检测和鉴别犬瘟热病毒强、弱毒株的复合反转录-套式聚合酶链式反应(RT-nPCR)的建立及初步应用

A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

根据GenBank上登录的犬瘟热病毒(Canine distemper virus,CDV)基因组全序列,选择CDV强、弱毒株间有区别保守区设计了一对通用引物P1和P4,并在该对引物跨越区域的内部设计了CDV强毒株特异性引物P2及弱毒株特异性引物P3,用引物P1/P4进行RT-PCR,然后用引物P2/P3/P4进行复合套式PCR,建立了一种能区分CDV强、弱毒株的复合反转录-套式聚合酶链式反应(RT-nPCR)的鉴别诊断方法。应用该方法从CDV强、弱毒株的基因组中分别扩增出了大小为247bp和177bp的特异性片段,从两种病毒基因组混合物中扩增出了大小为247bp和177bp的两条特异性片段,与犬细小病毒、犬腺病毒、犬冠状病毒、狂犬病病毒、新城疫病毒的细胞培养物以及正常细胞对照组进行复合RT-nPCR扩增时均为阴性。对从黑龙江省和吉林省采集的20份疑似CDV病料进行的检测结果表明,有15份类似CDV强毒,5份类似CDV弱毒。本研究建立的复合RT-nPCR可以有效检测CDV感染,能够将强、弱毒株区分开,可用于临床快速检测、流行病学监测以及追踪疫苗免疫效果等。

A multiplex reverse transcription-nested polymerase chain reaction （RT-nPCR） was developed for detection and differentiation of wild-type and vaccine strains of canine distemper virus （CDV）. A pair of primers P1 and P4 corresponding to the highly conserved regions of CDV genome was used as common primers in the first round PCR. Primers P2 or P3 specific for CDV wild-type strain or vaccine strain respectively were used as the forward primer together with the common reverse primer P4 in the second round PCR. A PCR fragment of 177 bp from vaccine strain genome and a fragment of 247 bp from wild-type strain genome were amplified by the RT-nPCR. Two fragments of 247 bp and 177 bp were amplified from mixed genomic samples of vaccine strain and wild-type strain. No PCR products were detected for uninfected cells, or cells infected with newcastle disease virus （NDV）, canine parvovirus （CPV）, canine coronavirus （CCV）, rabies virus （RV）, canine adenovirus （CAV）. The RT-nPCR method was successfully used to detect for suspected CDV infection in 20 clinical samples from Heilongjiang and Jilin Provinces, of which 15 samples were found infected by wild type CDV, and 5 by vaccine strains. This method was effective for detecting and differentiating wild-type CDV and vaccine strains in dogs, thus could be useful for the clinical detection and the epidemiological studies. It also can be used for monitoring the vaccinal immunity.