Establishment and application of multiplex RT-PCR methods for akabane virus, bovine herpesvirus 1 and bovine viral diarrhea virus

ZHANG Fanfan; WEI Jiazhen; LI Jiemao; TAN Meifang; HU Lizhen; ZENG Yanbing; GONG Xiaoqi; YANG Qun; XU Jun

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ZHANG F F, WEI J Z, LI J M, et al. Establishment and application of multiplex RT-PCR methods for akabane virus, bovine herpesvirus 1 and bovine viral diarrhea virus [J]. Fujian Journal of Agricultural Sciences，2024，39（X）：1−6

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ZHANG F F, WEI J Z, LI J M, et al. Establishment and application of multiplex RT-PCR methods for akabane virus, bovine herpesvirus 1 and bovine viral diarrhea virus [J]. Fujian Journal of Agricultural Sciences，2024，39（X）：1−6

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Establishment and application of multiplex RT-PCR methods for akabane virus, bovine herpesvirus 1 and bovine viral diarrhea virus

ZHANG Fanfan^{1, 2
,},
WEI Jiazhen^3
,,
LI Jiemao⁴,
TAN Meifang^{1, 2},
HU Lizhen^{1, 2},
ZENG Yanbing^{1, 2},
GONG Xiaoqi⁵,
YANG Qun^{1, 2
,
,},
XU Jun^{1, 2
,
,}

1.
Institute of Animal Husbandry and Veterinary Medicine, Jiangxi Academy of Agricultural Sciences, Nanchang, Jiangxi　330200, China
2.
Jiangxi Provincial Key Laboratory of Green and Healthy Breeding of Livestock and Poultry, Nanchang, Jiangxi　330200, China
3.
Jiangxi Biological Vocational College, Nanchang, Jiangxi　330200, China
4.
School of Life Sceinces, Jinggangshan University, Ji, an, Jiangxi　343009, China
5.
Comprehensive Administrative Law Enforcement Team of Xiangtang Township, Nanchang, Jiangxi　330201, China

Received Date: 2024-05-17
Rev Recd Date: 2024-10-01

Available Online: 2024-11-12

Abstract

Abstract

Objective To establish a multiplex RT-PCR method for the rapid and simultaneous detection of akabane virus （AKAV）, Bovine herpesvirus 1 （BoHV-1） and bovine viral diarrhea virus （BVDV）, so as to lay a foundation for the differential diagnosis, prevention and control of bovine reproductive disorder epidemics. Method Three pairs of primers were designed for the conservative regions of AKAV S gene, BoHV-1 gH gene and BVDV 3'UTR gene, and a multiplex RT-PCR assay for AKAV, BoHV-1 and BVDV was established through the optimal of reaction condition parameters. Result The specificity results showed that the method was positive only for AKAV, BoHV-1 and BVDV, and negative for the nucleic acids of bovine parvovirus （BPV）, bovine respiratory syncytial virus （BRSV）, bovine parainfluenza virus 3 （BPIV3）, foot-and-mouth disease virus （FMDV）, and bovine ephemeral fever virus （BEFV）. The sensitivity results showed that the lower threshold of this method for AKAV, BoHV-1 and BVDV recombinant plasmids were all 1.0×10³ copies/μL. The results of the reproducibility test showed good intra- and inter-batch reproducibility. The established method was used to determine 143 whole blood/tissue samples of cattle with reproductive disorders and compared with the existing local standards to verify the efficacy of this test for practical clinical application. The results showed that the positivity rates of AKAV, BoHV-1 and BVDV were 2.80% （4/143）, 21.68% （31/143） and 38.46% （55/143）, respectively, with the presence of several mixed infections; and the conformity rates of this method with the local standards for AKAV, BoHV-1 and BVDV were 99.3%, 98.6% and 97.2%, respectively. The Kappa coefficients of the two were 0.885, 0.960 and 0.942 respectively, indicating that the method established in this study was in good agreement with the local standards. Conclusion In this study, we pioneered a multiplex RT-PCR assay with high specificity, sensitivity and low cost for the differential detection of AKAV, BoHV-1 and BVDV.
- arkaban virus,
- Bovine herpesvirus 1,
- bovine viral diarrhea virus,
- multiplex RT-PCR,
- reproductive disorders

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References(22)

References

[1]	ASMARE K, SIBHAT B, AYELET G, et al. Serological evidence of Bovine herpesvirus-1, Bovine Viral Diarrhea virus and Schmallenberg virus infections in relation to reproductive disorders in dairy cattle in Ethiopia [J]. Acta Tropica, 2018, 178: 236−241.
[2]	OGAWA Y, FUKUTOMI T, SUGIURA K, et al. Comparison of Akabane virus isolated from sentinel cattle in Japan [J]. Veterinary Microbiology, 2007, 124(1/2): 16−24.
[3]	朱杰, 杨才俊, 祁明普, 等. 我国牛群中牛病毒性腹泻病毒流行的Meta分析 [J]. 华中农业大学学报, 2023, 42(2):48−62. ZHU J, YANG C J, QI M P, et al. Meta analysis of prevalence of bovine viral diarrhea virus(BVDV)in cattle in China [J]. Journal of Huazhong Agricultural University, 2023, 42(2): 48−62. (in Chinese)
[4]	NANDI S, KUMAR M, MANOHAR M, et al. Bovine herpes virus infections in cattle [J]. Animal Health Research Reviews, 2009, 10(1): 85−98. doi: 10.1017/S1466252309990028
[5]	MAHMOUD H, ALI A. Epidemiological investigation on Bovine alphaherpesvirus 1 and bovine viral diarrhea virus in cattle and camels in southern Egypt[J]. Veterinaria Italiana, 2022, 58(4). doi: 10.12834/VetIt.2361.14459.2.
[6]	KUMAR N, CHANDER Y, RIYESH T, et al. Isolation and characterization of bovine herpes virus 5 (BoHV5) from cattle in India [J]. PLoS One, 2020, 15(4): e0232093. doi: 10.1371/journal.pone.0232093
[7]	YANG G H, ZOU Y J, YANG R J, et al. A bovine viral diarrhea virus type 1c strain in China: Isolation, identification, and assessment of pathogenicity in rabbits [J]. Current Microbiology, 2022, 79(12): 356. doi: 10.1007/s00284-022-03069-z
[8]	WANG J, YIN J H, WANG S H, et al. Development and application of an indirect ELISA for the serological detection of bovine viral diarrhea virus infection based on the protein E2 antigen [J]. Molecular Biology Reports, 2023, 50(5): 4707−4713. doi: 10.1007/s11033-022-08226-y
[9]	BERTOLOTTI L, MURATORE E, NOGAROL C, et al. Development and validation of an indirect ELISA as a confirmatory test for surveillance of infectious bovine rhinotracheitis in vaccinated herds [J]. BMC Veterinary Research, 2015, 11: 300. doi: 10.1186/s12917-015-0612-5
[10]	DONG S J, FENG M, YU R S, et al. Establishment and application of visual LAMP detection method of infectious bovine rhinotracheitis virus [J]. Chinese Journal of Biotechnology, 2018, 34(10): 1587−1595.
[11]	KAMATA H, INAI K, MAEDA K, et al. Encephalomyelitis of cattle caused by akabane virus in southern Japan in 2006 [J]. Journal of Comparative Pathology, 2009, 140(2/3): 187−193.
[12]	BROCK J, LANGE M, GUELBENZU-GONZALO M, et al. Epidemiology of age-dependent prevalence of Bovine Herpes Virus Type 1 (BoHV-1) in dairy herds with and without vaccination [J]. Veterinary Research, 2020, 51(1): 124. doi: 10.1186/s13567-020-00842-5
[13]	卞志标, 郭怡德, 席振军, 等. 非洲猪瘟病毒防污染双重荧光定量PCR检测方法的建立 [J]. 动物医学进展, 2023, 44(1):19−26. doi: 10.3969/j.issn.1007-5038.2023.01.004 BIAN Z B, GUO Y D, XI Z J, et al. Establishment of ASFV double fluorescent quantitative PCR assay for pollution prevention [J]. Progress in Veterinary Medicine, 2023, 44(1): 19−26. (in Chinese) doi: 10.3969/j.issn.1007-5038.2023.01.004
[14]	白泉阳, 徐磊, 傅光华, 等. 检测猪源牛病毒性腹泻病毒荧光RT-PCR方法的建立与监测分析 [J]. 福建农业学报, 2017, 32(8):828−832. BAI Q Y, XU L, FU G H, et al. Establishment and monitoring analysis of fluorescence RT-PCR for detection of bovine viral diarrhea virus in swine [J]. Fujian Journal of Agricultural Sciences, 2017, 32(8): 828−832. (in Chinese)
[15]	孙映雪, 宋建德, 郑雪光, 等. 2020—2022年全球牛结节性皮肤病流行状况 [J]. 中国动物检疫, 2023, 40(5):1−4. doi: 10.3969/j.issn.1005-944X.2023.05.001 SUN Y X, SONG J D, ZHENG X G, et al. Global prevalence of lumpy skin disease from 2020 to 2022 [J]. China Animal Health Inspection, 2023, 40(5): 1−4. (in Chinese) doi: 10.3969/j.issn.1005-944X.2023.05.001
[16]	HOU P L, ZHAO G M, WANG H M, et al. Prevalence of bovine viral diarrhea virus in dairy cattle herds in Eastern China [J]. Tropical Animal Health and Production, 2019, 51(4): 791−798. doi: 10.1007/s11250-018-1751-z
[17]	ZHANG F F, LI H Q, LIN C, et al. Detection and genetic diversity of subgroup K avian leukosis virus in local chicken breeds in Jiangxi from 2021 to 2023 [J]. Frontiers in Microbiology, 2024, 15: 1341201. doi: 10.3389/fmicb.2024.1341201
[18]	ZHANG F F, XIE Q, YANG Q, et al. Prevalence and phylogenetic analysis of Gyrovirus galga 1 in Southern China from 2020 to 2022 [J]. Poultry Science, 2024, 103(3): 103397.
[19]	HOU P L, XU Y R, WANG H M, et al. Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay [J]. BMC Veterinary Research, 2020, 16(1): 114. doi: 10.1186/s12917-020-02330-6
[20]	HWANG S, LEE W, LEE Y. Development of a nucleic acid detection method based on the CRISPR-Cas13 for point-of-care testing of bovine viral diarrhea virus-1b [J]. Journal of Animal Science and Technology, 2024, 66(4): 781−791. doi: 10.5187/jast.2023.e77
[21]	鲍显伟, 李小龙, 石亚楠, 等. 牛传染性鼻气管炎病毒和牛病毒性腹泻病毒双重PCR检测方法的建立与应用 [J]. 中国动物检疫, 2023, 40(1):115−120. doi: 10.3969/j.issn.1005-944X.2023.01.021 BAO X W, LI X L, SHI Y N, et al. Establishment and application of a dual PCR assay for detection of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus [J]. China Animal Health Inspection, 2023, 40(1): 115−120. (in Chinese) doi: 10.3969/j.issn.1005-944X.2023.01.021
[22]	MENG W X, CHEN Z H, JIANG Q F, et al. A multiplex real-time fluorescence-based quantitative PCR assay for calf diarrhea viruses [J]. Frontiers in Microbiology, 2024, 14: 1327291. doi: 10.3389/fmicb.2023.1327291