摘要
为对基因Ⅰ型和Ⅱ型牛病毒性腹泻病毒(BVDV)进行鉴别检测,本研究根据GenBank中已登录的基因Ⅰ型和Ⅱ型BVDV 5'非编码区(5'-UTR)的参考序列,设计了一对BVDV Ⅰ型和Ⅱ型通用引物和2条鉴别探针,建立了Ⅰ型和Ⅱ型BVDV双重TaqMan荧光定量PCR检测方法,并利用该检测方法进行临床样品检测。特异性试验结果显示,除Ⅰ型和Ⅱ型BVDV外,该方法对猪瘟、牛传染性鼻气管炎、口蹄疫病毒等均无扩增曲线。敏感性试验结果显示,该方法对Ⅰ型BVDV检测下限为10拷贝/μL,Ⅱ型BVDV检测下限为100拷贝/μL,敏感性高。重复性试验结果显示,该方法组内和组间变异系数均小于1%。临床样品检测结果显示,Ⅰ型和Ⅱ型BVDV双重TaqMan荧光定量PCR检测方法可鉴别Ⅰ型和Ⅱ型BVDV,检测结果与OIE认可的BVDV普通PCR扩增产物测序结果一致。本研究所建立的Ⅰ型和Ⅱ型BVDV双重TaqMan荧光定量PCR具有快速、准确、可鉴别等优点,可用于Ⅰ型和Ⅱ型BVDV的快速鉴别检测。
To develop an assay for simultaneously detecting bovine viral diarrhea virus genotype Ⅰ（BVDV-Ⅰ） and BVDV-Ⅱ,one pair of universal primers and two specific probes of BVDV-Ⅰ and BVDV-Ⅱ were designed according to the 5＇ noncoding region（5＇ UTR） of BVDV-Ⅰ and BVDV-Ⅱ to establish the duplex Taq Man real-time PCR for BVDV-Ⅰ and BVDV-Ⅱ detections.The results indicated that the assay was specific for BVDV-Ⅰ and BVDV-Ⅱ detection, which had no any amplification for other virus such as classic swine fever virus, infectious bovine rhinotracheitis virus, foot and mouth disease virus etc. The sensitivity of this assay was up to 10 copies/μL for BVDV-Ⅰ and 100 copies/μL for BVDV-Ⅱ, respectively. The coefficient of variation of this assay was less than 1% in both intro-and intra-groups. Additionally, the results of clinical sample detections using this method were identical to the conventional PCR detection results that accepted by OIE, which demonstrated that the established duplex Taq Man real-time PCR could be used to identify BVDV-Ⅰ and BVDV-Ⅱ. In conclusion, this assay was convenient, strong-specific and high-sensitive, which could be applied to a rapid detection and identification of BVDV-Ⅰ and BVDV-Ⅱ.
作者
王艳杰
王秀明
张宸
王云凌
陈君彦
张贵刚
范秀丽
张妍
魏学峰
刘国英
WANG Yan-jie;WANG Xiu-ming;ZHANG Chen;WANG Yun-ling;CHEN Jun-yan;ZHANG Gui-gang;FAN Xiu-li;ZHANG Yan;WEI Xue-feng;LIU Guo-ying(The Spirit JinYu Biological Pharmaceutical Co.Ltd,Hohhot 010030,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2018年第8期692-696,共5页
Chinese Journal of Preventive Veterinary Medicine
