）和122 μl MT Buffer（MT缓冲液）。
注意1：因为FastPrep®仪器的剧烈运动，在Lysing Matrix E管内会形成巨大的压力，样品和基质的总体积不能超过管体积的7/8；留一些空间也会提高混匀效果。
裂解：加入978 μl Sodium Phosphate Buffer（磷酸钠缓冲液）和122 μl MT Buffer（MT 缓冲液）。
2.在FastPrep®中处理上述离心管，速度5.5，30秒。
3.Lysing Matrix E管离心14000g×10分钟。
注意2：对于大样品可延长振荡时间到15分钟。
4.将上清液转移到一个新的2ml离心管（自备）中，加入250μl PPS，并用手摇10次进行混合。
5.离心14000g×5分钟，至形成球状沉淀物，将上清液转移到一个新的5ml离心管（自备），并加入1ml Binding Matrix Suspension（在用之前重悬Binding Matrix Suspension）。
6.放到旋转体或用手颠倒2分钟，使DNA附着到基质上，把离心管放到架子上静置3分钟。
7.小心去除500μl上清，避免扰动沉淀的Binding Matrix，重悬剩余的上清；转移大约600μl的混合液到一个SPIN TM Filter中，离心14000g×1分钟；将SPIN TM Filter下面catch tube 中的液体倒掉；重复上述过程直到剩余混合液全部转移离心完（1.5mL污泥样品一般要转移三次）。
8.加入500μl SEWS-M到SPIN TM Filte r中，离心14000g×1分钟；将SPIN TM Filter下面catch tube中的液体轻轻地倒掉，再离心14000g×2分钟，去除基质中的SEWS-M。
不对称PCR扩增检测土壤中的肠道病原菌①
不对称PCR是指反应体系中两条浓度不一样的引物,在PCR 扩增的后期,当其中量少的一条引物被消耗完以后,另一条继续以线性扩增的方式,产生大量的扩增产物单链 DNA。
部队称PCR 扩增检测技术可以应用的范围相对较为广泛:如配合RT-PCR扩增技术研究真核DNA外显子、制备单链探针、DNA序列分析等,在寡核苷酸基因芯片的检测应用方面,不对称PCR在标记单链样品方面比常规PCR更有优势,与寡核苷酸芯片杂交效率也较高。
1 材料与方法1.1 土壤样品研磨过筛(20目),-80°C保存备用。
1.2 主要试剂及试剂盒主要试剂及试剂盒:引物、dNTP、50bp DNA Ladder,100bp DNA Ladder、Taq DNA聚合酶、琼脂糖、FastDNA SPIN Kit for Soil试剂盒(MP Biomedicals,Solon,OH,USA)。
1.3 主要仪器凝胶成像系统(Bio-Rad Laboratories,Segrate,Italy),PTC-100 PCR仪(MJ Research,Waltham,MA,USA),Mini-BeadBeater-16TM 核酸提取仪(Biospec products,USA),DYY-8B型电泳仪(北京市六一仪器厂)FastPrep TM FP120核酸提取仪(Bio 101,Carlsbad,USA),高速离心机(上海安亭)。
1.4 DNA提取利用FastDNA SPIN Kit for Soil试剂盒与Mini-BeadBeater-16T M 核酸提取仪提取土壤DNA。
主要过程参见FastDNA SPIN Kit for Soil试剂盒。
1.5 引物合成参照文献[1-2]合成引物:UP1:GAAGTCATCATGACCGT T C T G C A Y G C N G G N G G N A A R T T Y G A ,P 2r :AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRT CNGTCAT(R指的是A或者G,Y指的是C或者T,N代表任何碱基)。
水体样本提取的方法
水体样本提取的方法手提（细菌细胞直径约0.5um，长度约0.5~5um）将300-500ml水样通过0.45um或者0.22um的滤膜如果水样中不可溶解的颗粒较多，需要使用2-5um孔径的滤膜将不可溶解的颗粒杂质滤去，滤膜孔径大于水体微生物细胞直径。
用于水体微生物富集的滤膜的选择：常用的滤膜孔径大小有45mm和22mm两种，孔径太小滤膜容易阻塞，45mm的孔径大小透水性较好，提样的时候可根据客户的需要，选择45mm或22mm孔径的滤膜。
滤膜材质有很多种，常用的三种为聚苯醚砜滤膜、混合纤维素酯薄膜、氧化铝薄膜。
聚苯醚砜滤膜（Polyethersulfone）：最结实的滤膜之一，可以过滤比其它滤膜更多的水样。
使用真空泵可快速抽干，易于折叠不易撕破。
能抵受真空泵长时间高压力的滤过。
若需要过滤大量低微生物含量的清亮水样，0.22mm滤膜的更合适。
在提取核酸时，得率可与PowerWater® DNA 和RNA Isolation Kits中自带的混合纤维素酯滤膜相媲美。
混合纤维素酯滤膜（膜醋酸纤维素、硝酸纤维素）: 0.45μm孔径最合适材质。
如果水样浑浊，使用0.22μm滤膜过滤缓慢容易堵塞时，建议使用0.45μm孔径的滤膜。
纤维素滤膜吸水性强，不好处理。
有文献显示杀虫剂和除草剂很容易吸附到纤维素滤膜上。
若样品含有杀虫剂和除草剂，最好避免使用这类滤膜。
聚碳酸酯滤膜（Polycarbonate）: 这种滤膜很薄且容易起褶皱，所以不太好用。
通常用0.45μm孔径的滤膜来预防过滤时发生阻塞。
不像聚醚砜膜（PES）和混纤膜（MCE），水样中微生物会停留在滤膜表面。
使得滤膜很容易阻塞，本该滤过的小颗粒也会截留下来。
实验证明，珠磨研磨破碎强度越小，获得DNA分子量越大。
如果样品只是用来做PCR，可以采用强力的研磨方法提高得率，暂时忽略片段破碎。
样品富集之后按照以下方法进行提取：1. 滤膜剪碎，溶于500ul水中，液氮反复冻融（或用超低温冰箱）（从-70℃拿出来后在65℃水中冻融，小心操作，防止管子炸裂）【裂解细胞】2. 将水样于13000rpm下离心，10min，收集沉淀3. 沉淀溶于100ul 1×TE，重悬4. 加入30ul溶菌酶（工作浓度50mg/ml）37℃1h5. 加入500ul SDS（10%），及10ul蛋白酶K（20mg/ml），37℃过夜6. 加入200ul 5mol/l 氯化钠7. 加入预热至65℃的 CTAB/NaCl 100ul，65℃ 1h8. 加入等体积酚氯仿 13000rpm 10min，两次，将上清移至新管9. 加入等体积异丙醇 -20℃防止1h，13000rpm 30min10. 70%乙醇清洗一次，自然晾干溶于30ul无菌水中试剂盒MOBIO PowerSoil® DNA Isolation KitFastDNA® Spin Kit for Soil（MP bio土壤基因组DNA提取试剂盒）。
氟磺胺草醚对不同豆科作物生长及根际固氮菌的影响
氟磺胺草醚对不同豆科作物生长及根际固氮菌的影响陈未, 李江叶, 刘丽珠, 童非, 戴群, 高岩引用本文:陈未,李江叶,刘丽珠,等. 氟磺胺草醚对不同豆科作物生长及根际固氮菌的影响[J]. 农业环境科学学报, 2021, 40(10): 2076-2085.在线阅读 View online: https:///10.11654/jaes.2021-0474您可能感兴趣的其他文章Articles you may be interested inZnO NPs对四种豆科种子发芽及幼苗生长的影响彭晴晴,杨静雅,钟民正,邢洋洋,李紫燕,毛晖,周莉娜农业环境科学学报. 2021, 40(6): 1174-1182 https:///10.11654/jaes.2020-1462绿肥作物对云南旱地土壤镉有效性的影响王赟,付利波,梁海,章子含,杨伟,何正海,高嵩涓,曹卫东农业环境科学学报. 2021, 40(10): 2124-2133 https:///10.11654/jaes.2021-0457畜禽粪污抗生素对土壤生物学效应的Meta分析曾悦,黄红英,吴华山农业环境科学学报. 2021, 40(5): 1043-1050 https:///10.11654/jaes.2020-1044太湖地区集约化农田氮素减排增效技术实践闵炬,孙海军,陈贵,姜振萃,陆扣萍,纪荣婷,施卫明农业环境科学学报. 2018, 37(11): 2418-2426 https:///10.11654/jaes.2018-12873种磺胺类兽药单一及复合污染对不同作物根尖细胞的微核效应研究金彩霞,毛蕾,司晓薇农业环境科学学报. 2015(4): 666-671 https:///10.11654/jaes.2015.04.009关注微信公众号，获得更多资讯信息陈未，李江叶，刘丽珠，等.氟磺胺草醚对不同豆科作物生长及根际固氮菌的影响[J].农业环境科学学报,2021,40（10）：2076-2085.CHEN W,LI J Y,LIU L Z,et al.Effects of fomesafen on plant growth and nitrogen-fixing bacteria in the rhizosphere of different species of legumes[J].Journal of Agro-Environment Science ,2021,40（10）：2076-2085.开放科学OSID氟磺胺草醚对不同豆科作物生长及根际固氮菌的影响陈未1，李江叶1，刘丽珠1，童非1，戴群2，高岩1*（1.江苏省农业科学院农业资源与环境研究所，南京210014；2.江苏第二师范学院生命科学与化学化工学院，南京210013）Effects of fomesafen on plant growth and nitrogen-fixing bacteria in the rhizosphere of different species oflegumesCHEN Wei 1,LI Jiangye 1,LIU Lizhu 1,TONG Fei 1,DAI Qun 2,GAO Yan 1*（1.Institute of Agricultural Resources and Environment,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;2.School of收稿日期：2021-04-22录用日期：2021-06-09作者简介：陈未（1988—），女，江苏南京人，博士，助理研究员，从事污染农田土壤修复相关研究。
FastDNA SPIN Kit for Soil(mpbio土壤DNA提取试剂盒产品说明书-北京毕特博生物)
FastDNA® SPIN Kit for Soil
Catalog # 6560-200 50 preps
General Information
Qbiogene is a pioneer in developing kits for molecular biology research. We introduced the GENECLEAN® Kits in 1986 and have since been manufacturing products to bring convenience into your research. Our goal is to make your life easier by simplifying the complexities of lab work.
3. DNA Purification and Elution Reagents
A GENECLEAN® procedure is them used to purify the genomic DNA. The procedure purifies DNA with a proprietary silica matrix and eliminates contaminants that inhibit sussequent reactions. * Binding Matrix Suspension * SEWS-M (Salt Ethanol Wash) * DES (DNA Elution Solution Ultra-pure water)
• Purifies PCR-ready Genomic DNA from Soil Samples • For use with the FastPrep® Instrument
建库筛库流程(修改稿)
nirK基因文库的构建实验要求：实验操作中一定要戴手套，并使用70%的酒精消毒；并且每间隔一定时间（5-10min）再对手套进行消毒。
实验台面用70%的酒精擦干净。
实验过程中根据中间结果检查实验的可靠性。
提前做好实验准备，灭好枪头、离心管等实验耗材。
在实验中所用冰冻试剂在冰上完全融化后再用。
实验过程中注意定量，如跑胶是上样量为3μl，Marker为3μl。
对公用试剂用完后请及时保存到-20度或4度冰箱，防止试剂变质。
该实验流程不准随便改动，若有需改动的请教党老师。
（一）宏基因组DNA的提取采用土壤DNA快速提取试剂盒（FastDNA Spin kit for soil）及Fast Prep-24核酸提取仪进行提取。
DNA提取的质量决定后续实验的准确性和可靠性。
每个站位均用0.3g （勿超0.3g，冰冻状态）的沉积物样品进行DNA提取。
具体操作过程如下：（1）向Lysing MatrixZ tube中加入0.3g沉积物样品（勿超0.3g），1100μl裂解液溶解。
裂解液组成为978μl磷酸钠缓冲液和122μlMT缓冲液。
（均质化土样，使提的DNA含RNA量最少）。
注意：离心机提前预冷15min，至4℃。
（2）将上述离心管放入FastPrep Instrment中，设定速度5，离心时间40s。
（3）4℃离心机，将上述离心管以14000×g离心10min。
（4）将上清移入新的1.5ml离心管中加入250μlP PS，并轻柔颠倒10次将其混匀。
（5）将上述混合液以14000×g 4℃离心5min，将上清移入另两个新的1.5ml离心管中（每管加500μl），将Binding Matrix Suspension摇匀，悬浮，向上述上清中加入500μl 的Binding Matrix Suspension。
（6）用手轻柔颠倒混匀2min，将管放置于管架上静置3min (4℃冰箱)。
（7）每管吸出300μl上清（即每个站点吸出300*2），将之丢弃（注意勿扰沉淀），使余下的上清与Binding Matrix 混匀，将600μl的混合液（任一管）移入纯化柱（spin fillter）中14000×g离心1min，倒掉收集管中的废液，将剩余的（另一管）Binding Matrix全部移入纯化柱中14000×g离心1min，倒掉收集管中的废液。
使用rps4基因定量分析生物土壤结皮中苔藓的生物量
Use of quantitative PCR with the chloroplast generps4to determine moss abundance in the early succession stage of biological soil crustsSongqiang Deng1&Chunzi Wang1&Roberto De Philippis2&Xiangjun Zhou1&Chaoran Ye1&Lanzhou Chen1Received:14December2015/Revised:28February2016/Accepted:16March2016#Springer-Verlag Berlin Heidelberg2016Abstract The quantitative PCR(qPCR)method was firstly used to measure moss abundance with moss chloroplast gene rps4as compared to that with cyanobacterial16S rRNA gene and fungal25-28S rRNA gene in the early succession stage of biological soil crusts(BSCs).Four sites with three BSC types collected from Hobq Desert of China,representing cyanobacterial-,lichen-,and moss-dominated BSCs were in-vestigated.The copies of the moss rps4gene,cyanobacterial 16S rRNA gene,and fungal25-28S rRNA gene,chlorophyll a content,and the community composition variated significant-ly.The moss rps4gene copies attained a significant positive correlation with chlorophyll a content and showed a cross-validation with relative moss biomass.Keywords Moss.Quantitative PCR.rps4.Biomass. Community compositionIntroductionBiological soil crusts(BSCs)are highly specialized commu-nities that include cyanobacteria,algae,bacteria,fungi,li-chens,and mosses in dryland ecosystems(Belnap et al.2003;Belnap2013)and are named cyanobacterial,lichen, and moss BSCs based on the dominant organism group along with the developmental sequence(Chen et al.2014;Navarro-Noya et al.2014).BSCs play an important role in a desert ecosystem through increasing C-and N-input in the soil,soil water-holding capacity,and stabilizing soil against wind and water erosion(De Caire et al.2000;Langhans et al.2009; Fang et al.2015).BSC development can be beneficial to soil fertility due to their C-input(Mager and Thomas2011;Xu et al.2013).However,the methods for determining the con-tribution of different microbial communities to BSC biomass entail improvement.Mosses are important for stabilizing soils,capturing and distributing nutrients,regulating seed germination,and colo-nizing disturbed areas(Belnap and Lange2002).Moss cover and biomass,as well as their ecological role in some mature, well-developed moss BSCs,have been studied(Weber et al. 2012;Smith and Stark2014;Ball and Guevara2015;Bu et al. 2015;Fang et al.2015).However,in the early stage of BSC succession or,particularly,at the cyanobacterial-and lichen-stages,the mosses are nearly invisible and their biomass is low,especially under dry conditions or in sporophyte-dominant crusts.Therefore,conventional methods,such as the spectral measurement(Karnieli and Sarafis1996)and di-rect counting(Fang et al.2015),provide inaccurate determi-nations due to their low accuracy and sensitivity.Recently, real-time fluorescence quantitative PCR(qPCR)has been widely used for determining relative abundance in BSCs through the use of universal primers targeting different groups of microorganisms,like EUB338and EUB518primers for bacteria(Marchesi et al.1998),CY A359F and CY A781A/B primers for cyanobacteria(Nübel et al.1997),NL1f and LS2r primers for fungi(Bates and Garcia-Pichel2009),and so on. However,the qPCR method has not been used for determin-ing moss biomass.*Lanzhou Chenchenlz@1School of Resource and Environmental Sciences,Hubei Key Laboratory of Biomass-Resources Chemistry and Environmental Biotechnology,Wuhan University,Wuhan430079,People’sRepublic of China2Department of Agrifood Production and Environmental Sciences, University of Florence,Piazzale delle Cascine24,Firenze I-50144, ItalyThe rps4gene is a chloroplast gene encoding the protein4 of the small chloroplastic ribosomal subunit.The gene is the third most frequently used DNA marker in bryophyte system-atics for phylogenetic reconstructions due to its relatively small size and good sequence variation(Souza-Chies et al.1997; Wynns and Lange2014).Goffinet et al.(2001)performed phy-logenetic analyses of nucleotide and amino acid sequences of the chloroplast gene rps4for225species of mosses, representing84%of families recognized by Vitt(1984),and they resolved the core of most moss families.Wynns and Lange(2014)have comparatively studied16DNA regions for phylogenetic markers in pleurocarpous mosses,which in-clude approximately half of all moss species(O’Brien2007), and showed that it was extremely easy to amplify rps4gene.This study was aimed to use qPCR method to determine the absolute number of gene copies and the relative contribution of moss to BSC biomass by using a pair of primers targeting the bryophyte chloroplast gene rps4.We also employed primers targeting25-28S rRNA gene of fungi and16S rRNA gene of cyanobacteria to evaluate contribution of fungal and cyanobacterial biomass to total biomass in the early suc-cession stage of BSCs.Materials and methodsStudy area and samplingFour sites with three BSC types were collected from Hobq Desert,Dalateqi County,Inner Mongolia,China(40°21′N; 109°50′E)in April2015.The study area has been described preciously by Chen et al.(2014).Four experimental sites char-acterized by different colors and morphological appearances of BSCs were chosen.Site1(S1)was cyanobacteria-dominated BSCs,site2(S2)was lichen-dominated BSCs,site 3(S3)and site4(S4)were moss-dominated BSCs,as assessed based on the description of Williams et al.(2013).For each experimental site,three samples were collected randomly and pooled by using a sterilized spatula and by digging soil pits and picking up samples with a thickness ranging from0.1to 1cm,depending on the structure of the crust.All the BSC samples were packed on dry ice and stored at−80°C before processing.The unconsolidated sand was used as control. Measurement of chlorophyll a contentAir-dried samples were manually grounded,and1g samples were extracted with5ml of95%ethanol at4°C for24h under dark conditions.Subsequently,the samples were centri-fuged at6000×g for10min.The supernatants were retained, and the chlorophyll a content was quantified according to Chen et al.(2006).DNA extraction and quantitationDNA was extracted from duplicated0.5g aliquots of each BSC samples using the FastDNA Spin Kit for Soil(MP Biomedical,LLC)according to the supplied protocols.DNA extracts were examined on0.9%agarose gels in1×Tris-Acetic acid-EDTA with GelRed and quantified using Nanodrop2000c(Thermo Scientific,USA).Subsequently, the duplicate DNA was pooled,and the concentration of the solution was brought to25ngμl−1.Quantitative PCR(qRCR)analysisQPCR analysis was carried out to determine the absolute number of gene copies of cyanobacteria,fungi,and moss on a StepOne Real-Time PCR System(Applied Biosystems, Foster City,USA).Twenty microliter reaction contained 10μl SYBR Green Premix(TianGen,China),0.4μl ROX, 25ng soil DNA,10μg BSA,and300nM of each primer.The u s e d p a i r p r i m e r s w e r e r p s5′(5′-ATGTCCCGTTATCGAGGACCT-3′)and trnS(5′-TACCGAGGGTTCGAATC-3′)(Nadot et al.1994; S o u z a-C h i e s e t a l.1997);C YA359F(5′-GGGGAATYTTCCGCAATGGG-3′)and CYA781A/B(5′-G A C TA C T G G G G TAT C TA AT C C C AT T-3′/5′-ACTACAGGGGTATCTAATCCCTTT-3′);and NL1f(5′-ATATCAATAAGCGGAGGAAAAG-3′)and LS2r(5′-ATTCCCAAACAACTCGACTC-3′);they were specific DNA primer pairs for the moss chloroplast rps4gene, cyanobacterial16S rRNA gene,and fungal25-28S rRNA gene,respectively.The thermal cycle conditions were as fol-lows:initial denaturation at95°C for15min followed by 40cycles of95°C for15s,60°C/60°C/57°C(moss, cyanobacterial and fungal cycles,respectively)for20s,and 72°C for45s.The cycle was followed by a melt cycle with a 0.3°C increment per minute from60to95°C.All qPCRs were performed in triplicate,and ddH2O was used instead of DNA templates as negative control.DNA quantitative controls were generated by amplifying the rDNA and rps4genes from soil DNA using each pair of primers.The PCR products were purified by the PCR Cleanup Kit(Axygen,USA)and cloned into the pMD19T vector (Takara,Japan).DNA standards were then obtained from the plasmid DNA with concentrations ranging from2.7×103to 2.7×108copies of DNA per reaction.The copy numbers of the target samples for each reaction were calculated from the standard curves.Statistical analysesStatistically significant differences were calculated by inde-pendent sample t tests.The Pearson’s correlation coefficients among measured parameters were determined by the SPSSBiol Fertil Soils21.0software.Principal component analysis (PCA)was car-ried out using CANOCO (Ter Braak and Smilauer 1998).ResultsGene copy numbers of moss chloroplast rps4,cyanobacterial 16S rRNA,and fungal 25-28S rRNA quantified by qPCR were shown in Fig.1.The target genes in the unconsolidated soil and negative control were both undetermined.The content of moss rps4gene ranged from 3.9×104to 1.7×108copies per gram of soil with significant differences (P <0.001)among sites.Cyanobacterial 16S rRNA gene and fungal 25-28S rRNA gene copies ranged from 6.7×108to 1.7×109and from 4.8×107to 4.3×108copies per gram of soil,respective-ly,with significant differences among S1,S2,and S3(P <0.05)but not between S3and S4(P >0.05).The percentage of cyanobacterial 16S rRNA gene copies ranged from 69to 97%of the total copies,whereas that of the fungal 25-28S rRNA gene ranged from 3to 20%.Meanwhile,that of the moss rps4gene ranged from and 0to 17%.From S1to S4,there was a declining trend of cyanobacterial and fungal relative biomass and an increase trend of moss relative biomass in different BSC types.The chlorophyll a content is considered as a biomass proxy for photosynthetic organisms in BSCs.As shown in Fig.2,the average chlorophyll a contents of different BSC types ranged from 0.968to 5.306μg g −1soil with significant differences (P <0.001)among sites;the average DNA content ranged from 12.32(S1)to 16.44(S4)μg g −1of soil with significant (P <0.05)differences between S1and S3.The ratios of chlo-rophyll a to DNA contents were 0.078,0.116,0.317,and 0.322in S1,S2,S3,and S4,respectively (Fig.2).PCA and calculation of the Pearson ’s correlation coeffi-cients were performed to display the differences of communi-ty composition and parameter correlation among these sites (Fig.3).The moss relative biomass was significantly and pos-itively correlated (P <0.001)with a total DNA and chloro-phyll a content.Fungal relative biomass was not significantly correlated (P >0.05)with DNA and chlorophyll a content.Cyanobacterial relative biomass was significantly and nega-tively correlated (P <0.01)with moss relative biomass and chlorophyll a content.The four BSC sites were segregated by PC1and PC2,which were virtual factors generated from all the parameters used for PCA analysis by the software CANOCO.DiscussionThis study was the first attempt to use qPCR method for de-termining moss rps4gene abundance in BSCs.There was a linear relationship between the log of plasmid DNA copy numbers and the calculated C T values across the concentration range (R 2=0.996,0.997,and 0.998for moss,fungi,and cyanobacteria,respectively),and this suggested high accuracy in determining microbial biomass (Larionov et al.2005).Amplification efficiencies were 0.91,0.94,and 0.94for moss,fungi,and cyanobacteria,respectively,as calculated by the E =10[−1/slope]−1equation (Pfaffl 2001).Moss rps4genes were also detected at the lowest template concentration (3.9×104copies,Fig.1a )in S1and S2,and this indicated that the method was sensitive.The DNA content did not show significant differences,while the chlorophyll a content increased significantly from S1toS4Fig.1Gene copy abundances determined by qPCR (a )and the percentage of total gene copies present as gene copies of moss chloroplast rps4,fungal 25-28S rRNA,and cyanobacterial 16S rRNA (b )in different BSC types.Means ±SDs are indicated by the error bars .CK,unconsolidated soil (control);S1,cyanobacterial crust;S2,lichen crust;S3,moss crust;and S4,moss crust.*P <0.05,values significantly different with other samples;#P >0.05,values that showed no significant difference with S3Biol Fertil Soils(Fig.2).The inconsistency between DNA and chlorophyll a content can depend on the fact that the chlorophyll a content cannot be used to estimate total soil biomass (Steven et al.2014),due to the presence of non-photosynthesizing microor-ganisms such as fungi and bacteria.The fungal biomass in-creased significantly (P <0.05)by the increasing cyanobacterial biomass and led to the morphological change from cyanobacteria crust to lichen crust in comparison with S1and S2as shown in Fig.1b (Belnap and Lange 2002;Williams et al 2013).However,the moss rps4gene copy number and chloro-phyll a content were significantly and positively correlated and this may be a further indirect validation of the method to deter-mine moss biomass.The rising ratio of chlorophyll a /DNA content from S1to S4also suggested an increasing C-input efficiency in BSCs because the mosses contain more chloro-phyll and a higher photosynthetic capacity compared with the lichen and cyanobacteria (Pojar and MacKinnon 1994).The fungal and cyanobacterial gene copy numbers and their percentages in total biomass confirmed that already re-ported (Bates and Garcia-Pichel 2009;Colica et al.2014;Steven et al.2014).The percentage of cyanobacterial,fungi,and moss biomass variated and showed a declining trend of cyanobacterial relative biomass and a rising trend of moss biomass from S1to S4(Fig.1).The community composition analyses proved that S1was dominated by cyanobacteria,S2was dominated by lichen,and S3and S4were dominated by mosses.The analyses also confirmed that moss and fungi colonize even in the early stage of BSC succession (Williams et al.2013).In conclusion,this study was the first attempt to use qPCR method for determining moss biomass in BSCs;the method was accurate,sensitive,and reproducible.The moss rps4gene copy number significantly and positively correlated with chlo-rophyll a content,indicating an indirect validation of the method to determining moss biomass.However,given the heterogeneity of BSC succession in different regions,we rec-ognize the need for further analysis of additional sites with broader sampling across a large geographic range before the method can be widely used to evaluate BSC development in the early succession stage.Acknowledgments This research was supported by the National Natural Science Foundation of China (31370421),Wuhan basic research plan (201406101010060),and the National Talent Plan of Science Foundation (Wuhan University,J1103409).ReferencesBall BA,Guevara JA (2015)The nutrient plasticity of moss-dominatedcrust in the urbanized Sonoran Desert.Plant Soil 389:225–235Bates ST,Garcia-Pichel F (2009)A culture-independent study of free-living fungi in biological soil crusts of the Colorado Plateau:theirFig.2Chlorophyll a (a )and extractable DNA contents (b )of different BSC types.Means ±SDs are indicated by the error bar±.CK,unconsolidated soil (control);S1,cyanobacterial crust;S2,lichen crust;S3,moss crust;and S4,moss crust.$P <0.001,values significantly different with other samples;&P <0.05,values significantly different fromS1Fig.3Sample-species explanatory variables triplot from PCA.The values on the x-and y-axes represent the percentages of the total variation explained by principal components 1(PC1)and 2(PC2).S1,cyanobacterial crust;S2,lichen crust;S3,moss crust;and S4,moss crustBiol Fertil Soilsdiversity and relative contribution to microbial biomass.Environ Microbiol11:56–67Belnap J(2013)Some like it hot,some not.Science340:2Belnap J,Büdel B,Lange OL(2003)Biological soil crusts:characteristics and distribution.In:Belnap J,Lange OL(eds)Biological soil crusts: structure,function,and management.Springer,New York,pp3–30 Belnap J,Lange OL(2002)Biological soil crusts:structure,function,and management;with30tables.Springer,BerlinBu CF,Wu SF,Han FP,Yang YS,Meng J(2015)The combined effects of moss-dominated biocrusts and vegetation on erosion and soil mois-ture and implications for disturbance on the Loess Plateau,China.PLoS ONE10,e0127394Chen L,Rossi F,Deng S,Liu Y,Wang G,Adessi A,De Philippis R (2014)Macromolecular and chemical features of the excreted extra-cellular polysaccharides in induced biological soil crusts of different ages.Soil Biol Biochem78:1–9Chen L,Xie Z,Hu C,Li D,Wang G,Liu Y(2006)Man-made desert algal crusts as affected by environmental factors in Inner Mongolia, China.J Arid Environ67:521–527Colica G,Li H,Rossi F,Li D,Liu Y,De Philippis R(2014)Microbial secreted exopolysaccharides affect the hydrological behavior of in-duced biological soil crusts in desert sandy soils.Soil Biol Biochem 68:62–70De Caire G,De Cano M,Palma R,De 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生防菌剂多黏类芽胞杆菌对辣椒根际土壤细菌群落的影响
生防菌剂多黏类芽胞杆菌对辣椒根际土壤细菌群落的影响作者：韩永琴陈新建罗路云来源：《植物保护》2020年第02期摘要多黏類芽胞杆菌对辣椒疫病有较好的防治效果，本文重点研究了其对辣椒根际细菌微生物的影响。
收集了109 cfu/g多黏类芽胞杆菌WP、25%嘧菌酯SC、68%精甲霜·锰锌WG及对照处理后的辣椒根际细菌，通过高通量测序分析了根际细菌种群多样性和群落结构。
多黏类芽胞杆菌处理后的辣椒根际细菌多样性最高，化学药剂处理后的辣椒根际细菌多样性也均高于对照。
通过分析高通量测序结果，发现辣椒根际细菌包括变形菌门Proteobacteria、奇古菌门Thaumarchaeota、酸杆菌门Acidobacteria、绿弯菌门Chloroflexi、放线菌门Actinobacteria等13个门。
变形菌门的丰度在对照（JD）中高达70.65%，而在嘧菌酯（JA）、精甲霜·锰锌（JB）和多黏类芽胞杆菌（JC）的处理中分别降至24.41%、25.64%和34.95%，但仍然是各处理的优势菌群。
在化学药剂和多黏类芽胞杆菌的3种处理中酸杆菌门和绿弯菌门菌群的丰度远高于对照，特别是奇古菌门菌在化学药剂处理中的丰度极显著高于多黏类芽胞杆菌和对照处理。
经多黏类芽胞杆菌处理后发现芽单胞菌门和芽单胞菌属远高于对照，分别为7.35%（JC）、0.11%（JD）。
研究结果表明：施用多黏类芽胞杆菌不仅可以有效防治辣椒疫病，还可以改变辣椒根际土壤微生物区系，提高土壤中细菌种群的多样性。
关键词辣椒疫病; 多黏类芽胞杆菌; 微生物群落结构; 微生物多样性中图分类号： S476 文献标识码： A DOI： 10.16688/j.zwbh.2018463Abstract Bacillus polymyxa showed high activity in controlling pepper blight. The effect of the strain on bacterial microbes in the rhizosphere of pepper was studied. The rhizosphere bacteria of pepper treated by B. polymyxa 109 cfu/g WP， azoxystrobin 25% SC， metalaxyl-M· mancozeb 68% WG and the control were collected， and the diversity and community structure of the rhizosphere bacterial population were analyzed by Illumina Miseq sequencing technology. The diversity of rhizosphere bacteria of the pepper treated with B. polymyxa was the highest， and the diversity of rhizosphere bacteria of the pepper treated with chemical agents was also higher than that of the control. The bacterial populations included 13 phyla such as Proteobacteria，Thaumarchaeota， Acidobacteria， Chloroflexi and Actinobacteria. Proteobacteria was the most dominant phylum. The abundance of Proteobacteria was as high as 70.65% in the control （JD），but decreased in the treatments of azoxystrobin （JA）， Metalaxyl-M mancozeb WG （JB） and B. polymyxa （JC）， which was 24.41%， 25.64% and 34.95%， respectively. In the three treatments of chemical agent and B. polymyxa， the abundances of Acidobacteria and Chloroflexi were much higher than that of the control. In particular， the abundance of Thaumarchaeota in the treatment with chemical agent was extremely higher than that with B. polymyxa and the control. After treatment with B. polymyxa， the abundance of Gemmatimonadetes was 7.35% higher than that of the control. B. polymyxa as a biological pesticide can improve bacterial species abundance in plant rhizosphere soils and make a great contribution to the prevention of Phytophthora blight.Key words pepper Phytophthora blight; Bacillus polymyxa; microbial community structure; microbial diversity辣椒疫病是由辣椒疫霉菌Phytophthora capsici引起的土传性病害[1]，该病害可造成辣椒成片死亡，甚至绝收[2-3]。
FastDNA SPIN Kit for Soil中文说明书
FastDNA SPIN Kit for Soil中文说明书简介FastDNA ®SPIN Kit for Soil的目的是从土壤样品中提取基因组DNA 供PCR使用。
在不到30分钟内，快速DNA提取方法排除使用有害的有机溶剂，如苯酚和氯仿。
该工具包可在土壤社区从所有的细菌，真菌，植物和动物的基因组DNA提取。
该试剂盒包括三个部分：1.裂解Lysing Matrix E tubes中含有的陶瓷和硅粒子，旨在有效地溶解所有不同来源的微生物，包括真细菌孢子和芽孢，革兰氏阳性菌，酵母菌，藻类，线虫和真菌。
2.均质化试剂MT Buffer和Sodium Phosphate Buffer都经过精心挑选和准备，使完整的样本同质化和蛋白增溶。
试剂能够以最小的RNA污染提取基因组DNA。
3.DNA纯化和洗脱试剂GENECLEAN ®过程是他们用来纯化基因组DNA。
程序纯化DNA用专门的硅胶基质的同时消除污染物，抑制并发反应。
* Binding Matrix悬浮* SEWS-M（盐乙醇洗，用前加100ml乙醇）* DES（DNA洗脱液超纯水）试验方案样品处理：1.添加500毫克的土壤到Lysing Matrix E Tube。
由于FastPrep ®仪器的移动，在管内看到大量的聚集。
相同Lysing Matrix不应超过管的体积的7 / 8。
留在管的空间，也提高了更好的同质化的机会。
[见注1]裂解：加入978μl Sodium Phosphate Buffer 和122μl MT Buffer。
[见注1]2.在FastPrep ®仪器中混匀40秒的速度6.0。
3.14000 XG离心Lysing Matrix E Tubes 5-10分钟。
[见注2]4.上清转移到一个2ml干净的管。
加入250μlPPS试剂和混合，用手摇动管的10次。
5.14000 XG离心5分钟沉淀沉淀。
猪场土壤中5种四环素抗性基因的检测和定量
猪场土壤中5种四环素抗性基因的检测和定量2009年第4卷第5期,705—710生态毒AsianJournal理ofEcotoxicologyV o1.4.2009No.5.705—710猪场土壤中5种四环素抗性基因的检测和定量吴楠,乔敏,朱永官中国科学院生态环境研究中心土壤环境研究室,北京100085摘要:近年来由于四环素在畜禽养殖业中的大量使用,诱导了环境中四环素抗性微生物的产生,然而有关四环素抗性基因在土壤中存在,迁移和扩散的研究目前还很少.论文提取了北京一规模化养猪场周边土壤的微生物DNA,利用普通PCR检测到5种四环素抗性基因(1et(B/P),f(M),let(0),let(T),tet(W)),这5种基因都属于编码核糖体保护蛋白的一类.进一步建立了定量PCR程序对这5种基因进行了定量.结果显示,除tet()和tet(M)含量接近外(p=O.367),其余几种基因含量之问均差异显着(p<0.05),其中tet(w)的含量最高((2.16±0.20)×10copies?g(干土)),比含量最低的tet(B/尸)((2.89_+0.54)×10copies?g.(干土))高出约两个数量级.抗性基因(W),tet(T),tet(),(D)含量均较高,为猪场土壤中优势抗性基因.这些基因曾被报道广泛存在于猪和牛的肠道中,显示抗性基因很可能是通过基因横向转移(HGT)等机制从养殖动物体内传播到周围土壤中土着微生物体内的.论文所建立的实验方法为进一步系统研究抗生素抗性基因在土壤中的环境行为及其生态风险提供了基础.关键词:四环素抗性基因;实时荧光定量PCR;土壤;猪场文章编号:16735897(2009)570506中图分类号:XI71.5,X131.3文献标识码:A QuantificationofFiveTetracyclineResistanceGenesinSoilfromaSwineFeedlotWUNan,QIAOMin,ZHUY ong-guanDepartmentofSoilEnvironment,ResearchCenterforEco—EnvironmentalSciences,ChineseAcademyofSciences,Beijing100085Received18ArIril2009accepted24May2009Abstract:Inrecentyears,theprevalenceoftetracycline—resistantbacterialinenvironmentareinfluencedbythewideuse oftetracyclinesinlivestockproductionfacilities.However,thesurvival,fateanddisseminati onoftetracyclineresistance(Tc)genesinsoilarestillpoorlyunderstood.Polymerase—chain—reaction(PCR)assayswereconductedforfiveTcgenes encodingribosomalprotectionproteins(tet(B/P),let(M),tet(0),tet(),tet(W)),whichwerefo undintotalDNAex~actedfromsoilaroundaswinefeedlotinBeijing.Furthermore,areal—timequantitativePCRmethodwasdevelopedfor quantifyingthesefivespecificTcgenes.Exceptf0rmesimilarcopynumbersbetweentet(T)a ndtet(M)(P=0.367),me copynumbersofotherTcgenesinthesoilweresignificantlydifferent(P<0.05).Thehighes tcopynumbersoftet(W)((2.16_+0.20)×10copies'g(drysoil))wereabouttwoordersofmagnitudehigherthanthelowestcopynumber softet(B/P)((2.89-+0.54)×10copies?g(drysoil)).1et(M),tet(0),tet(),let(W)werepredominantinsoilandthese geneshadbeenreportedtobedominantinthegastrointestinaltractsofpigsandcaries,indicati ngthattheswinefeedlot hadaneffectonthesurroundingsoilandtheseTcgenesmightbetransferredfromanimalstothe indigenousbacterialinsoilthroughhorizontalgenetransfer(HGT).Theestablishedmethodinthisstudyprovidesthe basisforfurtherstudying theenvironmentalbehaviorandtheassociatedriskofantibioticresistancegenesinsoil. Keywords:tetracyclineresistance(Tc)genes;real—timequantitativePCR;soil;swinefeedlot收稿日期:2009—04—18录用日期:2009—05—24基金项目:中国科学院知识创新工程重大项目(No.KZCX1一Yw—O6—03);国家重点基础研究发展计划(973)项目(No2007CB407304;No.2007CB407301)作者简介:吴楠(1984一),女,硕士研究生,E—mail:*******************;通讯作者(Correspondingauthor),E—mail**************.cn7O6生态毒理第4卷1引言(Introduction)四环素是一类常见的抗生素,除广泛用于医疗外,还作为兽药和促生长剂大量用于养殖业(孔维栋等,2007).研究证明大部分抗生素难以被动物吸收利用,约25～75%的抗生素未经代谢而以母体化合物形式随粪尿排出体外(Chee—Sanfordeta1.,2001),因此如果将这些残留有四环素的粪便作为有机肥施入农田,就会对土壤环境中的微生物的耐药性产生选择压力,诱导四环素抗性基因(Te~acycfineresistancegenes,Tcgenes)的产生(Schmitteta1.,2006).抗性基因可以整合到一些可移动基因元件上,如质粒,转座子,整合子等,进而能够在共生微生物之间,革兰氏阳性菌和革兰氏阴性菌之间,甚至致病菌和非致病菌之间相互传播(Prudeneta1.,2006;Ansarieta1.,2008).通过这种基因横向转移(HorizontalGeneTransfer, HGT),抗性基因可以在土壤,地下水及各个环境介质中迁移,转化,并很可能随抗性质粒等被带入食物链并最终进人人体,增加人体的抗生素耐药性.因此,环境中具有HGT这种内在机制的微生物可能变成抗性基因的一个储存库(Martinez, 2008).更重要的是,这些媒介生物体能够以超过亲代菌株的效率来扩散这种抗生素抗性.一旦这些抗性基因从环境微生物进入到致病菌中,就会对人类健康构成潜在威胁(周肩星等,2007).近年来有许多关于微生物对四环素和其他类抗生素表型抗性(Phenotypicresistance)的研究(Guerraeta1.,2003),然而目前研究所采用的方法只能研究可培养微生物的抗性,而对不可培养微生物的抗性及微生物体内导致这些抗性的特定基因无法检测.也有一些研究利用普通PCR—DGGE 等技术来对环境中的四环素抗性基因进行直接检测(Chee—Sanfordeta1.,2001),但是还缺少定量结果.因此近年来越来越多的研究采用实时荧光定量PCR(Real—timequanti~tivePCR)技术作为研究手段,从数量上更为直观地探讨环境中抗性基因的变化,而且由于这种方法不依赖于微生物培养, 因此也能检测不可培养微生物所携带的抗性基因,使最终得到的量化结果更为全面和可信(Smitha1.,2004).目前国外对环境中四环素抗性基因的定量研究主要集中于水环境(包括沉积物) (Smitheta1.,2004;Peieta1.,2006;Peaketa1.,2007;Engemanneta1.,2008),而对土壤研究较少.国内关于环境中抗生素抗性基因的研究才刚刚起步(罗义等,2008),有关土壤中抗性基因存在情况的报道更是几乎为零,因此研究我国土壤环境中抗性基因的污染水平,抗性基因种类及主要污染区域是十分必要的.猪场是四环素等抗生素使用较为频繁的地方(Campagnoloeta1.,2002;Sengeloveta1.,2003),残留的四环素可能会诱导土壤微生物抗性基因的产生,使猪场土壤成为一个四环素抗性基因潜在的储存库.因此本试验选择猪场土壤作为研究对象,利用普通PCR对其中可能存在的一些四环素抗性基因进行了检测,并在此基础上设计了定量PCR程序,对已检测出的5种抗性基因进行了定量. 2材料与方法(Materialsandmethods)2.1供试材料土壤采自北京周边一规模化养猪场,该猪场建于1990年,养猪5000余头.采集猪场堆肥池周边土壤(表层0～10cm),共1,采回后,充分混匀,取部分鲜土提取DNA.测定土壤基本理化性质(鲁如坤,2000):含水量(80.1±1.0)%,pH7.98,有机质7.47g?～.2.2土壤DNA提取采用试剂盒FastDNASpinkitforSoil( Biomedicals),依照生产商说明的方法提取土壤微生物基因组DNA.提取的DNA用微量核酸蛋白质分析仪(Nanodrop)检测含量以及纯度(A260/A280值在1.8～2.0之间),表明用试剂盒提取的DNA纯度较高.2.3普通PCR反应程序本试验选择了14种常见的四环素抗性基因(f(/尸),let(A),let(),let(D),let(G),let(),let(M),let(O),tet(Q),tet(S),let(),let(),let(x),f(z)),对猪场土壤进行检测,这些抗性基因的引物设计主要来源于已发表的文献(Amilloveta1.,2001).在猪场土壤中一共检测到了5种抗性基因(1et(/P),let(),let(O),let(),f()),引物,片段大小及退火温度见表1.第5期吴楠等:猪场土壤中5种四环素抗性基因的检测和定量707表1四环素抗性基因普通PCR的引物和定量PCR反应程序Table1PrimerstargetingtetracyclineresistancegenesandPCRconditionsusedforreal—timePCRPCR反应体系体积为25t~L,包括0.1255U?LExTaqDNA聚合酶(TaKaRaBiotechnology),2.51zL缓冲液10xExTaqbuffer(含Mg),2txL浓度为2mmol?L的dNTPs,浓度为20txmol?L的引物各0.251xL,0.51xL的稀释10倍的DNA样品.PCR反应条件为:94℃下预变性4min,94℃45s,退火45s(退火温度见表1),72℃1min,35个循环,最后72℃下延伸6min.PCR产物与1/6体积6~loadingbufferdye(TaKaRa)混匀,用经EB染色的琼脂糖凝胶电泳检测.根据检测目标片段的大小(168～171bp),选择浓度为2%的琼脂糖凝胶,50bp的Marker,在80～90V电压下电泳35～45min.2.4目的片段测序及序列比对采用DNA凝胶纯化试剂盒(Promega)纯化扩增后的目的基因片段,将片断连接在pGEM—T Easy载体上(Promega,Corp.,Madison,Wis.),然后转化感受态大肠杆菌细胞(JM109,Biomed).涂平板后挑选阳性克隆子,用LB培养液扩大培养(约5mL),取lmL菌液送北京诺赛基因组研究中心测序插入基因片断,其余2-4mL菌液用来提取质粒, 稀释标线备用.测序结果通过使用NCBI网站(/blast/)的BLAST进行序列同源性检索比对.2.5实时荧光定量PCR(Real—timequantitative PCR)2.5.1制作标准曲线质粒用试剂盒MiniBESTPlasmidPurification KitV er.2.0(TaKaRa)依照生产商说明的方法提取, 提取的质粒用微量核酸蛋白质分析仪(Nanodrop) 检测含量以及纯度,准备稀释标线备用.基因拷贝数(Peieta1.,2006)按以下公式计算:拷贝数=(质量/分子量)x6.02×1,例如pGEM—T载体长3015bp,插入的tet()目的片断长168bp,每个碱基的平均分子量是330(每对碱基/bp是660),质粒原液xng?L～,阿弗加德罗常数(每mol的微粒数)是6.02x10mol～,那么每L的绝对模板数量是:(X/(3015+168)x660)xl0×6.02x10,从而计算出tet()基因的拷贝数.按10倍为稀释梯度稀释成标准曲线,根据标准曲线计算出样品中的每种抗性基因的拷贝数.在实时荧光定量PCR技术中,ct值(ThresholdCycle)表示每个反应管内的荧光信号到达设定的域值时所经历的循环数,每个模板的ct值与该模板的起始拷贝数的对数存在线性关系,起始拷贝数越多,ct值越小.利用已知起始拷贝数的标准品可做出标准曲线,其中横坐标代表起始拷贝数的对数,纵坐标代表ct值.因此,只要获得未知样品的ct值,即可从标准曲线上计算出该样品的起始拷贝数.数据分析由iCyclersoftware(version1.0.1384.0CR)完成.2.5.2定量PCR反应体系及程序定量PCR反应在iQ5(Bio—rad,USA)仪器上进行.反应体系为251xL,其中包含21xL(约1.27ng? L)模板DNA,12.5L荧光染料SYBRPremix ExTaq(TaKaRaBiotechnology),引物各200nmol?L一,反应在200tzL圆顶PCR管中进行.定量PCR 反应程序见表1.溶解曲线程序为55至95℃之间,每0.5℃读数,其问停留30s.每个样品均做3次重复,起始模板浓度由ct值确定.定量PCR产物用2%琼脂凝胶电泳检测.708生态毒理第4卷2.6统计方法数据分析采用SPSS13.0(SPSS,Inc.Chicago,IL)统计软件进行,组间比较采用One—way ANOV A,多重比较采用LSD检验.3结果(Results)3.1普通PCR结果检测通过普通PCR一电泳可以清晰显示目标基因片段(图1),同时没有观测到非特异性条带,说明所试土壤中存在四环素抗性基因,且试验所采用的PCR体系和程序是适宜的.500bp'......_.——250bp.____---?———Marker12345图1猪场土壤中四环素抗性基因的PCR电泳图(Marker:50bp;l:tet(W);2:tet(M);3:tet(O);4:tet(T);5:tet(B/P)) Fig.1EleclrophoresisofTcgenesbyPCRamplification (Marker:50bp;1:tet(W);2:tet(M);3:tet(O);4:tet(T);5:tet(B/P)) 3.2标准曲线及定量PCR结果定量四环素抗性(Tc)基因的标准曲线如图2所示.在由5或6个点确定的标准曲线中,Ct值和LogmTc基因拷贝数具有很强的线性关系(R在0.992—0.994之间),扩增效率E在91.8%～105.0%菩季苫鋈蓬之间,斜率在一2.9～3.5之间.根据标准曲线计算出样品中的各种Tc基因拷贝数分别为:tet(B/e)为(2.89+0.54)×106copies?g(干土),tet()为(4.45±0.25)×107copies?g(干土),tet(M)为(3.92±0.54)x107copies?g(干土),tet(O)为(1.65±0.07)×10copies?g(干土),tet(w)为(2.16_+0.20)×10.copies?g(干土)(图3).从图3可以看到tet(W)的含量最高,比含量最低的tet()高出约两个数量级,为主要优势基因,另外3种基因tet(),tet(M),tet(0)的含量也较高.各基因含量之间两两比较,除,()和()含量接近外(p=O.367),其余几种基因含量之间均差异显着(p<0.05).4讨论(Discussion)到目前为止,已有40多种四环素类抗药基因被发现和命名,包括近40种四环素抗药基因f和3种土霉素抗药基因otr(Levyeta1.,1999).大部分抗药基因编码四环素外排泵蛋白,少数编码核糖体保护蛋白,个别基因编码四环素类分子的修饰或破坏酶(Aminoveta1.,2001).编码核糖体保护蛋白的一类主要包括tet(0),tet(a),tet(),tet(M),tet(B/P),tet(),f()和otrA,在北京猪场周边土壤中发现的5种四环素抗性基因都属于这一类.与之前的研究报道比较发现,该猪场周边土壤中出现的几种优势基因f(),f(T),tet(M),tet(0),在其他许多养殖场(猪场,牛场等)周围环境中也常被检测到(mminoveta1.,2001;Chee—Sanfordeta1.,2001;Smitheta1.,2004;Peieta1.,Logl0Tc基因拷贝数LogStartingQuantity,copynumbers图2四环素抗性基因定量PCR的标准曲线Fig.2Stand~dcurveforreal-timePCRofTcgenes第5期吴楠等:猪场土壤中5种四环素抗性基因的检测和定量709 耙f(B/P)tet(0)tet(M)tet(丁)tet(w)四环素抗性基因Tcgenes图3猪场土壤中四环素抗性绝对基因拷贝数(相同字母表示差异不显着(p>0.05);不同字母表示差异显着(p<0.05).LSD多重比较)Fig.3Tcabsolutegenecopynumbersinswinefeedlotsoil (Differentlettersindicatedsignificantdifferences(p<0.05))2006;Schmitteta1.,2006;Peaketa1.,2007),说明这几种四环素抗性基因在养殖场周围环境中是广泛存在的;而tet(B/P)只在少数报道中被检测出(Chee—Sanfordeta1.,2001;mminoveta1.,2001),表明tet(B/尸)在环境中存在水平可能较低,这与本实验定量检测的结果也一致.另外,tet(),tet(D),()等,曾被报道广泛存在于猪和牛的肠道中(Scotteta1.,2000; Aminoveta1.,2001),因此猪场中养殖动物肠道内的抗性基因很可能作为抗性基因的污染源,随粪尿等途径进入土壤环境,并通过基因横向转移(ttGT)和重组等机制将抗性基因传播到周围土壤中土着微生物体内.许多研究表明,这些四环素抗性基因往往与转座子等基因移动原件相关联(ShowshandAndrews,1992;ChopraandRobe~, 2001;Melvilleeta1.,2004),具有在微生物种群间传播扩散的能力(Billingtoneta1.,2002);而且抗性基因进入土着微生物体内后,更利于其在环境中的存在和移动(Chee—Sanfordeta1.,2001).因此,抗性基因具备在环境中迁移传播的能力和倾向,Chee—Sanford等(2001)在距猪场化粪池下游250m处的地下水中仍检测到了抗性基因,表明抗性基因能够渗透到地下水中,证实了抗性基因在环境中的传播.由于不同种类的抗生素对应存在着不同的抗药基因,而每种抗生素的抗药基因往往又有很多种,再加上环境条件,检测方法等因素的影响,使得单一的环境调查很难完全系统的将各种抗药基因进行详细统计.虽然本实验只基于一个样本研究了猪场土壤中存在的部分抗生素抗性基因,但从一定程度上证实了四环素抗性基因在土壤环境中存在的多样性以及在动物养殖系统与周围环境之间传播的可能性.本项研究结果显示养殖场周围土壤是抗性基因的一个重要存储库,为抗性基因进入食物链提供了一个可能的来源,因此具有一定的生态与健康风险.本研究所建立的定量检测四环素抗性基因的方法也为今后进一步系统研究土壤中抗生素抗性基因的环境行为提供了基础.通讯作者简介:朱永官(1967一),男,理学博士.2001年入选中科院"百人计划",2002年获得国家基金委杰出青年基金,现任中国科学院生态环境研究中心中澳联合土壤环境研究室主任,研究员,博士生导师.主要从事土壤生态和土壤一植物系统中微量元素和重金属迁移积累的化学和生物一学调控机制和技术的研究.ReferencesAminovRI,Garrigues—JeanjeanN,MackieRI.2001.Molecular ecologyoftetracyclineresistance:developmentandvalidationof primersfordetectionoftetracyclineresistancegenesencoding ribosomalprotectionproteinslJJ.AppliedandEnvironmental Microbiology,67(1):22—32AnsariMI,GrohmannE,MalikA.2008.Conjugativeplasmids inmulti—resistantbacterialisolatesfromIndiansoil[J].Journal ofAppliedMicrobiology,104(6):1774-1781BillingtonSJ,SongerJG,JostBH.2002.Widespread distributionofaTetWdeterminantamongtetracycline—resistant isolatesoftheanimalpathogenArcanobacteriumpyogenes[J]. 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Occurrenceofthenewtetracyclineresistancegenetet(w)in bacteriafromthehumangut[J].AntimicrobialAgentsand Chemotherapy,44(3):775—777SengelovG,AgersoY,Halting—SorensenB,BalodaSB,Andersen JS,JensenLB.2003.Bacterialantibioticresistancelevelsin Danishfarmlandasaresultoftreatmentwimpiglnamlreslurry [J].EnvironmentInternational,28(7):587—595 ShowshSA,AndrewsRE.1992.TetracyclineenhancesTn916- mediatedconjugaltransfer[J].Plasmid,28(3):213—224 SmithMS,Y angRK,KnappCW,NiuYF,PeakN, HanfeltMM,GallandJC,GrahamDW.2004.Quantification oftetracyclineresistancegenesinfeedlotlagoonsbyreal—time PCR[J].AppliedandEnvironmentalMicrobiology,70(12):7372—7377ZhouQX,LuoY,WangME.2007.Environmentalresidues andecotoxicityofantibioticsandtheirresistancegenepollution: Areview[nJournalofEcotoxicology,2(3):243—251 (inChinese)中文参考文献孔维栋,朱永官.2007.抗生素类兽药对植物和土壤微生物的生态毒理学效应研究进展[J].生态毒理,2(1):l一9鲁如坤.2000.土壤农业化学分析方法[M].北京:中国农业科技出版社,13—110罗义,周启星.2008.抗生素抗性基因(ARGs)——一种新型环境污染物[J].环境科学,28(8):1499—1505周启星,罗义,王美娥.2007.抗生素的环境残留,生态毒性及抗性基因污染[J].生态毒理,2(3):243—251◆。
城市绿地中立枯丝核菌和齐整小核菌的qPCR快速检测方法
城市绿地中立枯丝核菌和齐整小核菌的qPCR 快速检测方法雒淑红1，骆玉珍1，赵莺莺2，张维维2,3，刘 文2，何山文2，安 磊2，王永杰1，韩继刚2（1. 上海海洋大学 食品学院，上海 200120；2. 上海市园林科学规划研究院，上海 200232；3. 东北大学 资源与土木工程学院，辽宁 沈阳 110819）摘要：【目的】土传病原真菌立枯丝核菌Rhizoctonia solani 和齐整小核菌Sclerotium rolfsii 严重威胁园林绿化植物正常生长。
建立针对这2种土壤病原真菌的快速定量检测方法。
【方法】通过筛选2种病原菌特异性引物，优化反应条件。
【结果】初步建立了2种病原菌的实时荧光定量PCR(qPCR)检测方法。
引物ST-RS1/ITS4和SRITSF/SRITSR 可以分别用于立枯丝核菌和齐整小核菌的qPCR 检测，其灵敏度分别达24×106和22×106拷贝·L −1，2次重复反应的变异系数分别为3.37%~4.61%和0.66%~8.61%。
对上海绿地土壤样品的检测结果表明：立枯丝核菌和齐整小核菌的检出率分别为100%和19%。
【结论】建立的qPCR 检测方法具有较强特异性、较高灵敏度和较强重复性，可以用于上海城市绿地土壤中立枯丝核菌和齐整小核菌的快速、有效定量检测。
图2表5参29关键词：城市绿地土壤；立枯丝核菌；齐整小核菌；qPCR中图分类号：Q949.32 文献标志码：A 文章编号：2095-0756(2022)05-1087-09Quantitative real-time PCR for rapid detection of Rhizoctonia solani andSclerotium rolfsii in urban green spaceLUO Shuhong 1，LUO Yuzhen 1，ZHAO Yingying 2，ZHANG Weiwei 2,3，LIU Wen 2，HE Shanwen 2，AN Lei 2，WANG Yongjie 1，HAN Jigang 2（1. College of Food Science and Technology, Shanghai Ocean University, Shanghai 200120, China; 2. Shanghai Academy of Landscape Architecture Science and Planning, Shanghai 200232, China; 3. College of Resources and Civil Engineering, Northeastern University, Shenyang 110819, Liaoning, China ）Abstract: [Objective ] This study aims to establish a rapid quantitative detection method for Rhizoctonia solani and Sclerotium rolfsii ，2 soil-borne pathogenic fungi that seriously threaten the normal growth of landscape plants in Shanghai. [Method ] The reaction conditions were optimized by screening 2 pathogen specific primers. [Result ] A quantitative real-time PCR (qPCR) method was established for the detection of the two pathogens. The primers ST-RS1/ITS4 and SRITSF/SRITSR could be used for qPCR detection of R. solani and S. rolfsii , with sensitivity of 24×106 and 22×106 copies ·L −1, respectively. The coefficients of variation of the 2repeated reactions were 3.37%−4.61% and 0.66%−8.61%, respectively. The detection results of soil samples in Shanghai green space showed that the detection rates of R. solani and S. rolfsii were 100% and 19%,respectively. [Conclusion ] The established qPCR method has high specificity, sensitivity and repeatability, and收稿日期：2021-12-14；修回日期：2022-05-28基金项目：上海市绿化和市容管理局科技攻关项目(G200201)；上海市科学技术委员会科研计划项目(19dz1203300)作者简介：雒淑红(ORCID: 0000-0001-8129-1927)，从事生物化学与分子生物学研究。
DNA提取方法
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其它文献
氯化钙-SDS-酶法可以高效去除腐殖酸，所提取的 DNA 纯度较高；
可选择基于的高盐提取法和透析袋回收法。
杨建采用FastPrep法的DNA得率最高，但其成本 较高；
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其它文献
Eichner 调整法 （Eicher C A, Erb R W, Timmis K N, et al. Appl Environ
醇
醇
醇
玻璃粉吸附法
TE 缓冲液 使用 Sephadex G-200 离心柱 玻璃粉吸附
2021/6/21
11
方法比较
SDS 的高盐提取 Tsai 报 道 的 方 Edgcomb 改 进 玻璃粉吸附法
法
法
法
方
产 量 低 , 且 电 泳 重 复 性 很 好 , 可 保证一定的提取 去除 PCR 抑制
结果不明显
与回收效率及
法
以满足大多数微 DNA 的提取质 剂，DNA 纯化过 生物分子生物学 量 , 提 取 和 纯 化 程中损失最少，
评
过程 DNA 不会 实验的工作需要 受到机械损伤， 提高了 PCR 的
价
适合从不同类型 敏感性，回收率 的土壤中提取总
DNA
达 75%-100%
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纯化
7
Tsai报 道方法
1g土壤，研碎
2mlPBS 37℃摇床 225r/min振荡30min
12000r/min 离心10min
1.5ml溶液I， 0.5ml溶菌酶， 37℃水浴2h，
2ml溶液II， -20℃冰浴， 65℃水浴循环三次
6000r/min离心10min，取上清， 等体积苯酚:氯仿:异戊醇25:24:1抽提两次
FASTDNASPINKITFORSOIL中文说明书
FASTDNASPINKITFORSOIL中文说明书
FastDNA TM SPIN Kit for Soil
1.称取500mg土壤样品到Lysing Matrix E Tube.不要超过管的体积的7/8
2.加入978μl Sodium Phosphate Buffer，涡旋10-15s
3.加入122μl MT Buffer.用力振荡，涡旋10-15s
4.在FastPrep仪器中以6.0的速度振荡40s
5.14000g离心5-10min
6.转移上清液到2ml干净的离心管中，加入250μl PPS液，用手摇动管子10
次，室温放置10min（不要涡旋）
7.14000g离心5min，转移上清液（600-800μl）到干净的2ml 离心管中。
加
入等量的Binding Matrix。
用手轻轻振荡混匀，上下颠倒3-5min
8.用移液器吸取数次以混匀溶液。
转移800μl混合物到SPIN Filter tube中，
14000g离心5min，倒空Catch Tube。
将剩余的混合物混匀后，重复第8步的过程（同一SPIN Filter tube），弃滤液
9.加入500μl SEWS-M，轻轻振荡混匀
10.14000g离心5min，倒空滤液，再次14000g离心5min，去除残留的乙醇
11.将SPIN TM Filter放到新的Catch Tube中，室温干燥5min
12.加入100μl DES，用手指轻轻弹SPIN TM Filter，悬浮管内物质
（55℃放置5min有助于增加DNA产量）
13.14000g离心2min，洗脱DNA。
-20℃保存。
土壤试剂盒操作手册和常见问题
土壤试剂盒操作手册和常见问题FastDNA Spin Kit for Soil实验步骤1.Add up to 500 mg of soil sample to a Lysing Matrix E tube.在裂解介质管E中最多加入500mg土壤样品。
注意：推荐最多加入500mg土壤样品，含水量比较多的土壤或者碎屑多的土壤可适量减少样品量。
2.Add 978 μL Solution Phosphate Buffer to sample in Lysing Matrix E tube.在裂解介质管E中加入978μl Sodium Phosphate Buffer3.Add 122 μL MT Buffer.What’s happening: Begin to solubilize membrane proteins with detergents as well asextra-cellular proteins and contaminations in soil.加入122μl MT Buffer发生的反应：用洗涤剂溶解细胞膜蛋白以及细胞外蛋白和土壤中的污染物。
注意：为了得到更好的样品处理效果，加入土壤样品及两个缓冲液后，在裂解介质管中仍能保留有250-500μl空间。
4.Homogenize in the FastPrep Instrument for 40 seconds ata speed setting of 6.0What’s happening: mechanical disruption of cell walls of soil organisms and releasing nucleic acids into the protective buffer.将样品置于FastPrep?仪器上匀浆40s，速度为6.0m/s发生的反应：机械破碎土壤微生物的细胞壁，将核酸释放入保护缓冲液中。
Fast系列产品试剂盒
Mp FastDNA® Kit---------------------美国Mpbio公司FastDNA®试剂盒配合FastPrep®仪使用，可在少于30分钟内裂解分离得到多达200mg的基因组DNA。
试剂盒可选择不同的试剂组合，提供3种不同的缓冲液用于处理不同类型的样品。
之后用基于硅吸附DNA原理的GENECLEAN®法进行纯化，得到的DNA可用于PCR和其它实验。
优点：配合FastPrep®，(包括Fastprep-24或FP120)样品的裂解处理更快、重复性更好对不同来源的样品，都可以在少于30分钟内裂解并分离出DNA灵活的规格，避免购买其它的DNA分离试剂盒不含有毒有害的试剂成分应用：从植物、动物、细菌、酵母、藻类与真菌中分离出基因组DNA成分：裂解研磨粉 A，纯化吸附硅砂 B，SEWS-M溶液，DES溶液，BBS溶液，CLS-VF溶液，PPS溶液，CLS-TC溶液，CLS-Y溶液lysing Matrix A,Binding Matrix,SEWS-M Solution,DES Solution,CLS-VF Solution,PPS Solution,CLS-TC Solution,CLS-Y SolutinFastDNA® SPIN Kit for Soil---------------------美国Mpbio公司FastDNA® SPIN土壤DNA试剂盒用于从土壤和其它环境样品中的细菌、真菌、动植物提取基因组DNA。
一个2ml管可裂解处理500mg土壤。
之后用基于硅吸附DNA原理的GENECLEAN®法进行纯化，得到的DNA可用于PCR和其它实验。
优点：配合FastPrep®，样品的裂解处理更快、重复性更好易于适用于不同的类型土壤中微生物基因组DNA的分离对不同来源的样品，都可以在60分钟内裂解分离出DNA不含有毒有害的试剂成分应用：从土壤或其它环境物质中细胞分离DNA成分：裂解研磨粉E，纯化吸附硅砂，SEWS-M溶液，DES溶液，BBS溶液，磷酸钠缓冲液，PPS 溶液，MT溶液，收集管，吸附柱Lysing Matrix E, PPS Solution, Binding Matrix, SEWS-M Solution, DES Solution, Sodium Phosphate Buffer, BBS Solution, MT Buffer, SPIN Filters, Catch Tubes产品信息：FastRNA® pro Soil Direct Kit---------------------美国Mpbio公司优点配合FastPrep®，样品的裂解处理更快、重复性更好适用于不同的类型土壤中微生物基因组RNA的分离对不同来源的样品，都可以在60分钟内裂解分离出RNA释放出的RNA用RNApro™处理纯化，保证了RNA质量不含有毒有害的试剂成分应用：从土壤或其它环境物质中细胞分离RNAFastRNA® Pro Blue Kit---------------------美国Mpbio公司FastRNA®Pro Blue RNA试剂盒用于从革兰氏阳性菌和阴性菌中高效分离RNA。
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FastDNA TM SPIN Kit for Soil
1.称取500mg土壤样品到Lysing Matrix E Tube.不要超过管的体积的7/8
2.加入978μl Sodium Phosphate Buffer，涡旋10-15s
3.加入122μl MT Buffer.用力振荡，涡旋10-15s
4.在FastPrep仪器中以6.0的速度振荡40s
5.14000g离心5-10min
6.转移上清液到2ml干净的离心管中，加入250μl PPS液，用手摇动管子10
次，室温放置10min（不要涡旋）
7.14000g离心5min，转移上清液（600-800μl）到干净的2ml离心管中。
加
入等量的Binding Matrix。
用手轻轻振荡混匀，上下颠倒3-5min
8.用移液器吸取数次以混匀溶液。
转移800μl混合物到SPIN Filter tube中，
14000g离心5min，倒空Catch Tube。
将剩余的混合物混匀后，重复第8步的过程（同一SPIN Filter tube），弃滤液
9.加入500μl SEWS-M，轻轻振荡混匀
10.14000g离心5min，倒空滤液，再次14000g离心5min，去除残留的乙醇
11.将SPIN TM Filter放到新的Catch Tube中，室温干燥5min
12.加入100μl DES，用手指轻轻弹SPIN TM Filter，悬浮管内物质
（55℃放置5min有助于增加DNA产量）
13.14000g离心2min，洗脱DNA。
-20℃保存。