猪圆环病毒3型TaqMan荧光定量PCR方法的建立和初步应用

doi:10.11843/j.issn.0366-6964.2018.06.026

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (6): 1320-1326.doi: 10.11843/j.issn.0366-6964.2018.06.026

• 研究简报 • 上一篇

猪圆环病毒3型TaqMan荧光定量PCR方法的建立和初步应用

李畅^1,2, 库旭钢^1,2, 王俊伟^1,2, 李静^1,2, 朱玲^1,2, 何启盖^1,2*

1. 华中农业大学农业微生物学国家重点实验室, 武汉 430070;
2. 华中农业大学动物医学院, 武汉 430070

收稿日期:2017-11-27 出版日期:2018-06-23 发布日期:2018-06-23
通讯作者: 何启盖,教授,博导,E-mail:he628@mail.hzau.edu.cn
作者简介:李畅(1992-),男,安徽宿州人,硕士,E-mail:liyaheng1113@foxmail.com
基金资助:
国家生猪产业技术体系资助项目（CARS-35）

Development and Application of a TaqMan-based Real-time Fluorescent PCR for Specific Detection of Porcine Circovirus Type 3

LI Chang^1,2, KU Xu-gang^1,2, WANG Jun-wei^1,2, LI Jing^1,2, ZHU Ling^1,2, HE Qi-gai^1,2*

1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China;
2. College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China

Received:2017-11-27 Online:2018-06-23 Published:2018-06-23

摘要/Abstract

摘要：

为了灵敏而便捷的检测猪圆环病毒3型（PCV3），根据PCV3 ORF2基因组保守序列设计合成4对引物，先使用荧光染料（SYBR）定量PCR法验证其能否有效扩增目的片段，并根据结果和引物对所扩增序列的交集进一步设计TaqMan探针，通过优化反应条件和反应体系建立快速检测PCV3的实时荧光定量PCR检测方法。结果显示，SYBR染料法和TaqMan探针法均能有效地扩增PCV3标准质粒以及其他阳性样品，并且对1.29×10²~1.29×10⁹拷贝·μL^-1的PCV3标准质粒（Pmd18-t-Cap）呈现良好的线性关系，该方法的检测灵敏度为1.29×10²拷贝·μL^-1，比常规PCR的灵敏度高100倍，该方法与猪圆环病毒2型、猪轮状病毒、猪繁殖与呼吸综合征病毒等病毒基因以及猪链球菌、猪肺炎支原体等细菌基因均无交叉反应，具有良好的特异性；批次内重复和批次间重复试验结果显示变异系数（CV）均小于1.5%，呈良好的可重复性。用该方法对2016年至2017年间收集的124份来自湖北省等地的猪病料DNA样品进行检测，结果显示本地区PCV3阳性率为8.06%（10/124），PCV2和PCV3共感染率为8.06%（10/124）。上述结果表明，本研究建立TaqMan荧光定量PCR能够灵敏特异的检测圆环病毒3型，为圆环病毒3型的临床检测提供有效手段。

Abstract:

To detect the Porcine Circovirus type 3 (PCV3) sensitively, rapidly and specifically, four pairs of primers were designed targeting the conserved region of PCV3 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 intersection 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 1.29×10²-1.29×10⁹ copies·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 respiratory syndrome virus(PRRSV), Porcine rotavirus(PoRV), Haemophilus parasuis(Hps), Actinobacillus pleuropneumoniae(App). And the limit detection of this assay was 1.29×10² copies·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.

中图分类号:

S852.659.2

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.

LI Chang, KU Xu-gang, WANG Jun-wei, LI Jing, ZHU Ling, HE Qi-gai. Development and Application of a TaqMan-based Real-time Fluorescent PCR for Specific Detection of Porcine Circovirus Type 3[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1320-1326.

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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

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