兔出血症病毒和巴氏杆菌双重PCR检测方法的建立及应用

参照GenBank公布的兔出血症病毒(RHDV)VP60、巴氏杆菌kmt基因序列,设计两对引物,分别用于扩增RHDV的VP60和巴氏杆菌kmt基因的目的片段。通过正交试验,对反应各组分浓度与组合、反应退火温度及反应参数进行优化,最后建立了RHDV、巴氏杆菌双重PCR检测方法并进行临床应用。结果显示:本试验建立的双重PCR检测方法能够特异性地检测RHDV及巴氏杆菌,最低核酸检出限分别达到70 pg和62pg。检测兔源大肠杆菌、葡萄球菌和链球菌,结果均为阴性。用本方法对临床送检的104份病料进行检测,结果检出RHDV与巴氏杆菌混合感染1份,巴氏杆菌单独感染10份,双重PCR检测结果与临床病原分离结果完全一致。表明本试验建立的兔出血症病毒和巴氏杆菌双重PCR检测方法具有良好的临床应用价值。

Establishment and application of a dual PCR assay for simultaneous detection of rabbit hemorrhagic disease virus and Pasteurella multocida

Based on the sequences of rabbit hemorrhagic disease virus(RHDV) VP60 and kmt gene of Pasteurella multocida(Pm) that have been published in GenBank,two pairs of primers were designed and used to amplify VP60 and kmt gene fragments,respectively.Orthogonal test was then designed to optimize the reaction parameters of dual PCR assay for simultaneous detection of RHDV and Pm.The results indicated that the dual PCR assay could be used in specific detection for RHDV and Pm,and the lowest detection limits of nucleic acid quantitation were 70 pg and 62 g,respectively.The detection results of E.coli,Staphylococcus and Streptococcus isolates from rabbits were negative.The detection results of 104 clinical samples showed one case of mixed infection of RHDV and Pm and ten cases of single infections of Pm,which were consistent with those obtained from clinical isolation.It suggests that the dual PCR assay for simultaneous detection of RHDV and Pm infections is of high clinical value.