猪流行性腹泻病毒TaqMan检测方法的建立及基于S基因的遗传变异分析

doi:10.11843/j.issn.0366-6964.2023.02.040

Abstract

Abstract: The present study established a universal TaqMan qPCR detection method that can quickly, efficiently and sensitively detect porcine epidemic diarrhea virus (PEDV), and further understand the genetic variation of PEDV in Hebei Province. Here we designed specific primers and probes with reference to the conservative region of M gene of the PEDV variant AJ1102, and optimized the primer concentration, annealing temperature and other conditions, a qPCR method for screening PEDV in pig farms was established, and the S gene of the positive samples was amplified and sequenced for genetic evolution analysis. The results showed that the specificity of this method was strong, and there was no cross reaction with common pig diseases such as porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3) and transmissible gastroenteritis virus (TGEV); The minimum detection limit of the established detection method for pMD19T-PEDV standard is 1.09×10¹ copy·μL^-1. It is about 100 times more sensitive than ordinary PCR and has high sensitivity; The coefficient of variation within and between groups was less than 1%, with good repeatability. The nine sequences successfully cloned based on the whole S gene were distributed in two subgroups of GⅡ, and the nucleotide homology of the nine cloned sequences were 96.6%-99.1%; The homology of amino acids were 94.6%-98.7%. The results showed that the epidemic strains of PEDV obtained in this study were closely related to the domestic strains isolated in recent years, but far related to the vaccine strains currently used in China and the European strains, which indicated that the current epidemic strains of PEDV in some areas of Hebei Province were more complex and had a trend of variation, suggesting the necessity of continuous detection of the variation dynamics of the epidemic strains of PEDV. This study not only provides technical support for the clinical diagnosis of PEDV, but also provides a reference for further understanding the genetic evolution of PEDV.

Key words: porcine epidemic diarrhea virus, qPCR, S gene, genetic variation analysis

CLC Number:

S852.659.6

ZHAI Gang, GU Wenyuan, LIU Tao, LIU Ying, ZHANG Shuai, FAN Jinghui, ZUO Yuzhu. Establishment of TaqMan Detection Method of Porcine Epidemic Diarrhea Virus and Analysis of Genetic Variation based on S Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 847-854.

References 25

[1]	HAVE P, MOVING V, SVANSSON V, et al. Coronavirus infection in mink (Mustela vision). Serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J]. Vet Microbiol, 1992, 31(1):1-10.
[2]	BI J, ZENG S L, XIAO S B, et al. Complete genome sequence of porcine epidemic diarrhea virus strain AJ1102 isolated from a suckling piglet with acute diarrhea in China[J]. J Virol, 2012, 86(19):10910-10911.
[3]	SUN R Q, CAI R J, CHEN Y Q, et al. Outbreak of porcine epidemic diarrhea in suckling piglets, China[J]. Emerg Infect Dis, 2012, 18(1):161-163.
[4]	DEBOUCK P, PENSAERT M. Experimental infection of pigs with a new porcine enteric coronavirus, CV777[J]. Am J Vet Res, 1980, 41(2):219-223.
[5]	王隆柏, 林裕胜, 车勇良, 等. 猪流行性腹泻病毒S、N和ORF3基因的遗传变异分析[J]. 畜牧兽医学报, 2014, 45(11):1830-1836.WANG L B, LIN Y S, CHE Y L, et al. Genetic variation analysis of S, N and ORF3 genes of porcine epidemic diarrhea virus[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(11):1830-1836. (in Chinese)
[6]	刘如月, 范京惠, 刘涛, 等. 携带猪流行性腹泻病毒S1抗原表位的乙肝核心抗原病毒样颗粒的构建与鉴定[J]. 畜牧兽医学报, 2021, 52(6):1760-1764.LIU R Y, FAN J H, LIU T, et al. Construction and identification of hepatitis B core antigen virus-like particles carrying PEDV S1 epitope[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6):1760-1764. (in Chinese)
[7]	PENSAERT M B, DE BOUCK P. A new coronavirus-like particle associated with diarrhea in swine[J]. Arch Virol, 1978, 58(3):243-247.
[8]	CHASEY D, CARTWRIGHT S F. Virus-like particles associated with porcine epidemic diarrhoea[J]. Res Vet Sci, 1978, 25(2):255-256.
[9]	万颖, 周改静, 麻园, 等. 猪流行性腹泻病毒N蛋白阻断ELISA抗体检测方法的建立及初步应用[J]. 畜牧兽医学报, 2022, 53(4):1173-1181.WAN Y, ZHOU G J, MA Y, et al. Development and application of N-protein blocking ELISA for detecting porcine epidemic diarrhea virus antibodies[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4):1173-1181. (in Chinese)
[10]	辛树阳, 严世杰, 宋涛, 等. 河北省PEDV流行毒株的分离鉴定及遗传演化分析[J]. 中国兽医学报, 2020, 40(7):1265-1271.XIN S Y, YAN S J, SONG T, et al. Isolation and identification and genetic evolution analysis of PEDV epidemic strains in Hebei province[J]. Chinese Journal of Veterinary Science, 2020, 40(7):1265-1271. (in Chinese)
[11]	朱海侠. 猪流行性腹泻病毒JS2017株的分离鉴定及S基因序列分析[J]. 中国畜牧兽医, 2019, 46(2):582-589.ZHU H X. Isolation and identification of porcine epidemic diarrhea virus strain JS2017 and its S gene sequence analysis[J]. China Animal Husbandry & Veterinary Medicine, 2019, 46(2):582-589. (in Chinese)
[12]	张辛铭. PEDV毒株的分离鉴定及抗体ELISA检测方法的建立[D]. 北京:中国农业科学院, 2020.ZHANG X M. Isolation and identification of PEDV strain with establishment of antibody ELISA detection methods[D]. Beijing:Chinese Academy of Agricultural Sciences, 2020. (in Chinese)
[13]	DORTMANS J C F M, LI W, VAN DER WOLF P J, et al. Porcine epidemic diarrhea virus (PEDV) introduction into a naive Dutch pig population in 2014[J]. Vet Microbiol, 2018, 221:13-18.
[14]	狄亚心, 谭飞, 秦松跃, 等. 猪流行性腹泻病毒RT-LAMP快速检测方法的建立及应用[J]. 中国预防兽医学报, 2021, 43(8):832-837.DI Y X, TAN F, QIN S Y, et al. Establishment and application of RT-LAMP rapid detection assay for porcine epidemic diarrhea virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(8):832-837. (in Chinese)
[15]	李飞, 曾喻兵, 江朝源, 等. 猪流行性腹泻病毒SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立与应用[J]. 中国兽医科学, 2021, 51(11):1355-1360.LI F, ZENG Y B, JIANG C Y, et al. Establishment and application of SYBR Green Ⅰ fluorescence quantitative RT-PCR method for detection of porcine epidemic diarrhea virus[J]. Chinese Veterinary Science, 2021, 51(11):1355-1360. (in Chinese)
[16]	陆文俊, 施开创, 屈素洁, et al. 猪脑心肌炎病毒抗体间接ELISA检测方法的建立及应用[J]. 动物医学进展, 2011, 32(12):6-19.LU W J, SHI K C, QU S J, et al. Development and application of an indirect ELISA for detection of antibody against porcine encephalomyocarditis virus[J]. Progr Vety Med, 2011, 32(12):6-19.(in Chinese)
[17]	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.
[18]	WANG X M, NIU B B, YAN H, et al. Genetic properties of endemic Chinese porcine epidemic diarrhea virus strains isolated since 2010[J]. Arch Virol, 2013, 158(12):2487-2494.
[19]	HSU W T, CHANG C Y, TSAI C H, et al. PEDV Infection generates conformation-specific antibodies that can be effectively detected by a cell-based ELISA[J]. Viruses, 2021, 13(2):303.
[20]	PINHEIRO A C A S, VOLPE E, PRINCIPI D, et al. Development of a multiplex RT-PCR assay for simultaneous detection of the major viruses that affect rainbow trout (Oncorhynchus mykiss)[J]. Aquac Int, 2016, 24(1):115-125.
[21]	刘政龙, 王金良, 沈志强. 猪流行性腹泻病毒研究进展[J]. 中国畜牧兽医, 2015, 42(9):2506-2511.LIU Z L, WANG J L, SHEN Z Q. Research progress on porcine epidemic diarrhea virus[J]. Chinese Animal Husbandry & Veterinary Medicine, 2015, 42(9):2506-2511. (in Chinese)
[22]	HAIN K S, JOSHI L R, OKDA F, et al. Immunogenicity of a recombinant parapoxvirus expressing the spike protein of porcine epidemic diarrhea virus[J]. J Gen Virol, 2016, 97(10):2719-2731.
[23]	YANG W H, MARTH J D. Protein glycosylation energizes T cells[J]. Nat Immunol, 2016, 17(6):613-614.
[24]	TIAN P F, JIN Y L, XING G, et al. Evidence of recombinant strains of porcine epidemic diarrhea virus, United States, 2013[J]. Emerg Infect Dis, 2014, 20(10):1735-1738.
[25]	董建国, 王瑞, 曲哲会, 等. 2014-2015年豫南地区猪流行性腹泻病毒S基因变异分析[J]. 畜牧兽医学报, 2018, 49(4):859-864.DONG J G, WANG R, QU Z H, et al. Genetic variations of S gene of porcine epidemic diarrhea virus in Sourthern Henan province from 2014 to 2015[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(4):859-864. (in Chinese)

Establishment of TaqMan Detection Method of Porcine Epidemic Diarrhea Virus and Analysis of Genetic Variation based on S Gene

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