兔轮状病毒、兔肠冠状病毒双重RT-PCR检测方法的建立及初步应用

Establishment and preliminary application of duplex RT-PCR for detection of lapine rotavirus and rabbit enteric coronavirus

兔轮状病毒(lapine rotavirus,LaRV)和兔肠冠状病毒(rabbit enteric coronavirus,RECV)是引起兔腹泻的重要病原,2种病毒引起的仔兔腹泻症状极为相似。为建立能同时鉴别LaRV、RECV的方法,根据LaRV和RECV基因组的保守区段设计2对特异性引物,预期特异性扩增LaRV和RECV的片段大小分别为291 bp和163 bp,经优化反应条件,成功建立了LaRV和RECV的双重RT-PCR检测方法。特异性检测结果显示,能特异性的扩增出LaRV和RECV的目的条带,与兔瘟、大肠杆菌、魏氏梭菌、沙门菌、泰泽菌、巴氏杆菌、金黄色葡萄球菌、绿脓杆菌、链球菌、支气管败血波氏杆菌均无交叉反应;LaRV和RECV的最低检出限分别为2.73×10^(5),1.22×10^(4)拷贝/μL,在相同条件下重复可获得一致的结果。对采自山东部分地区的107份兔腹泻样品的检测结果显示,LaRV和RECV的阳性率分别为10.28%和7.48%,两者与单一RT-PCR检测结果相一致。结果表明,所建立的双重RT-PCR方法能够快速、准确的对LaRV和RECV单一或混合感染的样品进行检测,为LaRV和RECV的鉴别诊断和流行病学调查提供新的技术手段。

Lapine rotavirus(LaRV)and rabbit coronavirus(RECV)are important pathogens causing diarrhea in rabbits.The symptoms of diarrhea caused by LaRV and RECV are similar.In order to establish a method for simultaneous detection of LaRV and RECV,two pairs of specific primers were designed according to the conservative sections of LaRV and RECV genomes.The specific amplification fragments of LaRV and RECV were expected to be 291 bp and 163 bp,respectively.After optimizing reaction conditions,a duplex RT-PCR method for detection of LaRV and RECV was successfully established.The specific detection results showed that the target bands of LaRV and RECV could be specifically amplified,and there was no cross reaction with rabbit hemorrhagic disease virus,Escherichia coli,Clostridium weissl,Salmonella,Tezechia coli,Pasteurella,Staphylococcus aureus,Pseudomonas aeruginosa,Streptococcus and Bordetella bronchiseptica.The minimum detection limits of LaRV and RECV were 2.73×10^(5) and 1.22×10^(4) copies/μL,respectively,consistent results could be obtained under the same conditions.The positive rates of LaRV and RECV were 10.28%and 7.48%respectively,which were consistent with the single RT-PCR test results.The results show that the established duplex RT-PCR method can rapidly and accurately detect the single or mixed infection of LaRV and RECV,providing a new technical means for the differential diagnosis and epidemiological investigation of LaRV and RECV.