鸡细小病毒与禽呼肠病毒二重PCR检测方法的建立

建立可同时检测鸡细小病毒(Chicken parvovirus,ChPV)与禽呼肠病毒(Avian reovirus,ARV)的二重PCR方法,为防控ChPV与ARV提供技术支撑。根据鸡细小病毒NS1基因和禽呼肠病毒σC基因的保守序列,设计合成两对引物用于检测ChPV和ARV,通过优化二重PCR的反应体系,特异性、敏感性试验评价建立的ChPV与ARV二重PCR。优化后的二重PCR反应体系为:2×PCR Mix 12.5μL,其中ChPV与ARV的上、下游引物各1.0μL,混合模板2.0μL,ddH2O补足25μL;最佳的反应程序为:95℃5min;95℃1min,56.1℃1min,72℃1min,35个循环;最后72℃延伸10min。结果显示,建立的二重PCR能够同时扩增出204bp ChPV和405bp ARV片段;该方法对ChPV与ARV的检测敏感性分别达到58fg和53fg,但对鸡新城疫病毒、H9亚型禽流感病毒、马立克病病毒、鸡传染性喉气管炎病毒、鸡传染性支气管炎病毒等病原体均无特异性扩增,对ChPV与ARV混合感染的临床阳性病料的检测结果与各病毒单项PCR检测结果符合率为94%以上。建立的二重PCR可用于ChPV与ARV感染的快速鉴别诊断。

Development of a Duplex PCR Assay for Detection of Chicken Parvovirus and Avian Reovirus

A duplex PCR assay for detecting both chicken parvovirus (ChPV)and avian reoviruses (ARV) was developed to provide technical support for effective control of ChPV and ARV.According to the con-served gene sequences of ChPV NS1 and ARVσC in GenBank,two sets of specific primers were designed. The duplex PCR for simutaneous detection of ChPV and ARV was established by optimizing the reaction conditions.The optimized PCR reaction system was 2×PCR Mix 12.5 μL,of which sense primer and anti-sense primer of ChPV and ARV was 1.0 μL each.The mixed template was 2.0 μL and added to 25.0 μL with ddH2 O.The optimal reaction procedure was 95 ℃ initial denaturation for 5 min;95 ℃ degeneration for 1 min,56.1 ℃ annealing for 1 min,72 ℃ for 1 min in 35 cycles;72 ℃ extension for 10 min.The result indicated that the duplex PCR assay successfully amplified 204 bp for ChPV,405 bp for ARV.The sensitiv-ity of the assay was 58 fg for ChPV and 53 fg for ARV,but it was not sensitive to other chicken pathogens, such as Newcastle disease virus,H9 subtype avian influenza virus,Marek′s disease virus,infectious bron-chitis virus,infectious laryngotracheitis virus.The results detected by the duplex PCR had a 94% coinci-dence rate with the results detected by single PCR for the ChPV and ARV respectively.The duplex PCR assay is a quick,sensitive and specific test for detection of ChPV and ARV.