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

doi:10.11843/j.issn.0366-6964.2023.06.028

摘要/Abstract

摘要： 旨在建立一种针对猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)的敏感性高、特异性强的快速临床诊断方法。以猪流行性腹泻病毒cDNA为模板,在重组酶和聚合酶的共同作用下实现对目的基因的扩增,本研究设计了用于重组酶聚合酶扩增的引物,优化了重组酶聚合酶扩增(recombinase polymerase amplification, RPA)反应体系,检测了反应的特异性和敏感性,确定了反应的最佳温度和时间,通过结合琼脂糖凝胶电泳和侧流层析试纸条的方法进行分析和鉴定。试验结果显示反应的最佳温度和时间为30 ℃、20 min,反应可以检测到的最低质粒拷贝数为10² copies·μL^-1,且在对猪的丁型冠状病毒、传染性胃肠炎病毒、轮状病毒和圆环病毒2型的检测中具有良好的特异性。综上所述,通过对RPA反应条件的摸索与优化,成功建立了猪流行性腹泻病毒重组酶聚合酶扩增结合琼脂糖凝胶电泳(Basic RPA)和侧流层析试纸条(LF-RPA)的检测方法,两种方法相比较于RT-PCR,有更短的检测时间且具有更高的敏感性,对仪器、场地和检测环境的要求更低,为今后PEDV的临床诊断及快速检测提供了更多的选择和更有效的技术支持。

关键词: 猪流行性腹泻病毒, 重组酶聚合酶扩增(RPA), 侧流层析(LFD), 快速诊断方法

Abstract: The purpose of the study was to establish a rapid clinical diagnostic method with high sensitivity and specificity for porcine epidemic diarrhea virus. Using the cDNA of porcine epidemic diarrhea virus as the template, the target gene was amplified by the combination of recombinant enzyme and polymerase. In this study, primers were designed for the amplification of recombinant enzyme polymerase, the reaction system of RPA was optimized, the specificity and sensitivity of the reaction were tested, and the optimum temperature and time were determined, analysis and identification were carried out by agarose gel electrophoresis and side-stream chromatography. The experimental results showed that the optimal temperature and time for the reaction were 30 ℃, 20 min, and the minimum number of plasmid copies that could be detected was 10² copies·μL^-1 also showed good specificity in the detection of porcine delta coronavirus, transmissible gastroenteritis virus, porcine rotavirus and porcine circovirus. The above results show that by exploring and optimizing the reaction conditions of RPA, a polymerase chain reaction combined with agarose gel electrophoresis (Basic RPA) and lateral flow chromatography (LF-RPA) were developed for the detection of porcine epidemic diarrhea virus, it has shorter detection time, higher sensitivity and lower requirement for instrument, site and detection environment, which provides more choice and more effective technical support for clinical diagnosis and rapid detection of PEDV in the future.

Key words: porcine epidemic diarrhea virus, recombinase polymerase amplification (RPA), lateral flow dipstick (LFD), rapid diagnosis

中图分类号:

S852.659.6

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.

ZHANG Senhao, WANG Xueying, CAI Limeng, XIE Weichun, KUANG Hongdi, WANG Xiaona, LI Jiaxuan, CUI Wen, JIANG Yanping, ZHOU Han, SHAN Zhifu, WANG Li, QIAO Xinyuan, LI Yijing, TANG Lijie. Establishment and Application of Rapid Diagnosis Method for RPA of Porcine Epidemic Diarrhea Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2498-2508.

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