呼吸道合胞病毒感染滴度测定方法的建立与比较

摘要

Objective Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection in infants.It is very important to quantitative assay of RSV titer in the study on RSV pathogenesis,candidate vaccine and antiviral treatment.Therefore,we develop Real-time Quantitative PCR (Q-PCR) assay and enzyme immunospots (EIS) for titrating RSV and compare them with traditional 50% tissue culture infectious doses (TCID_(50)).Methods Q-PCR,based upon the RSV-L genes,and EIS were utilized to titrate samples from RSV culture supernatants and RSV infected mouse lungs.Then,the results were compared with TCID_(50).Results For the samples from RSV culture supernatants,the ratio of Q-PCR and EIS (plaque forming unit,pfu) was 10:1 and the ratio of EIS and TCID_(50) was 10:1 when TCID_(50) was converted as pfu.For the samples from RSV infected mouse lungs,the ratio of Q-PCR and EIS was 1000:1 and the ratio of EIS and TCID_(50) was 5:1.Conclusion We have successfully established Q-PCR and EIS to titrate RSV and compared them with TCID_(50).We concluded EIS is a cost-effective method to titrate RSV.%目的 建立稳定的呼吸道合胞病毒(RSV)感染滴度测定的实时荧光定量PCR(Realtime Quantitative PCR,Q-PCR)检测方法和酶免疫斑点法(Enzyme Immunospots,EIS),并与半数定量方法(50% tissue culture infectious doses,TCID_(50))进行比较.方法 分别采用Q-PCR、EIS和TCID_(50)分析RSV感染的细胞和RSV攻毒后小鼠肺脏标本中的病毒滴度.结果 RSV感染细胞的上清中,三种分析方法检测的病毒滴度值之比为:Q-PCR和EIS(pfu)的比约为10:1,EIS(pfu)和TCID_(50)(TCID_(50) 换算成pfu)比约为10:1;RSV攻毒后小鼠肺脏标本中,三种分析方法检测的病毒滴度值之比为,QPCR和EIS(pfu)比约为1000:1,EIS(pfu)和TCID50(TCID50换算成pfu)比约为5:1.结论 成功建立了RSV感染滴度测定的Q-PCR和EIS分析方法,通过与TCID_(50)方法的比较分析,我们认为EIS法分析RSV滴度具有成本低和较高的灵敏度,有较好的应用前景.