逆转录套式聚合酶链反应检测呼吸道合胞病毒

Detection of reverse transcriptase-polymerasc chain reaction and Nested polymerase chain reaction for respiratory syncytial virus

目的建立一种敏感、特异和快速诊断人呼吸道合胞病毒(RSV)感染的方法。方法选择RSV基因组中高度保守的 F 基因作为扩增靶序列,独立设计合成了两对引物,建立了一个完整的逆转录套式聚合酶链反应(PCR)检测 RSV RNA 的方法。结果对引物进行特异性实验,证明其具有高度特异性;对引物进行敏感度实验,测定其敏感度为10TCID_(50)/ml(TCID_(50)为半数组织培养感染量)。对101例急性下呼吸道感染患儿的鼻咽部分泌物标本进行检测,发现48例阳性,阳性率为47.5%;同时用碱性磷酸酶抗碱性磷酸酶桥联酶标法进行检测,发现40例阳性,阳性率为39.6%。两种方法对比,差异无显著性(P>0.05)。结论该方法具有快速、敏感、特异和观察结果客观等优点,为国内逆转录套式 PCR 用于临床快速诊断 RSV 感染、试剂盒的研制和开发奠定了基础。

Objective To establish methods for sensitive,specific and rapid diagnosis of respiratory syneytial virus.Methods Two pairs of primers which are highly sensitive and specific for the F gene (fusion)of respiratory syncytial virus have been designed in this study.A complete set of methodology for detection of RSV RNA by the reverse transeriptase-polymerase chain reaction(RT-PCR)and the Nested polymerase chain reaction(Nested PCR)has been established.Results The RT-PCR and the Nested PCR was tested on the 101 NPS samples collected from the children with acute lower respiratory infection from December 1994 to February 1995.Forty-eight were positive.Meanwhile,alkaline phos- phatase-anti-alkaline phosphatase(APAAP)staining method was tested on these samples,40 were po- sitive.No significant difference between the two methods could be found(P>0.05).Conclusions It has laid a foundation for clinical applications for the rapid detection for RSV and kit's exploition in Chi- na.