猪流行性腹泻病毒锁核酸探针荧光定量 PCR检测方法的建立

doi:10.19978/j.cnki.xmsy.2024.02.05

广东畜牧兽医科技 ›› 2024, Vol. 49 ›› Issue (2): 30-36.DOI: 10.19978/j.cnki.xmsy.2024.02.05

猪流行性腹泻病毒锁核酸探针荧光定量 PCR检测方法的建立

李艳，卞志标，翟少伦*，李春玲*

（广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站，广东广州 510640；岭南现代农业科学与技术广东省实验室茂名分中心，广东茂名 525000）

出版日期:2024-04-24 发布日期:2024-04-18
通讯作者: 李春玲，翟少伦
基金资助:
广东省重点领域研发计划项目（2020B0202080004）；广州市重点研发项目（202206010192）；广东省科技计划项目（2023B0202010009）；广东省农业科学院大广食品专家工作站建设项目（2023 工作站 16）；江门市科技计划项目（江科【2021】183 号）；广东省农业农村厅科技计划项目（2023KJ119）

Methodology Development of a Locked Nucleic Acid-based Fluorescent Quantitation RT-PCR for Detecting Porcine Epidemic Diarrhea Virus

LI Yan，BIAN Zhibiao，ZHAI Shaolun*，LI Chunling*

（Institute of Animal Health，Guangdong Academy of Agricultural Sciences/Key Laboratory of Livestock Disease Prevention of Guangdong Provincel/Scientific Observation and Experiment Station of Veterinary Drugs and DiagnosticTechniques of Guangdong Province，Ministry of Agriculture，Guangzhou Guangdong 510640； Maoming Branch center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology，Maoming Guangdong 525000）

Online:2024-04-24 Published:2024-04-18

摘要/Abstract

摘要： 为建立一种快速、灵敏的猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）早期痕量检测方法，该研究以 PEDV 的 M 基因为靶基因，设计了特异性引物和锁核酸(LockedNucleic Acid，LNA)?TaqMan 探针，经过条件优化，建立了基于 LNA?TaqMan 探针的 PEDV 荧光定量 PCR 方法，并与常规 TaqMan 探针法进行了比对。结果显示，所建立的 PEDV 的 LNA?Taq?Man 探针荧光定量 PCR 方法最低检测限为 3.2 拷贝/微升，较常规 TaqMan 探针法提高了 10 倍；与猪传染性胃肠炎病毒、猪轮状病毒和猪德尔塔冠状病毒等多种病原不存在交叉反应，特异性良好；批内和批间重复性结果的变异系数均小于 1%，重复性较好。应用该方法对 103 份临床样品进行检测，结果显示，共检出 PEDV 阳性样品 47 份，阳性率 45.63%，与常规 TaqMan 探针法检测结果的阳性符合率为 90.38%。本研究建立的基于 LNA?TaqMan 探针的荧光定量 PCR 方法为 PEDV 的精准检测和流行病学调查提供了一种新的技术选择。

关键词: 猪流行性腹泻病毒, M 基因, LNA?TaqMan 探针荧光定量 PCR

Abstract: In this study，aLNA ? based fluorescent quantitation PCR method was developed for the rapid，accurate and trace detection of porcine epidemic diarrhea virus（PEDV）by targeting the M gene. Special primers and LNA ? TaqMan probe were designed according to the M gene，and the reaction conditions have been optimized. The LNA?based fluorescent quantitation PCR method was compared with the conventional TaqMan probe method. Results showed that the detection limit of the method was 3.2 copies/μL，improved by 10 times compared to conventional TaqMan probe method. The method was specific of PEDV and no cross reaction to TGEV，PoRV，and PDCoV. The method had a good repeatability with variation coefficient of intra? and inter?group less than 1%. The method was used to detect 103 clinical samples，the results showed that 47 samples were positive for PEDV，with a positive rate of 45.63%，and the positive coincidence rate with the conventional TaqMan probe PCR assay was 90.38%. This LNA? based fluorescent quantitation PCR method provides a new technical for accurate detection and epidemiological investigation of PEDV.

Key words: PEDV, Mgene, LNA?TaqMan real?time PCR

中图分类号:

S852.65+1

李艳, 卞志标, 翟少伦, 李春玲. 猪流行性腹泻病毒锁核酸探针荧光定量 PCR检测方法的建立[J]. 广东畜牧兽医科技, 2024, 49(2): 30-36.

LI Yan, BIAN Zhibiao, ZHAI Shaolun, LI Chunling. Methodology Development of a Locked Nucleic Acid-based Fluorescent Quantitation RT-PCR for Detecting Porcine Epidemic Diarrhea Virus[J]. Guangdong Journal of Animal and Veterinary Science, 2024, 49(2): 30-36.

[1]	史大庆，徐少珠，张国丽，肖永珍，齐冬梅*. 猪流行性腹泻病毒检测技术研究进展[J]. 广东畜牧兽医科技, 2024, 49(1): 51-56.
[2]	邵建宏 1，曾俊霞 2，赵福振 1，邱民希 2，苏子由 2，廖秀云 1，罗宝正 1*. 荧光 RT⁃LAMP 法检测猪流行性腹泻病毒方法的建立[J]. 广东畜牧兽医科技, 2019, 44(2): 42-45.

猪流行性腹泻病毒锁核酸探针荧光定量 PCR检测方法的建立

Methodology Development of a Locked Nucleic Acid-based Fluorescent Quantitation RT-PCR for Detecting Porcine Epidemic Diarrhea Virus

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