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

Abstract

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

CLC Number:

S852.659.6

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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Establishment and Application of Rapid Diagnosis Method for RPA of Porcine Epidemic Diarrhea Virus

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