检测牛5种腹泻病毒的多重RT-PCR方法的建立及应用

doi:10.11843/j.issn.0366-6964.2022.01.021

Abstract

Abstract: Bovine group A rotavirus (BRVA), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine norovirus (BNoV) and bovine nebovirus (BNeV) are common viruses that cause calf diarrhea, and there are mixed infections in clinic. The purpose of this experiment was to establish a multiplex PCR method that can simultaneously detect the above five viruses. The multiplex PCR method for simultaneous detection of BRVA, BCoV, BVDV, BNoV and BNeV was successfully established by designing primers, optimizing reaction system and conditions. This method only specifically detected the target virus, but did not amplify the diarrhea pathogens such as bovine astrovirus, bovine kobuvirus, bovine parvovirus, enterotoxigenic Escherichia coli, Salmonella and Campylobacter jejuni, which had good specificity and stability. The lowest detection limits for each virus were as follows:BRVA 1.63×10⁵copies·μL^-1, BCoV 2.0×10⁶copies·μL^-1, BVDV 1.9×10⁵copies·μL^-1, BNoV 1.96×10⁵copies·μL^-1 and BNeV 2.0×10⁵copies·μL^-1. The comparison experiment showed that the coincidence rate between the multiplex RT-PCR method and the RT-PCR method for detecting single virus was 100%. The detection results of 220 yak diarrhea samples collected from northwest Sichuan grassland from 2019 to 2020 showed that the detection rates of BRVA, BNoV and BNeV were 40.5%, 5.9% and 4.5%, respectively, while BVDV and BCoV were not detected. The multiplex RT-PCR method established in this study can simultaneously detect five common diarrhea viruses in fecal samples of calf diarrhea, providing technical support for the rapid diagnosis of bovine diarrhea.

Key words: bovine group A rotavirus, bovine coronavirus, bovine viral diarrhea virus, Norvirus, nebovirus, multiple RT-PCR

CLC Number:

S855.3

SUN Ji, YUE Hua, TANG Cheng. Establishment and Application of Multiplex RT-PCR for Detection of Five Bovine Diarrhea Viruses[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 209-218.

References 41

[1]	CHO Y L, YOON K J. An overview of calf diarrhea-infectious etiology, diagnosis, and intervention[J]. J Vet Sci, 2014, 15(1):1-17.
[2]	MANUJA B K, PRASAD M, MANUJA A, et al. A novel genomic constellation (G10P
[3]	) of group A rotavirus detected from buffalo calves in northern India[J]. Virus Res, 2008, 138(1-2):36-42.
	BOILEAU M J, KAPIL S. Bovine coronavirus associated syndromes[J]. Vet Clin North Am Food Anim Pract, 2010, 26(1):123-146.
[4]	RIDPATH J F, BOLIN S R, DUBOVI E J. Segregation of bovine viral diarrhea virus into genotypes[J]. Virology, 1994, 205(1):66-74.
[5]	GUO Z J, HE Q F, YUE H, et al. First detection of Nebovirus and Norovirus from cattle in China[J]. Arch Virol, 2018, 163(2):475-478.
[6]	GOMEZ D E, ARROYO L G, POLJAK Z, et al. Detection of bovine coronavirus in healthy and diarrheic dairy calves[J]. J Vet Intern Med, 2017, 31(6):1884-1891.
[7]	SMILEY J R, CHANG K O, HAYES J, et al. Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus[J]. J Virol, 2002, 76(20):10089-10098.
[8]	SEID U, DAWO F, TESFAYE A, et al. Isolation and characterization of coronavirus and rotavirus associated with calves in central part of Oromia, Ethiopia[J/OL]. Vet Med Int, 2020, doi:10. 1155/2020/8869970.
[9]	CASTELLS M, CAFFARENA R D, CASAUX M L, et al. Phylogenetic analyses of rotavirus A from cattle in Uruguay reveal the circulation of common and uncommon genotypes and suggest interspecies transmission[J]. Pathogens, 2020, 9(7):570.
[10]	GOMEZ D E, WEESE J S. Viral enteritis in calves[J]. Can Vet J, 2017, 58(12):1267-1274.
[11]	KOMOTO S, TACHAROENMUANG R, GUNTAPONG R, et al. Reassortment of human and animal rotavirus gene segments in emerging DS-1-Like G1P
[8]	rotavirus strains[J]. PLoS One, 2016, 11(2):e0148416.
[12]	ZHU W, DONG J B, HAGA T, et al. Rapid and sensitive detection of bovine coronavirus and group a bovine rotavirus from fecal samples by using one-step duplex RT-PCR assay[J]. J Vet Med Sci, 2011, 73(4):531-534.
[13]	CLARK M A. Bovine coronavirus[J]. Br Vet J, 1993, 149(1):51-70.
[14]	姚火春,杜念兴,徐为燕,等.牛冠状病毒的血清流行病学调查[J].南京农业大学学报, 1990, 13(2):117-121.YAO H C, DU N X, XU W Y, et al. Seroepizootiological survey of bovine coronavirus in cattle, human and other animals[J]. Journal of Nanjing Agricultural University, 1990, 13(2):117-121.(in Chinese)
[15]	COLLETT M S, LARSON R, BELZER S K, et al. Proteins encoded by bovine viral diarrhea virus:The genomic organization of a pestivirus[J]. Virology, 1988, 165(1):200-208.
[16]	BAKER J C. The clinical manifestations of bovine viral diarrhea infection[J]. Vet Clin North Am Food Anim Pract, 1995, 11(3):425-445.
[17]	GAO J F, LIU M Y, MENG X R, et al. Seroprevalence of bovine viral diarrhea infection in yaks (Bos grunniens) on the Qinghai-Tibetan Plateau of China[J]. Trop Anim Health Prod, 2013, 45(3):791-793.
[18]	DENG M L, JI S K, FEI W T, et al. Prevalence study and genetic typing of bovine viral diarrhea virus (BVDV) in four bovine species in China[J]. PLoS One, 2015, 10(4):e0121718.
[19]	TURAN T, IŞIDAN H, ATASOY M O, et al. Detection and molecular analysis of bovine enteric norovirus and nebovirus in Turkey[J]. J Vet Res, 2018, 62(2):129-135.
[20]	DI FELICE E, MAUROY A, POZZO F D, et al. Bovine noroviruses:A missing component of calf diarrhoea diagnosis[J]. Vet J, 2016, 207:53-62.
[21]	MAUROY A, SCIPIONI A, MATHIJS E, et al. Epidemiological study of bovine norovirus infection by RT-PCR and a VLP-based antibody ELISA[J]. Vet Microbiol, 2009, 137(3-4):243-251.
[22]	CANDIDO M, ALENCAR A L F, ALMEIDA-QUEIROZ S R, et al. First detection and molecular characterization of Nebovirus in Brazil[J]. Epidemiol Infect, 2016, 144(9):1876-1878.
[23]	OLIVER S L, ASOBAYIRE E, DASTJERDI A M, et al. Genomic characterization of the unclassified bovine enteric virus Newbury agent-1(Newbury1) endorses a new genus in the family Caliciviridae[J]. Virology, 2006, 350(1):240-250.
[24]	郭紫晶,何琪富,岳华,等.引起犊牛腹泻的Nebovirus[J].畜牧兽医学报, 2020, 51(3):433-442.GUO Z J, HE Q F, YUE H, et al. Nebovirus:a diarrhea-causing virus in calves[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3):433-442.(in Chinese)
[25]	张亮,张云飞,蒋仁新,等. 2017-2018年山东省奶牛场犊牛腹泻相关病毒病原学检测[J].中国动物检疫, 2020, 37(6):16-20.ZHANG L, ZHANG Y F, JIANG R X, et al. Pathogenic detection of viruses related to calf diarrhea in dairy farms in Shandong province during 2017 to 2018[J]. China Animal Health Inspection, 2020, 37(6):16-20.(in Chinese)
[26]	傅彩霞,靳兴军,郑瑞峰,等.北京地区规模化奶牛场三种病毒性腹泻病的血清学调查[J].动物医学进展, 2012, 33(5):85-88.FU C X, JIN X J, ZHENG R F, et al. Serological survey of three viral diarrheal diseases in Beijing large-scale dairy cow farms[J]. Progress in Veterinary Medicine, 2012, 33(5):85-88.(in Chinese)
[27]	王玥琳,郭紫晶,岳华,等.部分地区奶牛腹泻粪便样中诺如病毒的检测和演化分析[J].畜牧兽医学报, 2019, 50(5):1048-1055.WANG Y L, GUO Z J, YUE H, et al. Detection and evolutionary analysis of bovine Noroviruses in diarrheic fecals of dairy calves in some areas[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(5):1048-1055.(in Chinese)
[28]	郭紫晶,何琪富,汤承,等.检测纽布病毒的Real-time RT-PCR方法的建立与应用[J].畜牧兽医学报, 2019, 50(4):893-900.GUO Z J, HE Q F, TANG C, et al. Establishment and application of a real-time RT-PCR assay for detecting bovine Nebovirus[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(4):893-900.(in Chinese)
[29]	MACKAY I M. Real-time PCR in the microbiology laboratory[J]. Clin Microbiol Infect, 2004, 10(3):190-212.
[30]	BASERA S S, SINGH R, VAID N, et al. Detection of rotavirus infection in bovine calves by RNA-PAGE and RT-PCR[J]. Indian J Virol, 2010, 21(2):144-147.
[31]	MONTEIRO F L, CARGNELUTTI J F, MARTINS B, et al. Detection of bovine pestiviruses in sera of beef calves by a RT-PCR based on a newly designed set of pan-bovine pestivirus primers[J]. J Vet Diagn Invest, 2019, 31(2):255-258.
[32]	师志海,王文佳,兰亚莉,等.牛诺如病毒实时荧光定量PCR检测方法的建立及应用[J].畜牧兽医学报, 2020, 51(7):1728-1736.SHI Z H, WANG W J, LAN Y L, et al. Establishment and application of a real-time PCR assay for detecting bovine Norovirus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7):1728-1736.(in Chinese)
[33]	王文佳,师志海,兰亚莉,等.牛诺如病毒和冠状病毒双重PCR检测方法的建立及初步应用[J].动物医学进展, 2019, 40(10):6-9.WANG W J, SHI Z H, LAN Y L, et al. Establishment and preliminary application of a duplex PCR for detection of bovine Norovirus and bovine coronavirus[J]. Progress in Veterinary Medicine, 2019, 40(10):6-9.(in Chinese)
[34]	林初文,沈志强. BVDV、BCV和BRV三重RT-PCR检测方法的建立及应用[J].畜牧与兽医, 2013, 45(7):61-64.LIN C W, SHEN Z Q. Establishment and application of triple RT-PCR detection methods for BVDV, BCV and BRV[J]. Animal Husbandry&Veterinary Medicine, 2013, 45(7):61-64.(in Chinese)
[35]	何琪富,郭紫晶,李然,等.牛冠状病毒RT-PCR检测方法的建立及应用[J].畜牧兽医学报, 2018, 49(10):2292-2298.HE Q F, GUO Z J, LI R, et al. Establishment and application of a RT-PCR assay for detecting bovine coronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(10):2292-2298.(in Chinese)
[36]	MARKOULATOS P, SIAFAKAS N, MONCANY M. Multiplex polymerase chain reaction:A practical approach[J]. J Clin Lab Anal, 2002, 16(1):47-51.
[37]	王玥琳.牛诺瓦病毒的分子检测和基因组研究[D].成都:西南民族大学, 2020.WANG Y L. Molecular detection and genomic research of bovine Norovirus[D]. Chengdu:Southwest Minzu University, 2020.(in Chinese)
[38]	周芳,岳华,张斌,等.牦牛轮状病毒VP6基因序列分析及RT-PCR检测方法的建立与应用[J].畜牧兽医学报, 2016, 47(7):1465-1473.ZHOU F, YUE H, ZHANG B, et al. Establishment and application of an RT-PCR assay for yak rotavirus based on the sequence analysis of yak rotavirus VP6 gene[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(7):1465-1473.(in Chinese)
[39]	赵霞玲,牛家强,次仁央金,等.牦牛腹泻病毒BVDV、BCV、BRV流行病学调查[J].高原农业, 2020, 4(3):298-302.ZHAO X L, NIU J Q, CIREN Y J, et al. Epidemiological survey of yak diarrhea virus BVDV, BCV and BRV[J]. Journal of Plateau Agriculture, 2020, 4(3):298-302.(in Chinese)
[40]	何美琳,张焕容,王永,等.川西北牦牛3种病毒性腹泻病血清学调查[J].中国畜牧兽医, 2014, 41(3):248-251.HE M L, ZHANG H R, WANG Y, et al. Serological survey on three viral diarrhea diseases of yaks in Northwest Sichuan province[J]. China Animal Husbandry&Veterinary Medicine, 2014, 41(3):248-251.(in Chinese)
[41]	GUO Z J, HE Q F, ZHANG B, et al. First detection of neboviruses in yak (Bos grunniens) and identification of a novel neboviruses based on complete genome[J]. Vet Microbiol, 2019, 236:108388.

Establishment and Application of Multiplex RT-PCR for Detection of Five Bovine Diarrhea Viruses

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 41

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LI Jiannan, YUAN Liming, HUA Jinlian. Progress on the Application of CD46 in Breeding of Livestock for Disease Resistance [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1866-1874.
[2]	HUANG Jin, LI Siyuan, MAO Li, CAI Xuhang, XIE Lingling, WANG Fu, ZHOU Hua, LI Jizong, LI Bin. Eukaryotic Expression of Bovine Coronavirus S1 Protein and Establishment and Application of Indirect ELISA [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2050-2060.
[3]	LIU Qiang, NIU Xiaoxia, FANG Min, LIU Yanling, GAO Hui, CHEN Jixiang, JIAHUA Cairang, ZHANG Sinong, LI Yong. Research Progress of Bovine Coronavirus Spike Protein [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 944-956.
[4]	YU Qisheng, ZHU Qing, ZHOU Qun, SONG Xin, ZHANG Jiaqi, CHEN Taoyun, XU Lin, ZHANG Chaohui, ZHANG Bin. Expression of BCoV Spike Protein by Baculovirus Expression System and Its Immunogenicity in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 640-648.
[5]	LI Siyuan, FU Xincheng, YUAN Xuesong, MAO Li, CAI Xuhang, SUN Xinru, HUANG Jin, XIE Lingling, WANG Fu, ZHOU Hua, ZHANG Qi, LI Jizong, LI Bin. Detection of Bovine Viral Diarrhea Pathogens and Evolution Analysis of Bovine Coronavirus in Langfang, Hebei [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 649-659.
[6]	HUANG Jiang, LI Chuang, CUI Yueqi, YUAN Xueying, ZHAO Zhicheng, LIU Yu, ZHOU Yulong, ZHU Zhanbo, ZHANG Zecai. Study on the Effect of Gut Microbiota Disturbance on Susceptibility to BVDV Based on a Mouse Model [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3466-3473.
[7]	LI Yicong, PU Feiyang, FENG Xili, WANG Mengzhu, ZHAO Zeyang, ZHANG Derong, MA Zhongren, ZHOU Jianhua. Research Progress on Immunological Characteristics of Bovine Viral Diarrhea Virus Protein and Vaccines [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1381-1391.
[8]	SUN Feiyan, YE Jingfei, WEI Yu, WANG Zixian, ZHANG Jinyu, BING Liyuan, MENG Tingting, WANG Shuai, ZHAO Lifeng, SUN Liang, GUO Li. Epidemiological Investigation and Analysis of Bovine Coronavirus in Beef Cattle in Jilin Province [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 673-682.
[9]	CHEN Junzhen, QUAN Ran, FU Qiang, GE Lijuan, YUAN Yuanyuan, ZHANG Chengyuan, LI Jianlin, SHI Huijun. Study on the Effect of Heat Shock Protein HSP90B1 on the Replication of Bovine Viral Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 683-693.
[10]	WANG Mengjiao, JIANG Qian, MA Xuejun, XIA Ruiyang, GUO Xueping, SUN Lei, ZHONG Qi, MA Xuelian, YAO Gang. Molecular Epidemiological Investigation of Bovine Coronavirus in Calf Diarrhea in Main Cattle Producing Areas of Xinjiang [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5125-5133.
[11]	ZHANG Jiaqi, JIA Xining, ZHOU Qun, SONG Xin, ZHU Chenxi, LI Gege, ZHANG Bin. Construction of BVDV-1 Virus-like Particles based on E2 Protein and Evaluation of Its Immune Efficacy in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5143-5153.
[12]	SHI Huijun, DONG Wenli, GUO Yanting, YUAN Yuanyuan, CHEN Junzhen, YANG Li, RAN Duoliang, FU Qiang. Correlation Analysis of Cell Signal Peptidase Complex Subunit 1 and Bovine Viral Diarrhea Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1870-1876.
[13]	CUI Yuchen, YUE Hua, TANG Cheng. Development and Application of an Insulated Isothermal RT-PCR for On-site Detecting Nebovirus [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1310-1316.
[14]	LI Yajun, RU Yi, HAO Rongzeng, JIANG Chenghui, WANG Wei, ZHANG Yue, ZHANG Guicai, LIU Huanan, LU Bingzhou, YANG Yang, TAO Shiyu, YANG Rui, SONG Xiangdong, CHEN Jiao, YU Sijiu, ZHENG Haixue. Expression of Bovine Viral Diarrhea Virus E2 Protein in CHO Cells and Immunogenicity Analysis [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4315-4324.
[15]	LI Siyi, YUE Hua, TANG Cheng. Sequences Analysis of Nebovirus VP1 and RNA-dependent RNA Polymerase in Fecal Samples from Calf with Diarrhea [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2354-2360.