猪圆环病毒Ⅱ型TaqMan荧光定量PCR检测方法的建立及应用  

Establishment and application of TaqMan fluorescent probe- based quantitative PCR assay for detection of Porcine circovirus type 2

作　　者：郝立沙 张璐[1] 佟杰[1] 于颖[1] 杨莹莹[1] 张武超[1] 涂亚斌[1] 蔡雪辉[1] 

机构地区：[1]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/动物疫病诊断中心,哈尔滨150001

出　　处：《黑龙江畜牧兽医》2015年第6期168-171,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国际科技合作与交流专项(2011DFA32210);公益性行业(农业)科研专项(201203056)

摘　　要：为了快速准确检测猪圆环病毒Ⅱ型（PCV-2）并对病毒拷贝数进行定量,试验根据Gen Bank中PCV-2保守序列（登录号为FJ644559.1）设计1对特异性引物和Taq Man探针,制备标准品,建立PCV-2的荧光定量检测方法,并对临床样品进行检测。结果表明：该方法在1×10^1~1×10^8拷贝/μL的模板范围内具有良好的线性关系,相关系数可达0.999;敏感性是常规PCR方法的100倍;对猪细小病毒（PPV）、猪伪狂犬病病毒（PRV）、猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）均为阴性,没有交叉反应;批内、批间重复试验变异系数均小于2.50%;在临床检测中,比常规PCR方法检测PCV-2的阳性检出率高出38%。说明试验成功建立了PCV-2 Taq Man荧光定量PCR检测方法,可用于临床检测PCV-2感染及对其拷贝数进行定量。To detect quickly and accurately detect Porcine circovirus type 2 （ PCV - 2 ） and quantify the number of viral copies, a pair of specific primers and TaqMan probes were designed according to the conservative sequence of PCV -2 in Genbank （accession number： FJ644559. 1 ）. The standards were prepared, and then the fluorescent quantitative PCR assay for detection of PCV - 2 was established and used to detect the clinical samples. The results showed that the assay had good linearity within the template ranges from 1×10^1copties/μL 1×10^8 copies/μL, the correlation coefficient was up to 0. 999, and its sensitivity was 100 times that of routine PCR assay; the assay was negative with Porcine parvovirus （PPV） , Porcine pseudorabies virus （PRV）, Calssical swine fever virus （CSFV） and Porcine reproductive respiratory syndrome virus （ PRRSV）, there was no cross - reaction. The intra and inter - assay coefficients of variation were less than 2.50%. The posi- tive detection rate of PCV - 2 using the fluorescent quantitative PCR assay was 38% higher than that using conventional PCR method in the clin- ical detection. The result indicates that a Taqman fluorescent probe - based quantitative PCR assay is successfully established which can be used for the clinical detection of PCV - 2 infection and quantification of the number of PCV - 2 copies.

关 键 词：猪圆环病毒Ⅱ型(PCV-2) TaqMan荧光定量PCR 应用 探针 标准品 拷贝数 

分 类 号：S852.659.2[农业科学—基础兽医学]