畜牧兽医学报2018，Vol.49Issue(6)：1320-1326,7.DOI:10.11843/j.issn.0366-6964.2018.06.026
猪圆环病毒3型TaqMan荧光定量PCR方法的建立和初步应用
Development and Application of a TaqMan-based Real-time Fluorescent PCR for Specific Detection of Porcine Circovirus Type 3
摘要
Abstract
To detect the Porcine Circovirus type 3(PCV3)sensitively,rapidly and specifically,four pairs of primers were designed targeting the conserved region of PCV 3 ORF3 gene.SYBR-based quantitative PCR results showed that Real-time PCR with all primer pairs can detect viral genome efficaciously.Then a TaqMan probe were designed according to the PCR products inter-section sequence,and a real-time quantitative PCR method was established by optimizing the reaction conditions and systems.The method presented a good linear relation with the Pmd18-t-Cap vector(Recombinant plasmid harboring PCV3 ORF2 gene)ranging from 1.29×102-1.29× 109copies· μL －1.The assay was highly specific for PCV3,without cross-reaction with other porcine virus and bacteria,such as Porcine circovirus 2(PCV2),Porcine reproductive and respira-tory syndrome virus(PRRSV),Porcine rotavirus(PoRV),Haemophilus parasuis(Hps),Acti-nobacillus pleuropneumoniae(App).And the limit detection of this assay was 1.29×102copies·μL－1,more sensitive than the conventional PCR.The CV(Coefficient of Variation)of intra-assay and inter-assay were less than 2％,showed that the assay was reproducible.The real-time qPCR was further used to detect the DNA sample extracted from clinical sample collected from 2016 to 2017,the PCV3 positive ratio was 8.06％(10/124),and the co-infection rate of PCV3/PCV2 was 8.06％(10/124).The real-time qPCR assay provides a high sensitivity and specificity,repeatability method for detection of PCV3.关键词
圆环病毒3型/SYBR荧光定量PCR/TaqMan荧光定量PCR/检测Key words
porcine circovirus type 3(PCV3)/SYBR qPCR/TaqMan qPCR/detection分类
农业科技引用本文复制引用
李畅,库旭钢,王俊伟,李静,朱玲,何启盖..猪圆环病毒3型TaqMan荧光定量PCR方法的建立和初步应用[J].畜牧兽医学报,2018,49(6):1320-1326,7.基金项目
国家生猪产业技术体系资助项目(CARS-35) （CARS-35）
