鸡细小病毒与H9亚型禽流感病毒二重PCR检测方法的建立

Development of a Duplex RT-PCR Assay for Detection of ChPV and AIV H9 Subtype

[目的]建立一种能够同时诊断鸡细小病毒(chincken parvovirus,ChPV)和H9亚型禽流感病毒(avian influenza virus,AIV)的检测方法。[方法]根据GenBank中已发表的ChPV NS基因、H9亚型AIV HA基因的保守序列,分别设计多对引物,并通过一系列不同组合对温度和反应体系的调整试验,最终筛选出2对最佳特异性引物组合,建立了ChPV和AIV二重PCR检测方法用于ChPV和H9亚型AIV的检测。[结果]特异性和敏感性的试验结果表明,建立的二重PCR方法特异性良好,可以在一管PCR反应同时检测ChPV和H9亚型AIV。针对ChPV NS基因和H9亚型AIV HA基因的扩增片段大小分别为558和367 bp,其他常见的禽病病原体均未见阳性反应;该法对ChPV和H9亚型AIV的检测下限均为5×10^4拷贝/μL质粒;临床样品检测结果表明,对140份临床样品检测结果与AIV分离测序及ChPV PCR阳性产物测序结果完全一致。[结论]建立的ChPV和H9亚型AIV二重PCR检测方法不仅特异性良好,而且检测的灵敏度高,对ChPV和H9亚型AIV的监测及有效防控具有重要意义。

[Objective]In order to establish a method to simultaneously detect chicken parvovirus(ChPV)and avian influenza virus(AIV).[Method]Two pairs of specific primers were designed according to the sequences of ChPV NS genes,H9 subtype AIV HA genes which has been published in GenBank.Multiple pairs of primers were designed respectively,and through a series of experiments to adjust the temperature and reaction system of different combinations,the optimal combination of two pairs of specific primers was finally selected,and the duplex PCR detection method of AIV and ChPV was established for the detection of ChPV and H9 subtype AIV.[Result]The results of specificity and sensitivity test showed that the established triple PCR method was specific and good,and could simultaneously detect ChPV and H9 subtype AIV in one tube of PCR reaction.The amplified fragment size of the NS genes sequences of ChPV and HA genes sequences of H9 were 558 and 367 bp.The lower limit of detection for ChPV and H9 subtype AIV was 5×10^4 copies/μL plasmid.The results of clinical samples test showed that 140 clinical samples were completely consistent with the results of AIV isolation sequencing and ChPV PCR positive products sequencing.[Conclusion]The duplex-PCR detection method for ChPV and H9 subtype AIV established in this study has not only good specificity,but also high detection sensitivity,which is of great significance for the monitoring and effective prevention and control of ChPV and H9 subtype AIV.