PDCoV与TGEV双重实时荧光定量PCR检测方法的建立及初步应用

doi:10.16431/j.cnki.1671-7236.2021.10.038

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (10): 3855-3863.doi: 10.16431/j.cnki.1671-7236.2021.10.038

PDCoV与TGEV双重实时荧光定量PCR检测方法的建立及初步应用

王征帆^1,2,3, 朱利塞^2,3, 王娟^2,3, 李祎云^1,2,3, 向蕊^1,2,3, 应碧云^1,2,3, 王贵平^2,3,4, 贾爱卿^2,3,4, 白挨泉¹

1. 佛山科学技术学院, 佛山 528231;
2. 广东海大畜牧兽医研究院, 广州 511400;
3. 广东省养猪与猪病防控技术研究企业重点实验室, 广州 511400;
4. 广东海大集团股份有限公司, 广州 511400

收稿日期:2021-04-11 出版日期:2021-10-20 发布日期:2021-09-30
通讯作者: 贾爱卿, 白挨泉 E-mail:59492815@qq.com;baiaiquan2008@163.com
作者简介:王征帆(1993-),男,贵州雷山人,硕士,研究方向:猪传染病诊断与防治,E-mail:15320520429@163.com
基金资助:
广东省重点领域研发计划项目（2019B020218004）；广州市科学研究计划重点项目（201804020006）；番禺区创新创业领军团队项目（2017-R02-4）；广东省科技计划项目（2020B1212070023）；猪用疫苗生产新工艺及产业化关键技术的研发项目（2017YFD0500604）；广东省教育厅预防兽医学重点实验室资助项目（2014KTSPT037）；广东省现代农业产业技术体系生猪创新团队项目（2020KJ126）

Establishment and Preliminary Application of Duplex Real-time Quantitative PCR for Detection of PDCoV and TGEV

WANG Zhengfan^1,2,3, ZHU Lisai^2,3, WANG Juan^2,3, LI Yiyun^1,2,3, XIANG Rui^1,2,3, YING Biyun^1,2,3, WANG Guiping^2,3,4, JIA Aiqing^2,3,4, BAI Aiquan¹

1. Foshan University, Foshan 528231, China;
2. Guangdong Haid Institute of Animal Husbandry & Veterinary, Guangzhou 511400, China;
3. Guangdong Provincial Key Laboratory of Pig Breeding and Pig Disease Prevention and Control Technology Research Enterprise, Guangzhou 511400, China;
4. Guangdong Haid Group Co., Ltd., Guangzhou 511400, China

Received:2021-04-11 Online:2021-10-20 Published:2021-09-30

摘要/Abstract

摘要： 试验旨在建立快捷、高效而准确的鉴别诊断猪德尔塔冠状病毒（Porcine deltacoronavirus，PDCoV）与传染性胃肠炎病毒（Transmissible gastroenteritis virus，TGEV）的双重实时荧光定量PCR方法。通过绘制双重实时荧光定量PCR的标准曲线，检验该方法的特异性、敏感性和重复性，并对临床样品进行检测。结果显示，双重实时荧光定量PCR方法的循环阈值与PDCoV和TGEV质粒拷贝数的对数之间存在良好的线性关系，且对应的相关系数分别为R_（P）²=0.9994和R_（T）²=0.996；能特异性地检测PDCoV和TGEV，而与PEDV、PRV、PRRSV、CSFV和RV无交叉反应，具有较强的特异性；检验PDCoV与TGEV质粒标准品的最低检测限度分别达到2和20拷贝/μL，且分别比常规RT-PCR高1 000和100倍，具有较高的敏感度；PDCoV与TGEV的批内和批间重复性检测的Ct均值基本相同，且变异系数（CV）均<2%，具有较好的重复性。用该方法对114份仔猪腹泻样品检测结果显示，PDCoV和TGEV的阳性率分别为5.6%（6/114）和8.8%（10/114），混合感染检出率为4.6%（5/114），比常规RT-PCR具有更高的检出率和敏感性。结果表明，本试验建立的双重实时荧光定量PCR方法具有特异性强、灵敏度高、重复性和稳定性好等优点，适用于病毒早期诊断和批量临床样品检测，为疾病防控、流行病学调查及相关性研究提供了技术支持及数据参考。

关键词: 猪德尔塔冠状病毒(PDCoV); 传染性胃肠炎病毒(TGEV); TaqMan探针; 双重实时荧光定量PCR

Abstract: This study was aimed to establish a fast, efficient and sensitive duplex Real-time quantitative PCR method for differential diagnosis of Porcine deltacoronavirus (PDCoV) and Transmissible gastroenteritis virus (TGEV). By drawing the standard curve of duplex Real-time quantitative PCR, the specificity, sensitivity and repeatability of the method were tested, and clinical samples were tested. The results showed that there was a good linear relationship between the cycle threshold of this method and the logarithm of the copy number of PDCoV and TGEV plasmids, and the corresponding correlation coefficients were R_(P)²=0.9994 and R_(T)²=0.996, respectively. It could specifically detect PDCoV and TGEV, but had nocross-react with PEDV, PRV, PRRSV, CSFV and RV, and had strong specificity. The minimum detection limits of the PDCoV and TGEV plasmid standard products reached 2 and 20 copies/μL, respectively, and were 1 000 and 100 times higher than conventional RT-PCR, respectively, with higher sensitivity. The average Ct values of intra-assay and inter-assay reproducibility of PDCoV and TGEV were basically the same, and the coefficient of variation (CV) was less than 2%, which had good reproducibility. The results of 114 piglet diarrhea samples tested by this method showed that the positive rates of PDCoV and TGEV were 5.6% (6/114) and 8.8% (10/114), respectively, and the detection rate of mixed infection was 4.6% (5/114), duplex Real-time quantitative PCR had a higher detection rate and sensitivity than conventional RT-PCR. The result indicated that the duplex Real-time quantitative PCR had the advantages of strong specificity, high sensitivity, good repeatability and stability, it was suitable for early virus diagnosis and batch clinical sample detection, and provided technical support and data reference for disease prevention and control, epidemiological investigation and correlation research.

Key words: Porcine delta coronavirus(PDCoV); Transmissible gastroenteritis virus(TGEV); TaqMan probe; duplex Real-time quantitative PCR

中图分类号:

S852.65⁺1

王征帆, 朱利塞, 王娟, 李祎云, 向蕊, 应碧云, 王贵平, 贾爱卿, 白挨泉. PDCoV与TGEV双重实时荧光定量PCR检测方法的建立及初步应用[J]. 中国畜牧兽医, 2021, 48(10): 3855-3863.

WANG Zhengfan, ZHU Lisai, WANG Juan, LI Yiyun, XIANG Rui, YING Biyun, WANG Guiping, JIA Aiqing, BAI Aiquan. Establishment and Preliminary Application of Duplex Real-time Quantitative PCR for Detection of PDCoV and TGEV[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3855-3863.

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PDCoV与TGEV双重实时荧光定量PCR检测方法的建立及初步应用

Establishment and Preliminary Application of Duplex Real-time Quantitative PCR for Detection of PDCoV and TGEV

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