猪传染性胃肠炎病毒半巢式RT-PCR检测方法的建立及应用

Establishment and application of semi-nest RT-PCR for detection of transmissible gastroenteritis virus

为建立一种高效、敏感、特异的用于检测猪传染性胃肠炎病毒(TGEV)的半巢式RT-PCR方法,本研究依据GenBank发表TGEV的N基因序列设计3条特异性引物,通过优化反应体系,建立了检测TGEV的半巢式RT-PCR方法,并对反应体系特异性和敏感性进行了检测。结果显示,该方法能够成功扩增483 bp和338 bp两条目的条带;对TGEV具有良好的特异性,与猪流行性腹泻病毒、猪轮状病毒、猪博卡病毒、猪德尔塔冠状病毒均无交叉反应;最低可检测到1.86×10^-1pg/μL的TGEV cDNA。用所建半巢式RT-PCR对临床50份猪腹泻样品进行检测,结果显示,阳性率为36%,高于普通RT-PCR阳性检出率,且阳性样品符合率为100%。本试验所建立的半巢式RT-PCR检测方法灵敏度高、特异性好,可准确地对TGEV感染做出诊断,为TGEV的临床检测、流行病学调查提供了有效手段。

In order to establish an efficient,sensitive and specific semi-nest RT-PCR method for the detection of Transmissible gastroenteritis virus(TGEV),three specific primers were designed according to the N gene published by Gen Bank,the reaction system was established and optimized,and specificity and sensitivity were detected.The results showed that the method could successfully amplify the bands of 483 bp and 338 bp,and had good specificity to TGEV,there is no cross reaction with PEDV,PRov,PBov and PDCov,and the lowest sensitivity was 1.86×10^-1 pg/μL.The semi-nest RT-PCR shown the positive rate was 36% in 50 samples of pig diarrhea,which was higher than that of common RT-PCR,and then the positive samples coincidence rate was 100%.This semi-nest RT-PCR method has high sensitivity and specificity,and can accurately diagnose TGEV infection,which provides an effective method for clinical detection and epidemiological investigation of TGEV.