猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2019.06.016

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (6): 1261-1267.doi: 10.11843/j.issn.0366-6964.2019.06.016

猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用

郑兰兰^1,2, 朱静静¹, 王盼¹, 舒燕¹, 梁青青^1,2, 李炳晓^1,2, 王超群³, 魏战勇^1,2*

1. 河南农业大学牧医工程学院, 郑州 450002;
2. 河南省动物性食品安全重点实验室, 郑州 450002;
3. 许昌海关, 许昌 461000

收稿日期:2018-12-20 出版日期:2019-06-23 发布日期:2019-06-23
通讯作者: 魏战勇,主要从事动物分子病毒与免疫学研究,E-mail:weizhanyong@henau.edu.cn
作者简介:郑兰兰(1982-),女,河南开封人,讲师,博士,主要从事动物分子病毒与免疫学研究,E-mail:lanlan@henau.edu.cn;朱静静(1997-),女,河南周口人,本科生,主要从事动物分子病毒与免疫学研究,E-mail:130059810@qq.com。郑兰兰、朱静静为同等贡献作者
基金资助:
国家重点研发计划（2018YFD0501205）；许昌市基础与前沿项目（JC2018005）

Development and Application of a TaqMan based Real-time Fluorescent RT-PCR for Specific Detection of Porcine Deltacoronavirus

ZHENG Lanlan^1,2, ZHU Jingjing¹, WANG Pan¹, SHU Yan¹, LIANG Qingqing^1,2, LI Bingxiao^1,2, WANG Chaoqun³, WEI Zhanyong^1,2*

1. College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China;
2. Henan Animal Food Safety Key Laboratory, Zhengzhou 450002, China;
3. Xuchang Customs, Xuchang 461000, China

Received:2018-12-20 Online:2019-06-23 Published:2019-06-23

摘要/Abstract

摘要： 本研究旨在建立一种灵敏、快速检测猪δ冠状病毒（porcine deltacoronavirus，PDCoV）的TaqMan荧光定量RT-PCR方法。参照GenBank中PDCoV有关基因序列，设计一对特异性引物用于扩增PDCoV M基因。将测序正确的基因片段克隆入pMD18-T载体，构建重组质粒作为建立标准曲线的病毒模板。设计合成一对特异性引物与TaqMan探针，进行反应条件和反应体系的优化，建立快速检测PDCoV的TaqMan实时荧光定量RT-PCR方法，并进行该方法的灵敏性、特异性与重复性验证。结果表明，该方法能有效扩增1.0×10¹~1.0×10⁹拷贝·μL^-1的PDCoV标准质粒，建立的标准曲线呈现良好的线性关系。该方法的检测灵敏度为1.0×10¹拷贝·μL^-1；对猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪伪狂犬病病毒等病原不发生交叉反应，具有很好的特异性；重复性试验结果显示变异系数（CV）小于1%，重复性良好。对2017—2018年期间收集的河南省不同猪场的100份腹泻病料进行检测，PDCoV的阳性检出率为23%（23/100），与PDCoV SYBR Green Ⅰ荧光定量RT-PCR检测方法的符合率为100%。人工感染PDCoV的仔猪，取攻毒后不同时间的粪便样品，应用本研究建立方法与常规RT-PCR方法对样品进行检测，TaqMan荧光定量RT-PCR检测方法的灵敏度远远优于常规RT-PCR。上述结果表明，本研究所建立的TaqMan荧光定量RT-PCR检测方法能够灵敏、特异地检测PDCoV，可用于临床PDCoV的检测。

Abstract: This experiment was conducted to develop a TaqMan based real-time fluorescent RT-PCR for the detection of PDCoV. Based on the M gene sequence of PDCoV in GenBank, a pair of primers was designed, and M gene of PDCoV was amplified and cloned into pMD18-T vector to get the recombinant plasmid. Then another pair of primers and a TaqMan probe was designed, and a TaqMan based real-time fluorescent RT-PCR assay was developed by optimizing the conditions and systems of reaction. Sensitivity, specificity and reproducibility assay were determined. The results showed that this assay could detect the PDCoV between 1.0×10¹-1.0×10⁹ copies·μL^-1 with a good linear relation, and the sensitivity limit was 1.0×10¹ copies·μL^-1. There were no cross-reaction with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), pseudorabies virus (PRV) and other porcine viruses. The CV (Coefficient of Variation) of intra-assay and inter-assay were no more than 1%. The TaqMan based real-time RT-PCR assay was then used to detect 100 diarrhea clinical samples of porcine that collected from 2017 to 2018, and the PDCoV positive ratio was 23%, and the coincidence rate with SYBR Green Ⅰ real-time PCR assay was 100%. Piglets were inoculated with PDCoV, and fecal samples were collected and detected by TaqMan real-time RT-PCR and common RT-PCR assay, the results showed that the sensitivity of the former was much better than the latter. This assay provides a high sensitivity, specificity and reproducibility method for detection of PDCoV.

中图分类号:

S852.659.6

郑兰兰, 朱静静, 王盼, 舒燕, 梁青青, 李炳晓, 王超群, 魏战勇. 猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用[J]. 畜牧兽医学报, 2019, 50(6): 1261-1267.

ZHENG Lanlan, ZHU Jingjing, WANG Pan, SHU Yan, LIANG Qingqing, LI Bingxiao, WANG Chaoqun, WEI Zhanyong. Development and Application of a TaqMan based Real-time Fluorescent RT-PCR for Specific Detection of Porcine Deltacoronavirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1261-1267.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2019.06.016

https://www.xmsyxb.com/CN/Y2019/V50/I6/1261

参考文献

[1] WOO P C Y, LAU S K P, LAM C S F, et al. Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and avian coronaviruses as the gene source of Gammacoronavirus and Deltacoronavirus[J]. J Virol, 2012, 86(7):3995-4008.
[2] SINHA A, GAUGER P, ZHANG J Q, et al. PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine[J]. Vet Microbiol, 2015, 179(3-4):296-298.
[3] WANG L Y, BYRUM B, ZHANG Y. Porcine coronavirus HKU15 detected in 9 US states, 2014[J]. Emerg Infect Dis, 2014, 20(9):1594-1595.
[4] HU H, JUNG K, VLASOVA A N, et al. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States[J]. J Clin Microbiol, 2015, 53(5):1537-1548.
[5] LEE S, LEE C. Complete genome characterization of korean porcine deltacoronavirus strain KOR/KNU14-04/2014[J]. Genome Announc, 2014, 2(6):e01191-14.
[6] SAENG-CHUTO K, LORSIRIGOOL A, TEMEEYASEN G, et al. Different lineage of porcine deltacoronavirus in Thailand, Vietnam and Lao PDR in 2015[J]. Transbound Emerg Dis, 2017, 64(1):3-10.
[7] DONG N, FANG L R, YANG H, et al. Isolation, genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014[J]. Vet Microbiol, 2016, 196:98-106.
[8] WANG Y W, YUE H, FANG W H, et al. Complete genome sequence of porcine deltacoronavirus strain CH/Sichuan/S27/2012 from mainland China[J]. Genome Announc, 2015, 3(5):e00945-15.
[9] MA Y M, ZHANG Y, LIANG X Y, et al. Origin, evolution, and virulence of porcine deltacoronaviruses in the United States[J]. MBio, 2015, 6(2):e00064-15.
[10] CHEN Q, GAUGER P, STAFNE M, et al. Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets[J]. Virology, 2015, 482:51-59.
[11] LI W T, LI H, LIU Y B, et al. New variants of porcine epidemic diarrhea virus, China, 2011[J]. Emerg Infect Dis, 2012, 18(8):1350-1353.
[12] THACHIL A, GERBER P F, XIAO C T, et al. Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies[J]. PLoS One, 2015, 10(4):e0124363.
[13] JUNG K, HU H, EYERLY B, et al. Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs[J]. Emerg Infect Dis, 2015, 21(4):650-654.
[14] HU H, JUNG K, WANG Q H, et al. Development of a one-step RT-PCR assay for detection of pancoronaviruses (α-, β-, γ-, and δ-coronaviruses) using newly designed degenerate primers for porcine and avian `fecal samples[J]. J Virol Methods, 2018, 256:116-122.
[15] SONG D, ZHOU X, PENG Q, et al. Newly emerged porcine Deltacoronavirus associated with diarrhoea in swine in China:identification, prevalence and full-length genome sequence analysis[J]. Transbound Emerg Dis, 2015, 62(6):575-580.
[16] 张利卫, 曹贝贝,李炳晓,等. 新发猪Delta冠状病毒和猪流行性腹泻病毒SYBR Green Ⅰ双重荧光RT-PCR检测方法的建立及应用[J]. 中国兽医学报, 2018, 38(4):618-624.
ZHANG L W, CAO B B, LI B X, et al. Establishment and application of a duplex SYBR Green Ⅰ real-time RT-PCR assay for simultaneous detection of emerging PDCoV and PEDV[J]. Chinese Journal of Veterinary Science, 2018, 38(4):618-624. (in Chinese)
[17] 韦学雷, 梁青青,曹贝贝,等. 猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用[J]. 中国兽医学报, 2018, 38(1):11-16.
WEI X L, LIANG Q Q, CAO B B, et al. Establishment and application of a multiplex RT-PCR assay for simultaneous detection of porcine delta coronavirus, porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J]. Chinese Journal of Veterinary Science, 2018, 38(1):11-16. (in Chinese)
[18] 逄凤娇, 俞正玉,何孔旺,等. 猪Deltacoronavirus RT-PCR检测方法的建立及其应用[J]. 中国预防兽医学报, 2015, 37(9):683-686.
PANG F J, YU Z Y, HE K W, et al. Development and application of a RT-PCR assay for detection of porcine deltacoronavirus[J]. Chinese Journal of Preventive Veterinary Medicine, 2015, 37(9):683-686. (in Chinese)
[19] 陈小金, 张霞,王玉玲,等. 猪德尔塔冠状病毒荧光定量PCR检测方法的建立[J]. 中国动物检疫, 2018, 35(9):95-100.
CHEN X J, ZHANG X, WANG Y L, et al. Development of fluorescence quantitative PCR assay for detection of porcine deltacoronavirus[J]. China Animal Health Inspection, 2018, 35(9):95-100. (in Chinese)
[20] 韩丽,周雍,李炳晓,等. 猪Delta冠状病毒和猪流行性腹泻病毒双重RT-PCR诊断方法的建立及初步应用[J]. 中国兽医科学, 2017, 47(5):557-562.
HAN L, ZHOU Y, LI B X, et al. Establishment and preliminary application of a duplex reverse transcription-PCR for the detection of porcine deltacoronavirus and porcine epidemic diarrhea virus[J]. Chinese Veterinary Science, 2017, 47(5):557-562. (in Chinese)

猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用

Development and Application of a TaqMan based Real-time Fluorescent RT-PCR for Specific Detection of Porcine Deltacoronavirus

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	宋雯妍, 张瀚文, 吴澳迪, 张丽燕, 刘照, 叶桐桐, 陈创夫, 盛金良. 猪繁殖与呼吸综合征病毒GP5蛋白纳米抗体的筛选及其对病毒复制的抑制效应[J]. 畜牧兽医学报, 2024, 55(1): 258-270.
[2]	王莹, 李佳康, 曾悦, 史开拓, 韦丽婷, 彭佳佳, 李秋燕, 曹龙龙, 周登元. 猫杯状病毒单克隆抗体的制备[J]. 畜牧兽医学报, 2024, 55(1): 401-405.
[3]	李惠惠, 董可儿, 刘馨博, 张春晓, 马超, 陈利苹, 钟旗, 姚刚, 马雪连. 牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用[J]. 畜牧兽医学报, 2024, 55(1): 406-412.
[4]	周建浩, 王东方, 刘影, 王淑娟, 马震原, 谢彩华, 赵雪丽, 杨海波, 冯桂丹, 康台生, 胡煜锋, 李博文, 闫若潜. 猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用[J]. 畜牧兽医学报, 2024, 55(1): 413-418.
[5]	张家祺, 贾浠宁, 周群, 宋鑫, 朱晨曦, 李格格, 张斌. 基于E2蛋白BVDV-1病毒样颗粒的构建及对小鼠的免疫效力评估[J]. 畜牧兽医学报, 2023, 54(12): 5143-5153.
[6]	周立坤, 董鑫媛, 李家辉, 崔志莹, 赵士杰, 徐婕, 陈静, 张宜娜, 夏平安. MID2对猪繁殖与呼吸综合征病毒复制的调控作用[J]. 畜牧兽医学报, 2023, 54(11): 4735-4744.
[7]	郑如雯, 黄涛, 吴道义, 禹光美, 李芳芳, 隋鑫, 闵婷玉. 牛病毒性腹泻病毒LAMP-LFD检测方法的建立及初步应用[J]. 畜牧兽医学报, 2023, 54(11): 4745-4753.
[8]	贾艺泉, 于梦婷, 刘卫容, 方守国, 章松柏. 黄芩苷通过促进Ⅰ型干扰素表达抑制鸡传染性支气管炎病毒复制[J]. 畜牧兽医学报, 2023, 54(11): 4839-4850.
[9]	陈璐, 常新宇, 沈嘉忱, 沈瑞廷, 赵振华, 侯晓林. 鸡传染性支气管炎病毒感染HD11细胞的转录组分析[J]. 畜牧兽医学报, 2023, 54(11): 4860-4865.
[10]	贺叶青, 左百姣, 李庆豪, 孙娟, 金鑫, 夏璐, 魏战勇. 猪传染性胃肠炎病毒感染仔猪免疫器官的组织病理学观察[J]. 畜牧兽医学报, 2023, 54(11): 4872-4879.
[11]	陈文哲, 张向乐, 顾峰幸, 赵振翔, 李康丽, 薛钊宁, 郑海学, 张小丽, 朱紫祥. 猪GRK2蛋白抗口蹄疫病毒作用分析[J]. 畜牧兽医学报, 2023, 54(10): 4350-4361.
[12]	邹宏, 夏应菊, 李玲, 徐璐, 赵俊杰, 王团结, 张乾义, 宋振辉. 猪瘟病毒和牛病毒性腹泻病毒双重荧光定量PCR检测方法的建立和初步应用[J]. 畜牧兽医学报, 2023, 54(10): 4422-4427.
[13]	袁丽, 孙杨杨, 张路捷, 张杰, 孙海凤, 白娟, 姜平. 猪繁殖与呼吸综合征病毒GP3蛋白单克隆抗体制备及抗原表位鉴定[J]. 畜牧兽医学报, 2023, 54(8): 3424-3434.
[14]	曹丽艳, 孔祥雨, 李想通, 索学鹏, 段月月, 袁聪, 施磊, 张宇, 马国祥, 郑海学, 王琦. 猪急性腹泻综合征冠状病毒核衣壳蛋白单克隆抗体的制备及其抗体序列的分析[J]. 畜牧兽医学报, 2023, 54(6): 2487-2497.
[15]	张森豪, 王雪莹, 蔡李萌, 解伟纯, 匡虹迪, 王晓娜, 李佳璇, 崔文, 姜艳平, 周晗, 单智夫, 王丽, 乔薪瑗, 李一经, 唐丽杰. 猪流行性腹泻病毒重组酶聚合酶扩增快速诊断方法的建立和应用[J]. 畜牧兽医学报, 2023, 54(6): 2498-2508.