9种鸭病毒病GeXP多重PCR检测方法的建立及其应用

doi:10.11843/j.issn.0366-6964.2016.12.016

摘要/Abstract

摘要：

旨在建立一种能够同时鉴别H5、H7和H9亚型禽流感病毒、基因A型鸭甲肝病毒、鸭坦布苏病毒、鸭瘟病毒、减蛋综合征病毒、新城疫病毒、鸭圆环病毒、番鸭呼肠孤病毒和番鸭细小病毒9种常见鸭病毒病的GeXP多重PCR检测方法。根据这些病原体在GenBank上公布的保守基因序列，设计合成了12对特异性GeXP引物。用单一病毒或混合病毒样品的cDNA/DNA 模板优化反应条件，同时以其他常见鸭病病原体及鸭组织器官核酸为对照，验证所建立的GeXP方法的特异性和准确性。以10⁶ 至10¹ 拷贝•μL^-1梯度稀释的克隆质粒或体外转录RNA来检测GeXP方法的敏感性。随机选取不同浓度的模板(10³和 10⁷拷贝•μL^-1)进行干扰性试验，并与单一病原体为模板的GeXP多重PCR的检测结果比较。最后用该方法检测150份临床样品，与普通PCR方法的结果作比较，所有阳性结果样品均进行测序，进一步验证所建立的GeXP检测方法的可靠性。结果显示，单重或多重模板的GeXP检测均能特异性扩增出相应片段，不能扩增其他常见鸭病病原体，并且可在10³拷贝•μL^-1水平上同时特异地检测出9种鸭病原体。GeXP干扰性试验结果显示，当3种不同浓度的模板组合时，本试验所建立的方法依然可同时检测出所有病原体，与单重检测比较，未影响其检出。比较GeXP多重PCR方法和普通PCR方法对150份临床样品的检测结果，结果显示GeXP方法更为敏感与准确。笔者建立的同时鉴别9种鸭病毒病的GeXP多重PCR检测方法，具有高通量、特异性强和灵敏度高的特点，为混合感染的鸭病毒病临床样品提供了快速分子诊断新方法。

Abstract:

This experiment was developed to simultaneously detect these nine viral pathogens that cause infections in ducks including avian influenza virus (AIV) subtypes H5, H7, and H9; duck hepatitis A virus (DHAV-A); duck Tembusu virus (DTMUV); duck enteritis virus (DEV); egg drop syndrome virus (EDSV); Newcastle disease virus (NDV); duck circovirus (DuCV); muscovy duck reovirus (MDRV); and muscovy duck parvovirus (MDPV). Twelve pairs of specific primers were designed according to the conserved sequences of the genes from each pathogen available in the GenBank database. Single, mixed pathogen cDNA/DNA templates, other common duck pathogens and duck organs nucleic acids were used to evaluate the specificity of the GeXP-multiplex assay. Serial dilution from 10⁶ to 10¹ copies•μL^-1 of in vitro transcript RNA of target genes and plasmids were used to test the sensitivity of GeXP multiplex PCR assay. Different amounts of the templates (10³ and 10⁷ copies•μL^-1) were selected at random, mixed and tested in the GeXP multiplex PCR assay. The results were compared with those of the single template GeXP multiplex PCR assay. The GeXP assay was evaluated using 150 clinical specimens and compared with the single PCR. All positive specimens in the GeXP multiplex PCR and conventional PCR were identified via sequencing to verify the accuracy and reliability of the GeXP assay. These results showed that the corresponding specific fragments of genes were amplified by the single and multiplex GeXP PCR assay. Other pathogens did not result in amplification products. The detection limit of GeXP was 10³copies•μL^-1 when all nine types of duck virus were present. The results of the interference assay showed that three specific amplification peaks can still be observed in the case of combination of three different concentration templates. The results of these experiments showed that mixed infections can be detected by GeXP multiplex PCR with minimal interference. Compared with the results of conventional PCR, the GeXP multiplex PCR method was more sensitive and accurate in the detection of 150 clinical samples. In conclusion, this GeXP-based multiplex PCR is a high-throughput, specific and sensitive test to detect nine duck viruses. This assay provides a new method in rapid molecular diagnosis for mix clinical duck virus samples.

中图分类号:

S858.325.3

张艳芳，谢芝勋，谢丽基，邓显文，谢志勤，罗思思，黄莉，黄娇玲，曾婷婷, 王盛. 9种鸭病毒病GeXP多重PCR检测方法的建立及其应用[J]. 畜牧兽医学报, 2016, 47(12): 2457-2468.

ZHANG Yan-fang, XIE Zhi-xun, XIE Li-ji, DENG Xian-wen, XIE Zhi-qin, LUO Si-si, HUANG Li, HUANG Jiao-ling, ZENG Ting-ting, WANG Sheng. Development and Application of a GeXP-multiplex PCR Assay for Detection of Nine Duck Viruses[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(12): 2457-2468.

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参考文献

［1］麦尔旦•吐尔孙, 闫建伟, 王雅鹏. 中国肉鸭产业的区域优势分析——基于全国21个水禽主产省（市、区）的研究［J］. 农业现代化研究, 2013（4）:477-481.
MAI E D•T E S, YAN J W, WANG Y P. Analysis on regional advantages of china’s meat duck industry-based on researches in 21 main waterfowl-producing provinces (cities and districts)［J］. Research of Agricultural Modernization, 2013(4): 477-481. (in Chinese)
［2］张大丙. 当前鸭病流行现状及防控对策［J］.中国家禽, 2012, 34(7):38-40.
ZHANG D B. The current epidemic status of duck disease and its prevention and control strategies［J］. China Poultry, 2012, 34:38-40. (in Chinese)
［3］曾婷婷, 谢芝勋, 谢丽基, 等. 4株广西鸭源坦布苏病毒分离及初步鉴定［J］. 中国动物检疫, 2013, 30(6):31-35.
ZENG T T, XIE Z Z, XIE L J, et al. Isolation and identification of duck tembusu virus from Guangxi［J］. China Animal Health Inspection, 2013,30(6):31-35. (in Chinese)
［4］HOTTA K, TAKAKUWA H, YABUTA T, et al. Antibody survey on avian influenza viruses using egg yolks of ducks in Hanoi between 2010 and 2012［J］. Vet Microbiol, 2013, 166(1-2):179-183.
［5］SCHMITZ A, LE BRAS M O, GUILLEMOTO C, et al. Evaluation of a commercial ELISA for H5 low pathogenic avian influenza virus antibody detection in duck sera using Bayesian methods［J］. J Virol Methods, 2013, 193(1):197-204.
［6］WANG G, QU Y, WANG F, et al. The comprehensive diagnosis and prevention of duck plague in northwest Shandong province of China［J］. Poult Sci, 2013, 92(11):2892-2898.
［7］谢芝勋, 谢志勤, 刘加波, 等. 用聚合酶链反应检测鸭瘟病毒的研究［J］.中国兽药杂志, 2000, 34(4):10-12.
XIE Z X, XIE Z Q, LIU J B, et al. Detection of duck plague virus using the polymerase chain reaction［J］. Chinese Journal of Veterinary Drug, 2000, 34(4):10-12. (in Chinese)
［8］WOZ＇NIAKOWSKI G, SAMOREK-SALAMONOWICZ E, KOZDRUN＇ W. Quantitative analysis of waterfowl parvoviruses in geese and Muscovy ducks by real-time polymerase chain reaction: correlation between age, clinical symptoms and DNA copy number of waterfowl parvoviruses［J］. BMC Vet Res, 2012, 8:29.
［9］谢丽基, 谢芝勋, 庞耀珊, 等. 鸭Ⅰ型肝炎病毒荧光定量RT-PCR方法的建立及初步应用［J］. 中国家禽, 2012,34(10):23-26.
XIE L J, XIE Z X, PANG Y S, et al. Development and application of Real-time RT-PCR assay for detection of duck hepatitis virus type Ⅰ［J］. China Poultry, 2012, 34(10):23-26. (in Chinese)
［10］XIE L, XIE Z, ZHAO G, et al. A loop-mediated isothermal amplification assay for the visual detection of duck circovirus［J］. Virol J, 2014, 11:76.
［11］谢芝勋, 谢丽基, 刘加波, 等. 禽流感和新城疫病毒二重荧光定量RT-PCR 检测方法的建立［J］. 生物技术通讯, 2008, 19(3):410-413.
XIE Z X, XIE L J, LIU J B, et al. Development of a multiplex real-time RT-PCR assay of avian influenza virus and Newcaste disease virus［J］. Letters in Biotechnology, 2008, 19 (3):410-413. (in Chinese)
［12］张艳芳, 谢芝勋, 谢丽基, 等. 鸭坦布苏病毒和鸭瘟病毒二重RT-PCR检测方法的建立［J］.南方农业学报, 2014, 45(2):314-317.
ZHANG Y F, XIE Z X, XIE L J, et al. Development of a duplex RT-PCR assay for detection of duck tembusu virus and duck plague virus［J］. Journal of Southern Agriculture, 2014, 45(2):314-317. (in Chinese)
［13］张艳芳, 谢芝勋, 谢丽基, 等. 鸭坦布苏病毒和鸭瘟病毒二重荧光定量 RT-PCR 方法的建立［J］. 中国畜牧兽医, 2014, 41 (3):77-82.
ZHANG Y F, XIE Z X, XIE L J, et al. Development of duplex real-time RT-PCR assay for detection of duck Tembusu virus and duck plague virus［J］. China Animal Husbandry & Veterinary Medicine, 2014, 41 (3): 77-82. (in Chinese)
［14］ZENG Z, LIU Z, WANG W, et al. Establishment and application of a multiplex PCR for rapid and simultaneous detection of six viruses in swine［J］. J Virol Methods, 2014, 208:102-106.
［15］卢昌, 吴国华, 颜新敏, 等. GeXP 多重基因表达分析系统及其应用［J］. 中国兽医科学, 2013, 43(2):216-220.
LU C, WU G H, YAN X M, et al. GeXP based on multiplex system and its application［J］. Chinese Veterinary Science, 2013, 43(2): 216-220. (in Chinese)
［16］HU X, XU B, YANG Y, et al. A high throughput multiplex PCR assay for simultaneous detection of seven aminoglycoside-resistance genes in Enterobacteriaceae［J］. BMC Microbiol, 2013, 13:58.
［17］何玢, 王环宇, 张晨, 等. 8种脑炎相关虫媒病毒GeXP检测方法的建立［J］.病毒学报, 2012, 28(1):57-62.
HE B, WANG H Y, ZHANG C, et al. Development of a GeXP based multiplex RT-PCR assay for simultaneous detection of eight arboviruses related to encephalitis［J］. Chinese Journal of Virology, 2012, 28(1):57-62. (in Chinese)
［18］QIN M, WANG D Y, HUANG F, et al. Detection of pandemic influenza A H1N1 virus by multiplex reverse transcription-PCR with a GeXP analyzer［J］. J Virol Methods, 2010, 168 (1-2):255-258.
［19］YANG M , LUO L, NIE K, et al. Genotyping of 11 human papillomaviruses by multiplex PCR with a GeXP analyzer［J］. J Med Virol, 2012, 84(6):957-963.
［20］HU X, ZHANG Y, ZHOU X, et al. Simultaneously typing nine serotypes of enteroviruses associated with hand, foot, and mouth disease by a GeXP analyzer-based multiplex reverse transcription-PCR assay［J］. J Clin Microbiol, 2012, 50 (2):288-293.
［21］XIE Z, LUO S, XIE L, et al. Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzer-based multiplex PCR assay［J］. J Virol Methods, 2014, 207:188-195.
［22］张民秀, 谢芝勋, 邓显文, 等. 8 种猪呼吸道和繁殖障碍病病原体GeXP 检测方法的建立［J］.中国农业科学，2015, 48(24):4996-5006.
ZHANG M X, XIE Z X, DENG X W, et al. Establishment of a GeXP analyser-based multiplex PCR assay for detection of eight reproductive and respiratory swine pathogens［J］. Scientia Agricultura Sinica, 2015, 48(24):4996-5006. (in Chinese)
［23］曾婷婷, 谢芝勋, 谢丽基, 等. 应用二重实时荧光定量 RT-PCR 鉴别坦布苏病毒与产蛋下降综合征病毒［J］. 中国家禽, 2015, 37(1): 17-21.
ZENG T T, XIE Z X, XIE L J, et al. Development of duplex Real-time fluorescence RT-PCR assay for differential detection of Tembusu virus and egg drop syndrome virus［J］. China Poultry, 2015, 37(1): 17-21. (in Chinese)
［24］张艳芳, 谢芝勋, 谢丽基, 等. 鸭坦布苏病毒与鸭圆环病毒二重RT-PCR方法的建立［J］. 黑龙江畜牧兽医, 2014 (12): 44-47.
ZHANG Y F, XIE Z X, XIE L J, et al. Establishment of a duplex RT-PCR assay for detection of duck Tembusu virus and duck circovirus［J］. Heilongjiang Animal Science and Veterinary Medicine, 2014 (12): 44-47. (in Chinese)
［25］胡奇林, 林锋强, 陈少莺, 等. 应用 RT-PCR 技术检测番鸭呼肠孤病毒［J］. 中国兽医学报, 2004, 24(3): 231-232.
HU Q L, LIN F Q, CHENG S Y, et al. Detection of muscovy duck reovirus by RT-PCR［J］. Chinese Journal of Veterinary Science, 2004, 24(3): 231-232. (in Chinese)
［26］谢丽基, 谢芝勋, 邓显文, 等. 鸭Ⅰ型肝炎病毒和番鸭细小病毒二重荧光定RT-PCR方法的建立［J］. 中国兽医学报, 2013, 33(8): 1184-1189.
XIE L J, XIE Z X, DENG X W, et al. Development of duplex real-time RT-PCR assay for detection of duck hepatitis virus typeⅠand muscovy duck parvovirus［J］. Chinese Journal of Veterinary Science, 2013, 33(8): 1184-1189. (in Chinese)
［27］李瑾, 毛乃颖, 秦萌, 等. GeXP多重PCR技术同时检测12种常见呼吸道病毒［J］. 病毒学报, 2011, 27(6):526-532.
LI J, MAO N Y, QIN M, et al. A GeXP based multiplex RT-PCR assay for simultaneous detection of twelve human respiratory viruses［J］. Chinese Journal of Virology. 2011, 27(6):526-532. (in Chinese)
［28］罗思思, 谢芝勋, 谢丽基, 等. 同时鉴别 6 种鸡病毒性呼吸道病 GeXP 检测方法的建立［J］. 病毒学报, 2013（3）: 250-257.
LUO S S, XIE Z X, XIE L J, et al. Development of a GeXP assay for simultaneous differentiation of six chicken respiratory viruses［J］. Chinese Journal of Virology, 2013(3): 250-257. (in Chinese)
［29］XIE Z, PANG Y S, LIU J, et al. A multiplex RT-PCR for type A influenza virus and differentiation of avian H5,H7 and H9 hemagglutinin subtypes［J］. Mol Cell Probes, 2006, 20(3-4):245-249.
［30］曾婷婷，谢芝勋，谢丽基，等.应用多重GeXP-PCR同时检测6种鸡免疫抑制病病毒的研究［J］.畜牧兽医学报，2016，47（6）：1198-1208.
ZENG T T， XIE Z X， XIE L J， et al. Simultaneous detecting six immunosuppressive chicken virus by a GeXP analyser-based multiplex PCR assay［J］. Acta Veterinaria et Zootechnica Sinica, 2016,47(6):1198-1208.(in Chinese)

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