人偏肺病毒基因型双重逆转录PCR检测方法的建立  

A duplex RT-PCR for identifying genotypes of human metapneumovirus from clinical samples

作　　者：王芳[1] 朱汝南[1] 钱渊[1] 邓洁[1] 孙宇[1] 赵林清[1] 廖斌[2] 黄荣研[2] 

机构地区：[1]首都儿科研究所病毒研究室北京市感染与免疫中心实验室,100020 [2]首都儿科研究所附属儿童医院呼吸内科

出　　处：《中华检验医学杂志》2010年第1期33-36,共4页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目（30570080,30872153）;北京市自然科学基金资助项目（7052020）;北京市科委新星计划资助项目（2004834）

摘　　要：目的建立一种鉴别不同基因型hMPV的RT—PCR方法。方法根据不同基因型的hMPVG蛋白基因序列设计合成A、B基因型的特异性引物，在一次双重PCR反应中根据扩增产物大小鉴别不同基因型。用该方法鉴别37份hMPV阳性的临床标本的基因型。结果用hMPVG蛋白编码基因的分型引物进行PCR反应，对已知的hMPV阳性标本直接进行分型得到的扩增产物大小易于区分；对常见的呼吸道病毒无非特异扩增，显示引物特异性良好。对37份临床标本进行基因分型结果显示，20份A基因型分型结果与M基因测序分型结果一致，17份经M基因测序分型为B基因型的阳性标本中有14份与M基因测序分型结果一致，3份未得到扩增产物从而不能分型，总符合率为91．9％[（20＋14）／37]。结论成功建立了一种可以鉴别不同基因型的hMPV的RT—PCR方法。Objective To develop a convenient reverse transcription PCR （RT-PCR） method for identifying genotypes of human metapneumovirus （ hMPV ） from clinical samples. Methods According to the gene sequences of hMPV G with different genotypes, the A and B genotype specific primers were designed. A diplex RT-PCR was applied to identify different genotypes according to the molecular weight of PCR products in agarose gel. 37 clinical samples were detected through this method. Results It was convenient to distinguish different genotypes of hMPV （383 bp for A and 284 bp for B） by the diplex RT- PCR, and there was no non-specific amplification for common respiratory viruses, so it meant that the specificity of primers was good. The results of genotyping 37 clinical samples showed that 20 samples were identified as genotype A by both sequence analysis of M gene and diplex RT-PCR, whereas 17 samples were identified as genotype B by sequence analysis of M gene, but in these 17samples 14 samples were identified as genotype B by the diplex RT-PCR and remaining 3 samples could not be genotyped because there was no PCR product after amplification. The consistency rate for these two methods was 91.9% [ （20 ＋ 14）/37]. Conclusion The method of diplex RT-PCR was developed successfully and can be used for identify genotypes of hMPV.

关 键 词：变性肺病毒 基因型 逆转录聚合酶链反应 

分 类 号：R4[医药卫生—临床医学]