环介导等温扩增检测甲型H1N1病毒核酸方法的建立  

Development of Loop-mediated Isothermal Amplification(LAMP) Mothod for Detection of Influenza A(H1N1) Virus.

作　　者：张永乐[1] 潘克女[2] 武静[1] 钟华燕 娄国强[4] 杨劲[5] 

机构地区：[1]杭州市第六人民医院分子诊断实验室,310014 [2]杭州市第一人民医院检验科 [3]杭州市优思达生物技术有限公司 [4]杭州市第六人民医院感染科,310014 [5]浙江省血液中心

出　　处：《医学研究杂志》2011年第6期144-147,共4页Journal of Medical Research

摘　　要：目的建立检测甲型H1N1病毒环介导等温扩增的方法（LAMP）。方法根据已公布的甲型H1N1流感病毒HA基因序列766—995bp的6个位点设计6条LAMP引物（2条内引物，2条外引物，2条环引物），优化并建立LAMP检测体系。并对36例确诊H1N1感染患者和20例健康体检者的咽式子同时采用实时荧光RT—PCR（Real—timeRT—PCR）和LAMP进行H1N1病毒核酸的检测。结果所建立的甲型H1N1病毒核酸LAMP反应体系具有良好的特异性，采用该体系检测结果与Real—timeRT—PCR检测结果一致，对H1N1核酸阳性的标本经LAMP扩增产物电泳图为阶梯状条带，健康对照标本未见条带产生。灵敏度试验，采用10^3复制的H1N1质粒进行10倍稀释后LAMP法仍能检出。特异性试验，20例体检标本经两种方法检测全阴性，对肺炎支原体和肺炎衣原体阳性标本采用该反应体系进行检测结果为阴性。结论甲型H1N1病毒核酸LAMP检测方法简便、快捷，对实验仪器设备要求不高，适合各级实验室和现场快速诊断使用。Objective To detect HIN1 infection rapidly by using loop -mediated isothermal amplification （LAMP）. Methods The H1N1 -specific LAMP primer set,including two inner, two outer, and two loop primers, was designed accroding to the conserved segment of HA gene, located between 766 and 995nt. The LAMP assay was further optimized. The oral swab samples, from 36 confirmed H1N1 patients and 20 healthy controls, were tested both by Real - time PCR and LAMP method. Results H1N1 - LAMP spcifically detected H1N1 virus strain, showing specific ladder- like pattern in eletrophoresis, while no positive signal could be observed in control group. The results of H1N1 - LAMP were eonsistant to those of Real- time RT- PCR. The sensitivity of H1N1 - LAMP assay was performed by 10 - fold dilution of the samples with the concentration of 103 Copies, which was confirmed by real - time RT - PCR. For the specificity experiment, 20 example physical examination specimen examined were all negative after two methods. Conclusion H1N1 - LAMP assay is easy to perform, no requring of sophisticated equipment. It is potential for widely application in clinical laboratory and point of care setting.

关 键 词：流感病毒A型 H1N1亚型 病毒 环介导等温扩增 实时荧光RT—PCR 

分 类 号：R373.13[医药卫生—病原生物学]