摘要
为了进行猪伪狂犬病病毒的早期诊断,根据gE基因的保守序列设计了1对引物,构建阳性重组质粒,建立TaqMan荧光定量PCR检测方法。结果显示,该方法特异性强,对Bartha-K61弱毒疫苗株(gE-株)及其他病毒不发生交叉反应;敏感性高,最低检测下限为10copies/μL,比常规PCR高100倍;重复性好,批内、批间重复试验的变异系数均小于2%。应用建立的方法与常规PCR分别对153份临床样品进行检测,检出率分别为67%、60%,二者的符合率是93.7%。结果表明,该检测方法可为伪狂犬病病毒的早期诊断提供技术手段。
A pair of primers and TaqMan probes were designed based on the pseudorabies virus gE sequence,and then the real-time PCR assay was established for the detection of pseudorabies virus. The resuits indicated that the real-time PCR was highly specific,and there were no cross-reaction with the Bartha- K61（gE attenuated strain） and the related pathogens for the other swine diseases. The detection limit of the assay was 1 × 10^1copies/μL,which was 100 fold higher than that of the routine PCR. The coefficient of variation was less than 2 percent for both interand intra-assay. When this test and a conventional PCR were used to detect 153 clinical samples. They gave 67 % and 60 % positivity respectively, in which 93.7 positive samples were tested by either methods. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of pseudorabies virus.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第4期396-400,共5页
Chinese Veterinary Science
基金
中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室基本业务费(SKLVBP201201)
