蓝舌病病毒群特异性RT-LAMP检测方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2021.08.017

Abstract

Abstract: The purpose of this study was to establish a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of bluetongue viruses (BTVs) prevalent in China. Specific primers were designed according to the highly conserved regions of the Seg-5 gene of BTV strains isolated in China. By optimizing the reaction condition, the RT-LAMP method was established, and the specificity and sensitivity of the method were evaluated. To verify the accuracy and reliability of the RT-LAMP method, 46 BTV strains belonging to 12 sero-types (BTV-1,-2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and -24) were tested, further 120 BTV nucleic acid-positive blood samples collected from BTV-infected animals and 60 blood samples collected from sentinel animals in 2020 were tested through RT-LAMP and qRT-PCR simultaneously. The optimal reaction temperature of the RT-LAMP was 64℃, and the optimal reaction time was 45 min, the optimal concentration and ratio of the outer primer:inner primer:loop primer in the reaction mixture was 0.2 μmol· L^-1:0.6 μmol·L^-1:0.4 μmol·L^-1. This method can specifically detect the nucleic acids of BTV belonged to the 12 epidemic serotypes in China without any cross-reaction with the nucleic acids of epidemic hemorrhagic virus (EHDV), Chuzan disease virus (CHUV), Akabane disease virus (AKAV), foot-and-mouth disease virus (FMDV) and African horse sickness virus (AHSV). The lower detecting limit of the method was 4.5 copi-es·μL^-1 of BTV genomes. The detection results of 46 BTV strains belonged to 12 serotypes were positive. There was no significant difference (McNemar test P>0.05) between the detection results of RT-LAMP and qRT-PCR for 120 BTV nucleic acid positive blood samples with a coincidence rate of 95.0%. The detection results of 60 blood samples were completely consistent between RT-LAMP and qRT-PCR. These data demonstrated that the RT-LAMP method established in this study was accurate and reliable to detect the BTV strains isolated in China and the clinical blood samples collected from animals. The BTV RT-LAMP method established in this study was rapid, visual, strong specific and highly sensitive to detect samples, which provided technical means for the detection, diagnosis and epidemiological investigation of the BTVs prevailing in China.

Key words: bluetongue virus, group specificity, RT-LAMP, application

CLC Number:

S852.659.4

LI Zhanhong, ZHU Pei, SONG Ziang, LI Zhuoran, YANG Zhenxing, LI Huachun, YANG Heng, LIAO Defang. Establishment and Application of a Group Specific Reverse Transcriptase Loop-mediated Isothermal Amplification Method of Bluetongue Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2244-2253.

References 27

[1]	MACLACHLAN N J, DREW C P, DARPEL K E, et al. The pathology and pathogenesis of bluetongue[J]. J Comp Pathol, 2009, 141(1):1-16.
[2]	MACLACHLAN N J. Bluetongue:history, global epidemiology, and pathogenesis[J]. Prev Vet Med, 2011, 102(2):107-111.
[3]	SAEGERMAN C, BERKVENS D, MELLOR P S. Bluetongue epidemiology in the European Union[J]. Emerg Infect Dis, 2008, 14(4):539-544.
[4]	World Organisation for Animal Health. Terrestrial animal health code[M]. 13th ed. Paris:Office International des Epizooties, 2004.
[5]	MAAN S, MAAN N S, NOMIKOU K, et al. Novel bluetongue virus serotype from Kuwait[J]. Emerg Infect Dis, 2011, 17(5):886-889.
[6]	ZIENTARA S, SAILLEAU C, VIAROUGE C, et al. Novel bluetongue virus in goats, Corsica, France, 2014[J]. Emerg Infect Dis, 2014, 20(12):2123-2125.
[7]	李占鸿, 王金萍, 杨恒, 等. 蓝舌病病毒血清9型毒株在我国的首次分离[J]. 畜牧兽医学报, 2019, 50(2):354-363.LI Z H, WANG J P, YANG H, et al. Isolation of bluetongue virus serotype 9 strain in China for the first time[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(2):354-363.(in Chinese)
[8]	李占鸿, 肖雷, 孟锦昕, 等. 中国蓝舌病病毒血清5型毒株的分离与鉴定[J]. 中国兽医科学, 2019, 49(1):36-43.LI Z H, XIAO L, MENG J X, et al. Isolation and identification of bluetongue virus serotype 5 strain in China[J]. Chinese Veterinary Science, 2019, 49(1):36-43.(in Chinese)
[9]	杨恒, 王金萍, 李乐, 等. 我国蓝舌病病毒血清24型毒株的分离与遗传特征分析[J]. 中国兽医学报, 2019, 39(1):31-37.YANG H, WANG J P, LI L, et al. Genetic characteristics of bluetongue virus serotype 24 strains isolated in China[J]. Chinese Journal of Veterinary Science, 2019, 39(1):31-37.(in Chinese)
[10]	YANG H, LV M N, SUN M F, et al. Complete genome sequence of the first bluetongue virus serotype 7 isolate from China:evidence for entry of African-lineage strains and reassortment between the introduced and native strains[J]. Arch Virol, 2016, 161(1):223-227.
[11]	ROY P. Bluetongue virus proteins[J]. J Gen Virol, 1992, 73(Pt 12):3051-3064.
[12]	RATINIER M, CAPORALE M, GOLDER M, et al. Identification and characterization of a novel non-structural protein of Bluetongue virus[J]. PLoS Pathog, 2011, 7(12):e1002477.
[13]	BOYCE M, CELMA C C P, ROY P. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis[J]. Virol J, 2012, 9(1):178.
[14]	HWANG G Y, CHIOU J F, YANG Y Y, et al. High-sequence conservation among the United States bluetongue viruses cognate M2 genes which encode the nonstructural NS1 tubule protein[J]. Virology, 1993, 192(1):321-327.
[15]	POLCI A, CAMMÀ C, SERINI S, et al. Real-time polymerase chain reaction to detect bluetongue virus in blood samples[J]. Vet Ital, 2007, 43(1):77-88.
[16]	ARADAIB I E, SCHORE C E, CULLOR J S, et al. A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17[J]. Vet Microbiol, 1998, 59(2-3):99-108.
[17]	NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000, 28(12):E63.
[18]	NAGAMINE K, HASE T, NOTOMI T. Accelerated reaction by loop-mediated isothermal amplification using loop primers[J]. Mol Cell Probes, 2002, 16(3):223-229.
[19]	POON L L M, LEUNG C S W, CHAN K H, et al. Detection of human influenza a viruses by loop-mediated isothermal amplification[J]. J Clin Microbiol, 2005, 43(1):427-430.
[20]	WIGMANN É F, MEYER K, CENDOYA E, et al. A loop-mediated isothermal amplification (LAMP) based assay for the rapid and sensitive group-specific detection of fumonisin producing Fusarium spp[J]. Int J Food Microbiol, 2020, 325:108627.
[21]	BATH C, SCOTT M, SHARMA P M, et al. Further development of a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of foot-and-mouth disease virus and validation in the field with use of an internal positive control[J]. Transbound Emerg Dis, 2020, 67(6):2494-2506.
[22]	李占鸿, 肖雷, 杨振兴, 等. 牛源流行性出血病病毒(EHDV)血清10型毒株在我国的分离鉴定[J]. 病毒学报, 2019, 35(1):118-126.LI Z H, XIAO L, YANG Z X, et al. Isolation and identification of the epidemic hemorrhagic fever virus serotype 10 strain from cattle in China[J]. Chinese Journal of Virology, 2019, 35(1):118-126.(in Chinese)
[23]	杨恒, 肖雷, 李占鸿, 等. 2012-2016年中国南方地区帕利亚姆血清群病毒的分离与序列特征分析[J]. 畜牧兽医学报, 2018, 49(4):761-770.YANG H, XIAO L, LI Z H, et al. Sequence analysis of palyam serogroup virus isolated in south China from 2012 to 2016[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(4):761-770.(in Chinese)
[24]	杨振兴, 朱建波, 李占鸿, 等. 蓝舌病病毒和流行性出血病病毒双重荧光定量RT-PCR检测方法的建立及应用[J]. 中国兽医科学, 2019, 49(9):1104-1111.YANG Z X, ZHU J B, LI Z H, et al. Establishment and application of a duplex real-time RT-PCR assay for simultaneous detection of bluetongue virus and epidemic haemorrhagic disease virus[J]. Chinese Veterinary Science, 2019, 49(9):1104-1111.(in Chinese)
[25]	LEE F, LIN Y L, TSAI H J. Comparison of primer sets and one-step reverse transcription polymerase chain reaction kits for the detection of bluetongue viral RNA[J]. J Virol Methods, 2014, 200:6-9.
[26]	MOHANDAS S S, MUTHUCHELVAN D, PANDEY A B, et al. Development of reverse transcription loop mediated isothermal amplification assay for rapid detection of bluetongue viruses[J]. J Virol Methods, 2015, 222:103-105.
[27]	MAAN S, MAAN N S, BATRA K, et al. Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India[J]. J Virol Methods, 2016, 234:65-74.

Establishment and Application of a Group Specific Reverse Transcriptase Loop-mediated Isothermal Amplification Method of Bluetongue Virus

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 27

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	WANG Jingyu, PAN Yangyang, XU Gengquan, ZHANG Rui, ZHANG Wenlan, WANG Xiaoshan, WU Rentaodi, ZHAO Rigetu, CUI Yan, YU Sijiu. Preparation and Preliminary Application of Yak (Bos Grunniens) Fas-associated Factor 1 Polyclonal Antibody [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3369-3382.
[2]	TIAN Yanhong, YU Jiangxu, JIAO Yuzhou, GAO Dongyang, CAI Xuwang. Research Progress on Structural Modification and Its Effects of Salmonella Lipopolysaccharides [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1392-1402.
[3]	SUN Dongxiao, ZHANG Shengli, ZHANG Qin, LI Jiao, ZHANG Guixiang, LIU Chousheng, ZHENG Weijie. Application Progress on Genomic Selection Technology for Dairy Cattle in China [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4028-4039.
[4]	SHAO Changxuan, DANG Ankai, ZHAN Zhaohan, LAI Zhenheng, ZHU Yongjie, SHAN Anshan. Development and Application Strategy of Beta Sheet Antimicrobial Peptides [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2490-2501.
[5]	JIANG Lingli, GAO Youling, HU Xingjuan, ZHANG Xian, YANG Yunkai, WEI Hua, FANG Weihuan. Development and Primary Application of Colloidal Gold-based Test Strip for Rapid Detection of Listeria monocytogenes LLO [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1841-1848.
[6]	HUANG Jian, LIU Yunjia, YANG Xiaonong, LI Yan. RPA-Cas12a-LFD Based Nucleic Acid Detection for Feline Herpesvirus-1 and Preliminary Application [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1638-1643.
[7]	DING Jun, FU Zilin, HE Junhao, DUAN Xiaowei, MA Lu, BU Dengpan. Research Progress of Milk-derived Exosomes [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1019-1029.
[8]	OUYANG Qingyuan, HU Shenqiang, WANG Jiwen. Current Genomics Research and Application of Important Traits in Poultry [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 663-679.
[9]	ZOU Huiying, LI Junliang, ZHU Huabin. Progress on Research and Application of Prime Editing System [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3721-3730.
[10]	LI Yaqi, WANG Xianzhong, ZHANG Jiaojiao. Research Progress of Cell Proliferation and Apoptosis Regulated by Non-thermal Plasma and Its Application in Animal Husbandry [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 1-10.
[11]	LI Zhanhong, SONG Ziang, LIAO Defang, YANG Zhenxing, XIE Jiarui, GAO Xiang, HU Zhongyan, LI Zhuoran, LI Huachun, YANG Heng. Whole Genome Sequence Analyses of Bluetongue Virus Serotype 7 Strain Isolated from Yunnan Province [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1337-1348.
[12]	SU Kai, FAN Tingli, SONG Qinye. Ferritin Nanocarrier and Its Application Potential in the Field of Biomedicine [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4): 909-919.
[13]	LI Shuo, ZOU Tiande, WANG Zirui, YOU Jinming. The Metabolic Mechanism and Nutritional Physiological Function of α-ketoglutarate and Its Application in Livestock Production [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 923-932.
[14]	LIU Siyuan, LU Dan, TANG Zhonglin. Research Progress on CRISPR/Cas9 and Its Application in Pigs Genome Editing [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 409-416.
[15]	YI Huashan, ZHAO Yao, MA Xianping, LI Huan, XIA Yu, ZHU Wenqing, LIU Qianru, CHEN Siqian, CAO Huizhen, YAO Ting, GAO Lixu, ZHANG Jinyang, YANG Fahui, WEI Xiaorong, LI Qianyong, XIE Yuanbing. Establishment and Preliminary Application of Indirect ELISA Based on Recombinant NS4 Protein for Detection of Bluetongue Virus Antibodies [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3133-3140.