摘要
为了建立一种方便快速、特异性强、灵敏度高的牛病毒性腹泻病毒(Bovine viral diarrhea virus, BVDV)的检测方法,试验以BVDV 5′UTR基因高度保守区为靶标,设计特异性引物,对反应时间、温度及引物浓度进行优化,建立了BVDV的逆转录重组酶聚合酶扩增(RT-RPA)检测方法,对该方法进行特异性、灵敏度及重复性检测,并采用该方法检测102份牛肛拭子样本的阳性率,分析与市售BVDV RT-PCR试剂盒的符合率。结果表明:该方法在37.5℃恒温反应15 min后通过琼脂糖凝胶电泳即可完成对病毒核酸的检测;与牛传染性鼻气管炎病毒(Infectious bovine rhinotracheitis virus, IBRV)、伪狂犬病病毒(Pseudorabies virus, PRV)、牛冠状病毒(Bovine corona virus, BCoV)、大肠杆菌、巴氏杆菌无交叉反应,特异性良好;最低检出限为3.0×10^(2) copies/μL;组间和组内重复性均良好;检测102份牛肛拭子样本的阳性率为10.8%,与市售BVDV RT-PCR试剂盒的符合率为99.0%。说明试验成功建立了特异性强、灵敏度高、重复性好的BVDV RT-RPA检测方法,该方法可用于临床样本的快速检测。
In order to establish a convenient,rapid,specific and sensitive method for the detection of Bovine viral diarrhea virus(BVDV),in the test,using the highly conserved region of BVDV 5′UTR gene as the target,designing specific primers,optimizing the reaction time,temperature and primer concentration,the study established RT-RPA method for the detection of BVDV.The specificity,sensitivity and reproducibility of the method were tested.The method was also used to detect the positivity rate of 102 bovine anal swab samples and analyze the compliance rate with the commercially available BVDV RT-PCR kit.The results showed that the detection of viral nucleic acids could be completed by agarose gel electrophoresis after 15 min of constant temperature reaction at 37.5℃.There was no cross-reactivity with Infectious bovine rhinotracheitis virus(IBRV),Pseudorabies virus(PRV),Bovine corona virus(BCoV),Escherichia coli,and Pasteurella sp;the specificity was good.The minimum detection limit was 3.0×10^(2) copies/μL,and the inter-and intra-group reproducibility was good.The positivity rate of 102 samples of bovine anal swabs was 10.8%,and the compliance rate with the commercially available BVDV RT-PCR kit was 90.9%.The results indicated that the test successfully established a BVDV RT-RPA assay with high specificity,high sensitivity and good reproducibility,which could be used for the rapid detection of clinical samples.
作者
刘艺
王慧荣
刘文俊
杨丽华
董春光
韩文儒
武守艳
LIU Yi;WANG Huirong;LIU Wenjun;YANG Lihua;DONG Chunguang;HAN Wenru;WU Shouyan(Shanxi Agricultural University,Taigu 030801,China)
出处
《黑龙江畜牧兽医》
北大核心
2025年第7期102-106,共5页
Heilongjiang Animal Science And veterinary Medicine
基金
山西省科技攻关项目(20150311017-3)
山西省重点研发计划项目(201903D211012)
山西省农业科学院农业科技创新研究课题(YCX2020104,YCX2020105)
山西省农业科学院农业科技创新工程项目(YGC2019TD01)。
关键词
牛病毒性腹泻病毒
牛病毒性腹泻
逆转录重组酶聚合酶扩增
检测方法
快速检测
Bovine viral diarrhea virus
bovine viral diarrhea disease
reverse transcription-recombinase polymerase amplification
detection method
rapid assay
