A 群猪轮状病毒微滴式数字PCR检测方法的建立及其应用

doi:10.11843/j.issn.0366-6964.2025.08.046

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (8): 4095-4100.doi: 10.11843/j.issn.0366-6964.2025.08.046

A 群猪轮状病毒微滴式数字PCR检测方法的建立及其应用

陈冰冰¹^,³(), 蔡蔚游¹^,²(), 刘玉彤¹, 王修武¹^,³, 孙守湖¹^,³, 贺东生¹^,³^,*()

1. 华南农业大学兽医学院, 广州 510642
2. 汕头市澄海区动物疫病预防控制中心, 汕头 515800
3. 岭南现代农业科学与技术广东省实验室肇庆分中心, 肇庆 526238

收稿日期:2024-10-25 出版日期:2025-08-23 发布日期:2025-08-28
通讯作者: 贺东生 E-mail:chbingb@126.com;1012871238@qq.com;dhe@scau.edu.cn
作者简介:陈冰冰(2000-)，女，壮族，广西南宁人，硕士生，主要从事动物疫病分子生物学和免疫学研究，E-mail：chbingb@126.com
蔡蔚游(1992-)，男，广东高州人，硕士生，主要从事动物传染病研究，E-mail：1012871238@qq.com
第一联系人：
陈冰冰和蔡蔚游为同等贡献作者
基金资助:
岭南现代农业科学与技术广东省实验室肇庆分中心项目(P20211154-0302)

Establishment and Application of Droplet Digital PCR Method for the Detection of Porcine Rotavirus A

CHEN Bingbing¹^,³(), CAI Weiyou¹^,²(), LIU Yutong¹, WANG Xiuwu¹^,³, SUN Shouhu¹^,³, HE Dongsheng¹^,³^,*()

1. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
2. Prevention and Control Center of Animal Diseases of Chenghaiqu Shantou, Shantou 515800, China
3. Zhaoqing Branch of Guangdong Laboratory for Modern Agricultural Science and Technology in Lingnan, Zhaoqing 526238, China

Received:2024-10-25 Online:2025-08-23 Published:2025-08-28
Contact: HE Dongsheng E-mail:chbingb@126.com;1012871238@qq.com;dhe@scau.edu.cn

摘要/Abstract

摘要：

旨在建立一种特异、灵敏的A 群猪轮状病毒(porcine rotavirus A, PoRVA)微滴式数字PCR(ddPCR)新型检测方法。本研究根据GenBank中PoRVA的VP6基因保守序列，设计猪轮状病毒特异性引物和探针，优化其反应条件，从而构建PoRVA的ddPCR检测方法，并测定其特异性、灵敏度、重复性以及临床样品试验。优化反应条件结果显示，最佳引物浓度为600 nmol·L^-1，最佳探针浓度为300 nmol·L^-1；退火温度在56 ℃时，病毒阳性液滴拷贝数最高；特异性结果显示，自行建立的检测方法仅能检出PoRVA，而猪丁型冠状病毒、猪流行性腹泻病毒等七种对照病毒核酸检测结果均为阴性；灵敏度结果显示，该方法最低检测限度为3.97 copies·μL^-1，比普通PCR(最低检测限度3 970 copies·μL^-1)和荧光定量PCR(最低检测限度39.7 copies·μL^-1)分别高1 000倍和10倍；重复性结果显示，组内和组间变异系数小于10%；对142份临床样品检测显示，ddPCR检出阳性率高于qPCR和PCR。综上，本研究自行建立的PoRVA ddPCR新型检测方法特异性强、灵敏度高、重复性好，适用于猪轮状病毒早期感染诊断和流行病学调查，可为日后持续性监控猪轮状疫情提供可靠的手段。

关键词: A 群猪轮状病毒, 微滴式数字PCR, 检测方法

Abstract:

The aim of this essay was to establish a specific and sensitive novel detection method for porcine rotavirus A (PoRVA) using droplet digital PCR (ddPCR). This study designed specific primers and probes for PoRVA based on the conserved sequences of the VP6 gene from GenBank, and successfully constructed a ddPCR detection method for PoRVA by optimizing the reaction conditions and determined the specificity, sensitivity, repeatability, and made some clinical sample testing. The optimization of the reaction conditions showed that the optimal concentrations of upstream and downstream primers and the corresponding probe were 600 nmol·L^-1and 300 nmol·L^-1, respectively, with the highest copy number of virus-positive droplets and the best effect at an annealing temperature of 56 ℃ The specificity results show that the self-established detection method can only detect PoRVA, while the nucleic acid detection results for seven control viruses such as porcine deltacorona virus and porcine epidemic diarrhea virus are all negative. Sensitivity results showed that the detection limit of this method was 3.97 copies·μL^-1, which is 1 000 times and 10 times higher than that of conventional PCR (3 970 copies·μL^-1) and quantitative fluorescent PCR (39.7 copies·μL^-1), respectively. Repeatability results showed that the intra- and inter-group coefficients of variation were less than 10%. Testing of 142 clinical samples showed that the positive detection rate by ddPCR was higher than that of qPCR and PCR. The results indicate that the novel ddPCR detection method for PoRV established in this study has strong specificity, high sensitivity, and good repeatability. It is suitable for early infection diagnosis and epidemiological investigation of PoRV, and can provide a reliable means for continuous monitoring of porcine rotavirus epidemics in the future.

Key words: porcine rotavirus A, droplet digital PCR, detection method

中图分类号:

S855.3

陈冰冰, 蔡蔚游, 刘玉彤, 王修武, 孙守湖, 贺东生. A 群猪轮状病毒微滴式数字PCR检测方法的建立及其应用[J]. 畜牧兽医学报, 2025, 56(8): 4095-4100.

CHEN Bingbing, CAI Weiyou, LIU Yutong, WANG Xiuwu, SUN Shouhu, HE Dongsheng. Establishment and Application of Droplet Digital PCR Method for the Detection of Porcine Rotavirus A[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 4095-4100.

图/表 3

参考文献 13

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质粒浓度/ (copies·μL^-1) Plasmid concentration	组内重复试验 (${\bar x}$±s) Intra-assay	组内变异系数 (CV)/% Intra-assay variability	组间重复试验 (${\bar x}$±s) Inter-assay	组间变异系数 (CV)/% Inter-assay variability
3.97×10³	3 942.2±47.6	1.2	3 748.3±70.2	1.9
3.97×10²	387.5±7.5	1.9	362.2±18.9	5.2
3.97×10¹	38.9±2.5	6.4	36.7±3.0	8.2
3.97×10⁰	3.7±0.3	8.1	3.8±0.4	9.8

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A 群猪轮状病毒微滴式数字PCR检测方法的建立及其应用

Establishment and Application of Droplet Digital PCR Method for the Detection of Porcine Rotavirus A

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