牛病毒性腹泻病毒反转录PCR快速检测方法的建立与初步应用

Establishment and Initial Application of RT-PCR for Quick Detectionof Bovine Viral Diarrhea Virus

根据GenBank中登录的牛病毒性腹泻病毒（BVDV）5＇非编码区基因序列,设计合成了1对特异性引物,建立了检测BVDV的反转录PCR快速检测方法.通过对该方法的特异性、敏感性和重复性进行试验,结果显示,该方法从BVDV标准毒株Oregon C24V中扩增出267 bp的特异性片段,该方法重复性好,反应批内检测结果相同.与牛轮状病毒、牛冠状病毒、猪瘟病毒和F4新生牛肾传代正常细胞无交叉反应,具有高度的特异性,而且敏感性高,最低检出限为10~1.84 TCID50/mL.利用该方法对42份临床腹泻病牛疑似粪便样品进行了检测,结果检出7份阳性,而同时利用IDEXX公司抗原检测试剂盒检出阳性只有6份.表明,建立的该方法具有快速、敏感、特异等优点,是牛病毒性腹泻病毒病的临床诊断和流行病学调查的有力工具.

A reverse transcription- coupled PCR（RT- PCR） assay for quick detection of bovine viral diarrhea virus（BVDV） was established using a pair of specific primers based on the 5＂UTR gene of BVDV published in GenBank. The results showed that this method could specifically amplify a 267 bp fragment from BVDV Oregon C24 V strain, reproducibility of this assay were reliable and the results were fully consistent. The specificity test proved that this assay had a high specificity which had no cross- reaction with bovine rotavirus, bovine coronavirus, classical swine fever virus and normal F4 NBPC cells. The assay also had good sensitivity, and the detection limit was up to 10-- 1.84 TCID50/mL. 7 positive of 42 samples from clinical diarrhea bovine were detected by RT- PCR and only 6 positive were detected by IDEXX diagnostic kit test at the same time. The results revealed that established RT - PCR assay possessed some advantages such as fast, sensitive and specific. It may be used for clinical diagnosis and the epidemiologic survey of bovine viral diarrhea/mucosal disease as a powerful tool.