应用逆转录聚合酶链反应鉴别呼吸道合胞病毒亚型

Identification of subgroups of respiratory syncytial virus by reverse transcriptionpolymerase chain reaction

目的为了使鉴别呼吸道合胞病毒（ｒｅｓｐｉｒａｔｏｒｙｓｙｎｃｙｔｉａｌｖｉｒｕｓ，ＲＳＶ）Ａ、Ｂ亚型的方法更为简单、特异、有效。方法根据ＲＳＶＧ蛋白编码基因的核苷酸序列设计一套引物，其中Ｐ１为亚型间通用的引物，Ｐ２和Ｐ３分别为Ａ、Ｂ亚型特异性引物。将这些引物用于同一逆转录聚合酶链（ＲＴＰＣＲ）反应，Ａ、Ｂ亚型株的扩增产物分别为２７７ｂｐ和８６３ｂｐ，根据ＰＣＲ产物的大小即可鉴别所测毒株的亚型。用这一方法对ＲＳＶ原型株和９株我国分离株进行亚型鉴定。结果分离株８株为Ａ亚型，１株为Ｂ亚型，分型结果与单克隆抗体检测和基因序列分析完全相符。结论ＲＴＰＣＲ方法鉴别呼吸道合胞病毒亚型具有快速、简便、敏感、特异等特点。

Objective To identify subgroups of respiratory syncytial virus (RSV) isolates.Methods A reverse transcriptionpolymerase chain reaction ( RTPCR) was developed by using primer pool in PCR reaction. Three primers were designed based on nucleotide sequence of G protein genes of RSV prototype strains of subgroups A and B (Long and CH18537), which contained one primer for both subtypes and two for either subgroup A or B. Using these three primers in the same RTPCR mixture, the strains tested could be easily differentiated as either subgroup A or B in agarose gel by the size of the PCR products ( 277 bp for subgroup A and 863 bp for subgroup B). The technique was used to determine the subgroups of 9 isolates of field strains of RSV. Results Eight of the 9 strains belonged to subgroup A and 1 was subgroup B, which was consistent with that obtained with the monoclonal antibody and gene sequencing of these strains. Conclusion The RTPCR technique described here is of high specificity and sensitivity and can be used for identification of RSV field strains isolated from clinical specimens.