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

摘要/Abstract

摘要： 本研究旨在建立针对中国流行蓝舌病病毒（bluetongue virus，BTV）的逆转录-环介导等温扩增（RT-LAMP）检测方法。根据我国分离BTV毒株Seg-5基因序列的高度保守区域，设计特异性引物，优化反应条件及反应体系，进行特异性及灵敏度验证，建立BTV RT-LAMP检测方法；对46株中国分离的12种血清型BTV（BTV-1、-2、-3、-4、-5、-7、-9、-12、-15、-16、-21与-24）代表毒株进行检测，进一步对120份BTV核酸阳性血液样品及60份2020年采集自监控动物的临床血液样品进行BTV的RT-LAMP及qRT-PCR检测，验证建立的RT-LAMP检测方法的准确性和可靠性。试验结果表明，本研究建立的RT-LAMP方法最佳反应条件为64℃，扩增45 min；反应体系中外引物：内引物：环引物的最佳浓度及比例为0.2 μmol·L^-1：0.6 μmol·L^-1：0.4 μmol·L^-1。该方法可特异性检测中国流行的12种血清型BTV核酸，与动物流行性出血病病毒（EHDV）、中山病毒（CHUV）、阿卡斑病毒（AKAV）、口蹄疫病毒（FMDV）及非洲马瘟病毒（AHSV）核酸均无交叉扩增现象；最低可检测4.5拷贝·μL^-1的BTV核酸。对46株分属12种血清型的BTV代表毒株的检测结果均为阳性；120份BTV核酸阳性血液样品的检测结果与qRT-PCR检测结果无显著差别（McNemar检验P>0.05），符合率为95.0%；60份临床血液样品的检测结果与qRT-PCR结果完全一致，以上结果表明本研究建立的RT-LAMP方法准确可靠，对中国分离的BTV毒株及临床血液样品均具有良好的检测效果。本研究建立的BTV RT-LAMP检测方法具有反应快速、结果可视化、特异性强和敏感度高等优点，为我国开展BTV检测诊断与流行病学研究提供了技术支持，具有较好的应用前景。

关键词: 蓝舌病病毒, 群特异性, RT-LAMP, 应用

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

中图分类号:

S852.659.4

李占鸿, 朱沛, 宋子昂, 李卓然, 杨振兴, 李华春, 杨恒, 廖德芳. 蓝舌病病毒群特异性RT-LAMP检测方法的建立与初步应用[J]. 畜牧兽医学报, 2021, 52(8): 2244-2253.

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.

参考文献 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.