牛病毒性腹泻病毒LAMP-LFD检测方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2023.11.028

Abstract

Abstract: In order to establish a specific, sensitive, rapid and simple detection method for bovine viral diarrhea virus (BVDV), four loop-mediated isothermal amplification (LAMP) specific primers and one DNA probe labeled with fluorescein isothiocyanate were designed according to the conserved segment of BVDV 5'UTR sequence by combining loop-mediated isothermal amplification (LAMP) with lateral flow dipstick(LFD). By optimizing the reaction temperature, reaction time, ratio of internal and external primer concentrations, Bst DNA 2.0 polymerase concentration, dNTP Mix concentration, Mg²⁺ concentration, etc., the specificity and detection sensitivity of this method were analyzed, and 52 clinical samples were tested. A LAMP-LFD method for detecting BVDV was established. LAMP-LFD detection technology can specifically detect BVDV, but foot-and-mouth disease virus, bovine epidemic fever virus, and bovine herpesvirus type 1 have not been detected. The sensitivity of LAMP-LFD is high, and the minimum detection concentration is 1.9×10¹ copies·μL^-1. Both LAMP-LFD and PCR detection methods detected 5 positive samples, with a coincidence rate of 100%. The LAMP-LFD method for rapid detection of BVDV was successfully established in this experiment. The detection method has the advantages of short detection time, strong specificity, simple operation and high sensitivity, and can be used as a new detection method for grassroots detection.

Key words: infectious disease, BVDV, loop-mediated isothermal amplification, transverse flow test strip

CLC Number:

S852.659.6

ZHENG Ruwen, HUANG Tao, WU Daoyi, YU Guangmei, LI Fangfang, SUI Xin, MIN Tingyu. Establishment and Preliminary Application of LAMP-LFD Detection Method for Bovine Viral Diarrhea Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4745-4753.

References 24

[1]	NELSON D D, DUPRAU J L, WOLFF P L, et al. Persistent bovine viral diarrhea virus infection in domestic and wild small ruminants and camelids including the mountain goat (Oreamnos americanus)[J]. Front Microbiol, 2016, 6:1415.
[2]	CHI S S, CHEN S, JIA W J, et al. Non-structural proteins of bovine viral diarrhea virus[J]. Virus Genes, 2022, 58(6):491-500.
[3]	DEREGT D, LOEWEN K G. Bovine viral diarrhea virus:biotypes and disease[J]. Can Vet J, 1995, 36:371-378.
[4]	LANYON S R, HILL F I, REICHEL M P, et al. Bovine viral diarrhoea:pathogenesis and diagnosis[J]. Vet J, 2014, 199(2):201-209.
[5]	BRODERSEN B W. Immunohistochemistry used as a screening method for persistent bovine viral diarrhea virus infection[J]. Vet Clin North Am Food Anim Pract, 2004, 20(1):85-93.
[6]	NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000, 28(12):E63.
[7]	VELDERS A H, SCHOEN C, SAGGIOMO V. Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection[J]. BMC Res Notes, 2018, 11:93.
[8]	DOMESLE K J, YANG Q R, HAMMACK T S, et al.. Validation of a Salmonella loop-mediated isothermal amplification assay in animal food[J]. Int J Food Microbiol, 2018, 264:63-76.
[9]	李家伟, 郭利, 杨勇, 等. 牛病毒性腹泻病毒LAMP检测方法的建立与应用[J]. 中国畜牧兽医, 2015, 42(12):3111-3118.LI J W, GUO L, YANG Y, et al. Establishment and application of LAMP detection method of bovine viral diarrhea virus[J]. China Animal Husbandry & Veterinary Medicine, 2015, 42(12):3111-3118. (in Chinese)
[10]	KIATPATHOMCHAI W, JAROENRAM W, ARUNRUT N, et al. Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick[J]. J Virol Methods, 2008, 153(2):214-217.
[11]	LETELLIER C, KERKHOFS P, WELLEMANS G, et al. Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 5' untranslated region[J]. Vet Microbiol, 1999, 64(2-3):155-167.
[12]	范晴, 谢芝勋, 刘加波, 等. 牛病毒性腹泻病毒实时荧光定量RT-PCR检测方法的建立[J]. 动物医学进展, 2010, 31(10):10-14.FAN Q, XIE Z X, LIU J B, et al. Establishment of real-time fluorescent quantitative PCR for detection of bovine viral diarrhea virus[J]. Progress in Veterinary Medicine, 2010, 31(10):10-14. (in Chinese)
[13]	孙吉, 岳华, 汤承. 检测牛5种腹泻病毒的多重RT-PCR方法的建立及应用[J]. 畜牧兽医学报, 2022, 53(1): 209-218.SUN J, YUE H, TANG C. 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. (in Chinese)
[14]	张晓梅, 姜晓霞, 孙晓波, 等. 牛病毒性呼吸道疾病主要病原多重PCR检测方法的建立与应用[J]. 中国兽医科学, 2022, 52(8):957-963.ZHANG X M, JIANG X X, SUN X B, et al. Development and application of a multiplex PCR method for major viruses causing bovine viral respiratory disease[J]. Chinese Veterinary Science, 2022, 52(8):957-963. (in Chinese)
[15]	张国华, 吕思文, 金振华, 等. 牛病毒性腹泻的流行特点、临床症状和防控[J]. 现代畜牧科技, 2021(7):80, 82.ZHANG G H, LV S W, JIN Z H, et al. Epidemic characteristics, clinical symptoms, prevention and control of bovine viral diarrhea[J]. Modern Animal Husbandry Science & Technology, 2021(7):80, 82. (in Chinese)
[16]	蒋颖. BVDV基因1型和2型RT-PCR鉴别诊断方法的建立和抗BVDV单克隆抗体的制备[D]. 扬州:扬州大学, 2007.JIANG Y. RT-PCR-based differential diagnosis for BVDV 1 and 2 and the development of a hybridoma cell strain secreting monoclonal antibody against BVDV[D]. Yangzhou: Yangzhou University, 2007. (in Chinese)
[17]	JENSEN J, AIKEN J, SCHULTZ R D. Detection of bovine viral diarrhea virus genome in leukocytes from persistently infected cattle by RNA-cDNA hybridization[J]. Can J Vet Res, 1990, 54(2):256-259.
[18]	BOCK R E, BURGESS G W, DOUGLAS I C. Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus[J]. Aust Vet J, 1986, 63(12):406-408.
[19]	KATZ J B, HANSON S K. Competitive and blocking enzyme-linked immunoassay for detection of fetal bovine serum antibodies to bovine viral diarrhea virus[J]. J Virol Methods, 1987, 15(3):167-175.
[20]	CHEN L, FAN X Z, WANG Q, et al. A novel RT-LAMP assay for rapid and simple detection of classical swine fever virus[J]. Virol Sin, 2010, 25(1):59-64.
[21]	柴方超, 周前进, 陈炯. 环介导等温扩增联合横向流动试纸条可视化检测迟缓爱德华菌[J]. 中国兽医学报, 2017, 37(6):1103-1110.CHAI F C, ZHOU Q J, CHEN J. Visual detection of Edwardsiella tarda based on loop-mediated isothermal amplification combined with a lateral flow dipstick[J]. Chinese Journal of Veterinary Science, 2017, 37(6):1103-1110. (in Chinese)
[22]	柴书军, 郭军庆, 刘运超, 等. 猪细小病毒7型LAMP-LFD快速检测方法的建立及应用[J]. 畜牧与兽医, 2022, 54(9):71-77.CHAI S J, GUO J Q, LIU Y C, et al. Establishment and application of an LAMP-LFD rapid detection method for porcine parvovirus type 7[J]. Animal Husbandry & Veterinary Medicine, 2022, 54(9):71-77. (in Chinese)
[23]	杨倩, 李召仁, 鲜思美, 等. 羊口疮病毒LAMP-LFD快速检测方法的建立与初步应用[J]. 中国兽医学报, 2022, 42(3):507-513.YANG Q, LI Z R, XIAN S M, et al. Rapid and sensitive diagnosis of Orf virus by loop-mediated isothermal amplification combined with a lateral flow dipstick[J]. Chinese Journal of Veterinary Science, 2022, 42(3):507-513. (in Chinese)
[24]	孙盼盼, 时建立, 彭喆, 等. 猪支原体肺炎LAMP-LFD快速检测方法的建立及初步应用[J]. 畜牧兽医学报, 2020, 51(6): 1419-1428.SUN P P, SHI J L, PENG Z, et al. Rapid and sensitive diagnosis of Mycoplasma hyopneumoniae by loop-mediated isothermal amplification combined with a lateral flow dipstick[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1419-1428.

Establishment and Preliminary Application of LAMP-LFD Detection Method for Bovine Viral Diarrhea Virus

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