五重荧光定量RT-PCR法检测呼吸道病毒  

Detection of respiratory virus by multiplex real-time RT-PCR assay

作　　者：崔大伟[1] 郑书发[1] 范剑[1] 楼滨[1] 余斐[1] 秦志梅[1] 邹伟华[2] 吴英萍[1] 陈凌晓[1] 陈瑜[1] 

机构地区：[1]浙江大学医学院附属第一医院检验科,杭州310003 [2]湖州市中心医院检验科,浙江湖州313000

出　　处：《临床检验杂志》2011年第5期333-335,共3页Chinese Journal of Clinical Laboratory Science

基　　金："十一五"国家科技重大专项(2009ZX10004-210)

摘　　要：目的建立一种五重荧光定量RT-PCR检测并鉴别流感病毒A(Flu A)、流感病毒B(Flu B)、呼吸道合胞病毒(RSV)以及新甲型H1N1流感病毒(nH1N1)的方法。方法以人细胞RNA酶P基因为内参,用Primer Express 3.0设计PCR引物和探针。用一系列不同滴度的病毒培养物和不同来源的呼吸道病毒分别进行灵敏度和特异性分析。结果该法灵敏度较好,可测半数组织培养感染剂量(TCID50/mL)为0.02～0.2的病毒量,特异性达100%,每对引物和探针只检测出相应的病毒,无交叉反应。已确诊的nH1N1、Flu B、RSV感染患者的样本用该法均成功地测出。结论建立的五重荧光定量RT-PCR法灵敏度和特异性都较高,可检测出多种呼吸道病毒,对临床呼吸道病毒感染的诊断有一定的价值。Objective To establish a multiplex real-time RT-PCR assay to detect and discriminate influenza A virus （FluA） , influen- za B virus （FluB）, respiratory syncytial virus （RSV） and novel H1N1 （nH1N1） virus in a single test tube. Methods Using the Primer Express 3.0 software, the primers and probes of PCR for the above-mentioned viruses were designed, and the primer and probe set specifically targeted to the human nucleic acid RNase P（RP） , which was used as the internal control, were designed. The analyti- cal sensitivity was determined by serial dilutions of each virus, and the specificity of multiplex assay was determined by testing different virus. Results The analytical sensitivities of the multiplex real-time RT-PCR assay for Flu A, Flu B, RSV and nH1N1 virus were ranged from 0.02 to 0.2 TCID50/mL, and the specificity was 100%. No cross-reaction was observed for the mixed viruses and common respiratory pathogens and commensal organisms. All of the specific target DNA showed strong positive signals. Flu B, RSV and nil1 N1, which were certified by other methods, were successfully detectable by the established multiplex assay. Conclusion A sensitive and specific multiplex real-time RT-PCR assay was developed to detect the most common pathogens causing respiratory tract infection, e. g. , Flu A, Flu B, RSV and nH1N1 virus. The multiplex RT-PCR should be valuable for clinical diagnosis of respiratory virus infection.

关 键 词：多重RT—PCR 新甲型Hl N1流感病毒 呼吸道合胞病毒 流感病毒 

分 类 号：R446.5[医药卫生—诊断学]