牛病毒性腹泻病毒分型一步法RT-PCR检测方法的建立及应用  

Development and Application of One-step RT-PCR Detection Method for Typing Bovine Viral Diarrhea Virus

作　　者：金艳 JIN Yan(Changchun Vocational and Technical College,Changchun 130000,China)

机构地区：[1]长春职业技术学院,长春130000

出　　处：《中国动物传染病学报》2023年第4期67-73,共7页Chinese Journal of Animal Infectious Diseases

摘　　要：为了建立牛病毒性腹泻病毒(BVDV)的分型检测方法,并掌握长春地区BVDV的流行规律。本研究根据BVDV-1型和BVDV-2型的5’-UTR基因序列差异设计鉴别检测引物,经一步法RT-PCR反应条件的优化,建立了BVDV分型一步法RT-PCR检测方法,并应用该方法对长春地区采集的380份样品进行检测。结果表明:该方法最佳退火温度为51℃,最佳模板含量为5.7μg,检测牛肠道病毒(BEV)、牛传染性鼻气管炎病毒(IBRV)、牛轮状病毒(BRV)、牛呼吸道合胞体病毒(BRSV)均为阴性;对BVDV-1型和BVDV-2型的检测灵敏度可以分别达到0.58 ng RNA和0.51 ng RNA,相对于病毒分离方法的符合率为92.31%,长春地区BVDV阳性率为24.21%,其中BVDV-1和BVDV-2阳性率分别为15.53%和7.37%。以上结果表明,本研究建立的BVDV分型一步法RT-PCR检测方法具有良好的特异性、敏感性和准确性,长春地区BVDV的流行优势基因型为BVDV-1型。In order to develop a typing detection method of bovine viral diarrhea virus(BVDV) and investigate its epidemiology in Changchun area,the primers were designed according to the difference of 5'-UTR gene sequence between BVDV-1 and BVDV-2.The one-step RT-PCR detection method was developed for BVDV typing after the optimization of reaction conditions.The optical reaction conditions were determined to be 51℃for annealing temperature and 5.7 μg for template.This one-step RT-PCR detection method had no reaction to bovine enterovirus,bovine infectious rhinotracheitis virus,bovine rotavirus and bovine respiratory syncytial virus.The sensitivity was 0.58 ng RNA for BVDV-1 and 0.51 ng RNA for BVDV-2.The coincidence rate with the virus isolation method was 92.31%.Total 380 samples were collected from Changchun area and tested for BVDV with this method.The general positive rate was 24.21%,of which the positive rates were 15.53% for BVDV-1 and 7.37% for BVDV-2.These results indicated that the one-step RTPCR method developed here had good specificity,sensitivity and accuracy for BVDV typing and BVDV-1 was the dominant epidemic genotype in Changchun area.

关 键 词：牛病毒性腹泻病毒 分型 一步法RT-PCR检测方法 流行优势基因型 

分 类 号：S855.3[农业科学—临床兽医学]