二重荧光RT-LAMP方法鉴别检测口蹄疫病毒和水泡性口炎病毒

doi:10.11843/j.issn.0366-6964.2018.05.014

摘要/Abstract

摘要：

口蹄疫和水泡性口炎是牛常见高度急性病毒传染病，在全球范围内广泛存在。本研究旨在建立一种可同时鉴别口蹄疫病毒和水泡性口炎病毒的二重荧光RT-LAMP检测方法。根据口蹄疫病毒（FMDV）3D基因和水泡性口炎病毒（VSV）N基因的保守序列，设计了2套特异性引物，在每条内引物的5'端标记荧光基团，通过扩增产物颜色判断检测结果。优化反应条件，建立了可同时检测口蹄疫病毒和水泡性口炎病毒的二重荧光RT-LAMP方法。结果显示，该方法灵敏性高，每个反应最低能够检测到100个拷贝混合模板；特异性好，能在同一个反应管里检测到两种病毒，对其他牛病原体无扩增；干扰性小，扩增效率不受模板浓度影响。本研究建立的口蹄疫病毒和水泡性口炎病毒二重荧光RT-LAMP方法具有简便、快速、特异、敏感等优点，可用于FMDV和VSV的临床检测和流行病学调查。

Abstract:

Foot-and-mouth disease and vesicular stomatitis are serious cute disease common in cattle, and prevalence in large parts of the word. The aim of this study was to establish a duplex fluorescence-based reverse transcript loop-mediated isothermal amplification assay for detection of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV). Two sets of specific primers were designed according to the conserved sequences of FMDV 3D gene and VSV N gene, and the inner primers were synthesized with two different fluorophores at its 5'ends. A duplex fluorescence-based reverse transcript loop-mediated isothermal amplification assay was developed and used for detection of FMDV and VSV. The results showed that this assay could detect at least 100 copies of premixed plasmid containing the targets gene of FMDV and VSV per tube. The specificity of this assay was high, and be able to detect FMDV and VSV without any cross-reactions with other known non-targeted bovine pathogens. Different concentration template of FMDV and VSV could be identified when mixed together without any interference. These findings indicate that this duplex RT-LAMP assay is a simple, rapid, specific, and sensitive method for detection of FMDV and VSV, and can be applied in clinical detection and epidemiological investigation of FMDV and VSV.

中图分类号:

范晴, 谢芝勋, 谢志勤, 谢丽基, 黄娇玲, 张艳芳, 曾婷婷, 王盛, 罗思思, 邓显文, 刘加波. 二重荧光RT-LAMP方法鉴别检测口蹄疫病毒和水泡性口炎病毒[J]. 畜牧兽医学报, 2018, 49(5): 996-1004.

FAN Qing, XIE Zhi-xun, XIE Zhi-qin, XIE Li-ji, HUANG Jiao-ling, ZHANG Yan-fang, ZENG Ting-ting, WANG Sheng, LUO Si-si, DENG Xian-wen, LIU Jia-bo. Development of a Duplex Fluorescence-based Reverse Transcript Loop-mediated Isothermal Amplification Assay for Detection of Foot-and-mouth Disease Virus and Vesicular Stomatitis Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(5): 996-1004.

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https://www.xmsyxb.com/CN/Y2018/V49/I5/996

参考文献

[1] DIVERS T J, PEEK S F. Rebhun's diseases of dairy cattle[M]. 2nd ed. Beijing:China Agricultural University Press, 2009.
[2] BRITO B P, RODRIGUEZ L L, HAMMOND J M, et al. Review of the global distribution of Foot-and-Mouth disease virus from 2007 to 2014[J]. Transbound Emerg Dis, 2017, 64(2):316-332.
[3] MEYER R F, KNUDSEN R C. Foot-and-mouth disease:A review of the virus and the symptoms[J].J Environ Health, 2001, 64(4):21-23.
[4] WERNERY U, KINNE J. Foot and mouth disease and similar virus infections in camelids:A review[J].Rev Sci Tech, 2012, 31(3):907-918.
[5] FERNÁNDEZ J, AGVERO M, ROMERO L, et al. Rapid and differential diagnosis of Foot-and-Mouth disease, swine vesicular disease, and vesicular stomatitis by a new multiplex RT-PCR assay[J].J Virol Methods, 2008, 147(2):301-311.
[6] 王永连, 徐立波. 牛水疱性口炎的流行、诊断和防控措施[J]. 饲料博览, 2017(4):66-67.
WANG Y L, XU L B. Epidemic, diagnosis and control measures of bull vesicular stomatitis[J]. Feed Review, 2017(4):66-67. (in Chinese)
[7] OIE. NB:Foot and mouth disease[M/OL]//OIE Terrestrial Manual, 2012:145-173.
[8] OIE. NB:Vesicular stomatitis[M/OL]//OIE Terrestrial Manual, 2015:1-14.
[9] NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res, 2000, 28(12):e63.
[10] JIANG Y S, BHADRA S, LI B L, et al. Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons[J].Anal Chem, 2015, 87(6):3314-3320.
[11] FAN Q, XIE Z X, XIE L J, et al. A reverse transcription loop-mediated isothermal amplification method for rapid detection of bovine viral diarrhea virus[J].J Virol Methods, 2012, 186(1-2):43-48.
[12] 庞耀珊, 谢芝勋, 谢丽基, 等. 对虾白斑综合征病毒可视化环介导等温扩增(LAMP)检测技术的建立与应用[J]. 农业生物技术学报, 2013, 21(8):1002-1008.
PANG Y S, XIE Z X, XIE L J, et al. Development and application of visual loop mediated isothermal amplification (LAMP) assay for shrimp (Penacus orientalis) White spot syndrome virus[J]. Journal of Agricultural Biotechnology, 2013, 21(8):1002-1008. (in Chinese)
[13] ZHAO Y X, CHEN F, LI Q, et al. Isothermal amplification of nucleic acids[J].Chem Rev, 2015, 115(22):12491-12545.
[14] 林文慧, 邹秉杰, 宋沁馨, 等. 多重环介导等温扩增技术研究进展[J]. 遗传, 2015, 37(9):899-910.
LIN W H, ZOU B J, SONG Q X, et al. Progress in multiplex loop-mediated isothermal amplification technology[J].Hereditas (Beijing), 2015, 37(9):899-910. (in Chinese)
[15] 赵军, 陈泽平, 李桢, 等. 二重LAMP检测鸡蛋污染致病菌[J]. 食品工业科技, 2016, 37(4):107-110, 116.
ZHAO J, CHEN Z P, LI Z, et al. Multiplex LAMP method for the detection of pathogenic bacteria in eggs[J].Science and Technology of Food Industry, 2016, 37(4):107-110, 116. (in Chinese)
[16] 刘宁伟, 邹大阳, 董德荣, 等. 多重环介导等温扩增快速检测沙门菌、副溶血弧菌和单核细胞增生性李斯特菌方法的建立[J]. 军事医学, 2016, 40(9):767-772.
LIU N W, ZOU D Y, DONG D R, et al. Development of multiplex loop-mediated isothermal amplification (mLAMP) for detection ofSalmonella, Vibrio parahaemolyticus and Listeria monocytogenes[J]. Military Medical Sciences, 2016, 40(9):767-772. (in Chinese)
[17] 陈兵, 孙洁, 陈书琨, 等. 三种家禽病毒多重RT-LAMP检测方法的建立与应用[J]. 上海畜牧兽医通讯, 2016(1):2-5.
CHEN B, SUN J, CHEN S K, et al. The development of duplex RT-LAMP assay for detection three poultry viruses[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine, 2016(1):2-5. (in Chinese)
[18] YAMAZAKI W, MIOULET V, MURRAY L, et al. Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection of foot-and-mouth disease virus[J].J Virol Methods,2013, 192(1-2):18-24.
[19] 姜侃, 张水峰, 张慧, 等. 两种致病性弧菌二重LAMP-熔解曲线检测方法的建立[J]. 现代食品科技, 2016, 32(10):246-251.
JIANG K, ZHANG S F, ZHANG H, et al. Development of a multiplex LAMP-dissociation curve method for the detection of two pathogenicvibrio species[J]. Modern Food Science and Technology, 2016, 32(10):246-251. (in Chinese)
[20] KOUGUCHI Y, FUJIWARA T, TERAMOTO M, et al. Homogenous, real-time duplex loop-mediated isothermal amplification using a single fluorophore-labeled primer and an intercalator dye:Its application to the simultaneous detection of Shiga toxin genes 1 and 2 in Shiga toxigenic Escherichia coli isolates[J]. Mol Cell Probes, 2010, 24(4):190-195.
[21] MAHONY J, CHONG S, BULIR D, et al. Multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of influenza A/H1, A/H3 and influenza B can provide a specimen-to-result diagnosis in 40 min with single genome copy sensitivity[J].J Clin Virol, 2013, 58(1):127-131.
[22] NURUL NAJIAN A B, ENGKU NUR SYAFIRAH E A, ISMAIL N, et al. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira[J]. Anal Chim Acta, 2016, 903:142-148.
[23] KUBOTA R, JENKINS D M. Real-time duplex applications of loop-mediated amplification (LAMP) by assimilating probes[J].Int J Mol Sci, 2015, 16(3):4786-4799.
[24] 王彩霞, 冯春燕, 王巧黎, 等. 同时检测贝类派琴虫和包纳米虫的双重LAMP方法的建立[J]. 中国畜牧兽医, 2015, 42(8):1935-1942.
WANG C X, FENG C Y, WANG Q L, et al. Establishment of duplex loop-mediated isothermal amplification method for simultaneous detection of shellfish infected withPerkinsus and Bonamia[J]. China Animal Husbandry & Veterinary Medicine, 2015, 42(8):1935-1942. (in Chinese)
[25] ISEKI H, ALHASSAN A, OHTA N. Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovineBabesia parasites[J]. J Microbiol Methods, 2007, 71(3):281-287.
[26] AONUMA H, YOSHIMURA A, KOBAYASHI T, et al. A single fluorescence-based LAMP reaction for identifying multiple parasites in mosquitoes[J].Exp Parasitol, 2010, 25(1):179-183.