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

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.