甲型H1N1流感病毒采用不同核酸提取方法的比较研究
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摘要
目的研究甲型H1N1流感病毒核酸提取的比较准确和有效的方法。方法选用2014年12月至2015年12月就诊的甲型H1N1流感患者的咽拭子共40份作为研究对象。对所获得的标本进行10-1~10-4的系列稀释,获得不同浓度的流感病毒标本备检。主要采用三种试剂盒进行实验:一种是Promega公司的Maxwell16全自动核酸提取仪以及该提取仪的配套试剂盒,为A组,一种是QIAGEN公司的Rneasy Mini Kit,为B组,第三种则是国产的H生物公司的RNA提取试剂盒,为C组。对三种方法的核酸提取情况进行比较。结果对同一份咽拭子的核酸进行提取,A组所提取的总RNA浓度最高,B组的总RNA浓度次之,C组的总RNA浓度最低。A组在各个稀释浓度的流感病毒提取率均较高,差异均具有统计学意义(P<0.05);C组和B组随着稀释浓度的增大,流感病毒的提取率逐渐降低,尤其是C组当稀释浓度为10-4时,提取不到流感病毒。结论全自动核酸提取仪对核酸的提取效果较好,即使在高倍稀释的情况下也能够达到较高的核酸提取率,避免了手工提取的误差,是一种快捷有效的检测方法,值得在临床上推广使用。
Objective To study the accurate and effective method for the extraction of influenza a H1N1 influenza virus.Methods A total of 40 pharyngeal swab points from patients with influenza A( H1N1) from December 2014 to December 2015 in our hospital were selected as the research object.The obtained specimens were 10~(-1)~ 10~(-4)series dilution,obtained with different concentrations of influenza virus samples preparation of inspection.This research mainly uses three kinds of reagent box to carry on the experiment.A was Maxwell16 Promega automatic nucleic acid extraction apparatus and the extractor kit,as group A;B was the Qiagen RNeasy Mini Kit;C was domestic H company RNA extraction kit.The nucleic acid extraction of the three methods was compared.Results The total RNA concentration of A group was the highest,followed by B group,in C group was the lowest.The concentration of extraction rate in Group A at each dilution were highest and the difference was statistically significant( P < 0.05).With increase of concentration,the influenza virus extraction rate decreased gradually in Group B and group C,especially in group C,when diluted to 10~(-4),influenza virus couldn't be extracted.Conclusion The effect of Automatic nucleic acid extraction apparatus for nucleic acid extraction is better,even under high dilution conditions,it can also achieve high nucleic acid extraction rate.It is a rapid and effective detection method to avoid the error of the manual extraction,which is worthy of promotion and application in clinical.
Objective To study the accurate and effective method for the extraction of influenza a H1N1 influenza virus.Methods A total of 40 pharyngeal swab points from patients with influenza A( H1N1) from December 2014 to December 2015 in our hospital were selected as the research object.The obtained specimens were 10~(-1)~ 10~(-4)series dilution,obtained with different concentrations of influenza virus samples preparation of inspection.This research mainly uses three kinds of reagent box to carry on the experiment.A was Maxwell16 Promega automatic nucleic acid extraction apparatus and the extractor kit,as group A;B was the Qiagen RNeasy Mini Kit;C was domestic H company RNA extraction kit.The nucleic acid extraction of the three methods was compared.Results The total RNA concentration of A group was the highest,followed by B group,in C group was the lowest.The concentration of extraction rate in Group A at each dilution were highest and the difference was statistically significant( P < 0.05).With increase of concentration,the influenza virus extraction rate decreased gradually in Group B and group C,especially in group C,when diluted to 10~(-4),influenza virus couldn't be extracted.Conclusion The effect of Automatic nucleic acid extraction apparatus for nucleic acid extraction is better,even under high dilution conditions,it can also achieve high nucleic acid extraction rate.It is a rapid and effective detection method to avoid the error of the manual extraction,which is worthy of promotion and application in clinical.
引文
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[12]刘小君,向华国,曾锦婷,等.荧光探针PCR与膜芯片技术在女性生殖道HPV分型的对比研究[J].现代检验医学杂志,2015,30(6):92-93.
[13]俞松山,叶惠英,韩珊珊.免疫荧光技术与荧光探针PCR技术用于检测小儿呼吸道合胞病毒的对比分析[J].实用预防医学,2016,23(6):750-752.
[14]白志军,胡林,李魁彪,等.交叉引物恒温扩增法检测甲型H1N1流感病毒及临床应用[J].中国人兽共患病学报,2015,(3):208-211.
[15]田棣,何静,刘祎,等.胶体金免疫层析试剂盒对甲型H1N1流感病毒(2009)抗原快速检测的敏感性分析[J].检验医学,2013,28(2):154-158.
[16]李文静,徐红星,陈昭华,等.基于Pre-NAT全自动核酸检测系统的HBV-DNA超敏检测试剂方法学性能验证[J].临床检验杂志,2015,33(7):532-534.
[17]李铁钢,李魁彪,肖新才,等.新型甲型H1N1流感监测体系的建立与应用分析[J].热带医学杂志,2015,15(1):91-94.
[2]占玲俊,鲍琳琳,李枫棣,等.Trizol RNA提取法在雪貂组织的流感病毒H3N2定量PCR检测中优于RNeasy mini kit(Qiagen)柱子法[J].中国比较医学杂志,2012,22(2):22-27.
[3]张险朋,董瑜,温贤诚,等.三种核酸提取方法检测禽流感病毒的比较[J].动物医学进展,2014,35(11):116-118,119.
[4]李玉香,汪杨,鲍万国,等.重症甲型H1N1流感病毒基因与特异性抗体检测的诊断意义[J].中华医院感染学杂志,2015,25(12):2641-2643.
[5]张继华,马磊,寸怡娜,等.H1N1亚型流感假病毒评价中和抗体的研究[J].中华实验和临床病毒学杂志,2015,29(1):35-37.
[6]陈帅帅,丁建祖,尤金彪,等.流感病毒M1单克隆抗体的制备及其生物学特征[J].国际流行病学传染病学杂志,2015,42(1):3-8.
[7]吕沁风,罗鹏,何蕾,等.甲型H1N1流感病毒FAST-LAMP检测方法的建立[J].生物技术通报,2015,31(5):78-83.
[8]胡贞.PCR技术及其在禽流感诊断中的应用[J].中国畜牧兽医文摘,2015,30(8):64-64.
[9]戴玉柱,崔大伟,杨先知,等.五重荧光定量RT-PCR法检测甲型流感病毒[J].临床检验杂志,2015,30(9):641-644.
[10]李艳霞,陈国南.基于HRP直接标记的流感病毒H1N1电化学免疫传感器[J].分析测试学报,2015,34(6):701-705.
[11]武海萍,刘云龙,童欢,等.高灵敏焦测序结合生物发光分析仪检测甲型H1N1流感病毒[J].现代生物医学进展,2014,14(35):6818-6822.
[12]刘小君,向华国,曾锦婷,等.荧光探针PCR与膜芯片技术在女性生殖道HPV分型的对比研究[J].现代检验医学杂志,2015,30(6):92-93.
[13]俞松山,叶惠英,韩珊珊.免疫荧光技术与荧光探针PCR技术用于检测小儿呼吸道合胞病毒的对比分析[J].实用预防医学,2016,23(6):750-752.
[14]白志军,胡林,李魁彪,等.交叉引物恒温扩增法检测甲型H1N1流感病毒及临床应用[J].中国人兽共患病学报,2015,(3):208-211.
[15]田棣,何静,刘祎,等.胶体金免疫层析试剂盒对甲型H1N1流感病毒(2009)抗原快速检测的敏感性分析[J].检验医学,2013,28(2):154-158.
[16]李文静,徐红星,陈昭华,等.基于Pre-NAT全自动核酸检测系统的HBV-DNA超敏检测试剂方法学性能验证[J].临床检验杂志,2015,33(7):532-534.
[17]李铁钢,李魁彪,肖新才,等.新型甲型H1N1流感监测体系的建立与应用分析[J].热带医学杂志,2015,15(1):91-94.