逆转录-聚合酶链反应-酶联夹心杂交法检测呼吸道合胞病毒及其亚型感染

The study of identification of RSV subgroups A and B by reverse transcription polymerase chain reaction enzyme linked sandwich hybridization assay

目的:建立一种呼吸道合胞病毒(RSV)及其亚型核酸检测方法并初步应用于临床,检测浙南地区RSV及其亚型的流行情况。方法:根据RSVG蛋白基因核苷酸序列设计引物和探针,通过逆转录-聚合酶链反应-酶联夹心杂交法(RT-PCR-ELSH)随机检测了浙南地区连续两年冬春季期间202例急性下呼吸道感染的婴幼儿鼻咽部分泌物中RSV及其亚型的病毒核酸;与病毒分离培养、直接免疫荧光法(DFA)、OligoDetect试剂盒比较探讨其临床实用性。结果:202例标本中RSV阳性123例,阳性检出率为60.9%,2002～2003年和2003～2004年冬春季分别为68.2%和52.6%;两个冬春季都以RSVA亚型为主,占78.9%,2002～2003年冬春季A亚型占RSV阳性标本中的76.7%,2003～2004年A亚型占82.0%;RT-PCR-ELSH阳性检出率与其他三种方法的阳性检出率差异无显著性(P>0.05)。结论:RT-PCR-ELSH法用于检测RSV及其亚型,是一种灵敏度高、特异性强,方便、快速的方法,非常适用于临床标本的大批量检测和流行病学调查;RSV是引起浙南地区冬春季婴幼儿急性下呼吸道感染的最主要的病原体,并以A亚型为主。

Objective: To establish a RT-PCR-enzyme-linked sandwich hybridization(RT- PCR-ELSH)method for the identification of RSV subgroups A and B and to analyze the epidemics and distributive situations of RSV subgroups A and B in infants with acute lower respiratory tract infections in the southern area of Zhejiang province. Methods:Primers and probes to RSV G protein genes were designed based on published sequence database from NCBI GeneBank. The nucleic acid(RNA)of RSV in the nasopharyngeal aspirates(NPAs)obtained from 202 infants with acute lower respiratory tract infection at Children’s Hospital of Wenzhou Medical College over two consecutive winters and springs from 2002～2003 and 2003～ 2004 were randomly detected with RT-PCR-ELSH,to analyze the diseased situation of RSV and its subgroups in the southern area of Zhejiang province. The value and feasibility of RT- PCR-ELSH were assessed by comparing with virus isolation in cell culture, DFA and OligoDetect Kit obtained from Chemicon International. Results:Of the 202 samples,123(60.9%)were positive for RSV with RT-PCR-ELSH. In winters and springs of 2002～2003 and 2003～2004 RSV accounted for 68.2% and 52.6%, respectively. RSV subgroup A(78.9%)predominated in this two consecutive winters and springs of 2002～2003 and 2003～2004 (76.7%,82.0% of RSV positive samples,respectively). The sensitivity of RT-PCR-ELSH was the same as the other three measurement ways. Conclusions:RT-PCR-ELSH,with high sensitivity,specificity,convenience and rapidity, was an alternative for detection of RSV nucleic acid, especially used in the epidemiological survey and routine diagnosis of samples in batch. RSV is the major pathogen of acute lower respiratory tract infections among infants in winter and spring in the southern area of Zhejiang province. And RSV subgroup A predominated during the two consecutive winters and springs of 2002～2003 and 2003～2004.