同时监测8种牛常见病原体的GeXP高通量快速检测技术

doi:10.11843/j.issn.0366-6964.2017.10.015

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (10): 1920-1931.doi: 10.11843/j.issn.0366-6964.2017.10.015

同时监测8种牛常见病原体的GeXP高通量快速检测技术

范晴, 谢芝勋*, 谢志勤, 谢丽基, 黄莉, 黄娇玲, 张艳芳, 曾婷婷, 王盛, 罗思思, 邓显文, 刘加波, 庞耀珊

广西兽医研究所广西兽医生物技术重点实验室, 南宁 530001

收稿日期:2017-04-07 出版日期:2017-10-23 发布日期:2017-10-23
通讯作者: 谢芝勋,E-mail:xiezhixun@126.com
作者简介:范晴(1982-),女,云南红河人,副研究员,主要从事牛病防治与病原分子生物学研究,Tel:0771-3120371,E-mail:fanqing1224@126.com
基金资助:
广西自然科学基金（2016GXNSFCA380003；2014GXNSFBA118105）；桂科（AB16380003）。

Development of a GeXP Assay for Simultaneous Differentiation of 8 Pathogens of Bovine Infectious Diseases

FAN Qing, XIE Zhi-xun*, XIE Zhi-qin, XIE Li-ji, HUANG Li, HUANG Jiao-ling, ZHANG Yan-fang, ZENG Ting-ting, WANG Sheng, LUO Si-si, DENG Xian-wen, LIU Jia-bo, PANG Yao-shan

Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China

Received:2017-04-07 Online:2017-10-23 Published:2017-10-23

摘要/Abstract

摘要：

本研究旨在建立一种可同时检测8种常见牛传染病病原体（口蹄疫病毒、蓝舌病病毒、水疱性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒大肠杆菌、牛传染性鼻气管炎病毒和小反刍兽疫病毒）的GeXP高通量快速检测方法。将多重PCR技术和毛细管电泳技术有效地结合，根据上述8种病原体的基因保守序列，设计并合成了8对特异性引物，在每对特异性引物的5'端加上一段通用引物，形成特异性嵌合引物。优化反应体系和反应条件，用单一病毒和混合病毒样品来验证所建立的GeXP方法的特异性，用克隆质粒稀释液来测试GeXP方法的灵敏度，检测305份临床样品，进一步验证该方法的可靠性。结果：优化后的GeXP检测体系，可扩增出各病原体对应的特异片段，能在100拷贝·μL^-1水平同时并特异地检测出8种牛病病原体核酸。305份临床样品的检测结果与荧光PCR检测结果一致，阳性样品测序结果表明无非特异性扩增的假阳性。结果显示，所建立的GeXP方法具有高通量、特异性好、灵敏度高、方便快速等优点，可用于临床鉴别诊断和流行病学调查，具有较高的临床应用价值。

Abstract:

The aim of this study was to develop a multiple PCR assay on a basis of Genome Lab Genetic Expression Analysis System (GeXP) for simultaneous detection of 8 pathogens of bovine infectious diseases, including food-and-mouth disease virus (FMDV), bluetongue virus (BTV) ,vesicular stomatitis virus (VSV), bovine viral diarrheal virus (BVDV), bovine rotavirus (BRV), enterotoxigenic E. coli (ETEC), bovine herpesvirus 1 (IBRV), Peste des petits ruminants virus (PPRV). The GeXP profilerutilisesgene-specific primers containing 5'-universal adaptor sequences. Eight gene-specific primers consist of a universal sequence fused to the 5'-end and a gene-specific sequence were designed according to the conserved sequence of each pathogen and used for amplification.The reaction system and condition were optimized. The specificity of GeXP were examined with samples of the single and mixture of 8 viruses' co-infection. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of cloned plasmids. And 305 clinical samples were detected by GeXP to further evaluate the reliability. The results showed that the corresponding specific fragments of genes were amplified respectively. The detection limit of GeXP was 100 copies·μL^-1 when all of 8 pre-mixed plasmids containing target genes of 8 bovine pathogens. In detection of 305 clinical samples, the results of GeXP were consistent with real-time PCR completely. Analysis of positive samples by sequencing demonstrated that the GeXP assay had no false positive samples of nonspecific amplification. In conclusion, this GeXP assay is a high throughput, specific, sensitive, rapid and simple method for the detection of 8 pathogens of bovine infectious diseases. It is an effective tool applied to differential diagnosis rapidly for clinical samples and epidemiological investigation.

中图分类号:

S858.235

范晴, 谢芝勋, 谢志勤, 谢丽基, 黄莉, 黄娇玲, 张艳芳, 曾婷婷, 王盛, 罗思思, 邓显文, 刘加波, 庞耀珊. 同时监测8种牛常见病原体的GeXP高通量快速检测技术[J]. 畜牧兽医学报, 2017, 48(10): 1920-1931.

FAN Qing, XIE Zhi-xun, XIE Zhi-qin, XIE Li-ji, HUANG Li, HUANG Jiao-ling, ZHANG Yan-fang, ZENG Ting-ting, WANG Sheng, LUO Si-si, DENG Xian-wen, LIU Jia-bo, PANG Yao-shan. Development of a GeXP Assay for Simultaneous Differentiation of 8 Pathogens of Bovine Infectious Diseases[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(10): 1920-1931.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2017.10.015

https://www.xmsyxb.com/CN/Y2017/V48/I10/1920

参考文献

[1] 目前我国肉牛养殖的现状以及前景分析[N]. 2015-10-29. http://www.ar114.com.cn/news/show.php?itemid=78926.
Current situation and prospect analysis on breeding cattle in China[N]. 2015-10-29. http://www.ar114.com.cn/news/show.php?itemid=78926.(in Chinese)
[2] 周作集, 刘克俊, 李芳芳, 等. 广西肉牛的发展现状和对策[J]. 广西畜牧兽医, 2014, 30(5): 233-236.
ZHOU Z J, LIU K J, LI F F, et al. The present situation and strategies of cattle industry in Guangxi province[J]. Guangxi Journal of Animal Husbandry & Veterinary Medicine, 2014, 30(5): 233-236. (in Chinese)
[3] SIERRA S, DÁVILA M, LOWENSTEIN P R, et al. Response of foot-and-mouth disease virus to increased mutagenesis: influence of viral load and fitness in loss of infectivity[J]. J Virol, 2000, 74(18): 8316-8323.
[4] CERNICCHIARO N, WHITE B J, RENTER D G, et al. Evaluation of economic and performance outcomes associated with the number of treatments after an initial diagnosis of bovine respiratory disease in commercial feeder cattle[J]. Am J Vet Res, 2013, 74(2): 300-309.
[5] 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.
[6] MARTÍN-ACEBES M A, GONZÁLEZ-MAGALDI M, ROSAS M F, et al. Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: a comparative study with foot-and-mouth disease virus and vesicular stomatitis virus[J]. Virology, 2008, 374(2): 432-443.
[7] NAYDUCH D, COHNSTAEDT L W, SASKI C, et al. Studying Culicoides vectors of BTV in the post-genomic era: resources, bottlenecks to progress and future directions[J]. Virus Res, 2014, 182: 43-49.
[8] 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.
[9] SANTMAN-BERENDS I M G A, MARS M H, VAN DUIJN L, et al. Evaluation of the epidemiological and economic consequences of control scenarios for bovine viral diarrhea virus in dairy herds[J]. J Dairy Sci, 2015, 98(11): 7699-7716.
[10] KHATIB L A, TSAI Y L, OLSON B H. A biomarker for the identification of cattle fecal pollution in water using the LTⅡa toxin gene from enterotoxigenic Escherichia coli[J]. Appl Microbiol Biotechnol, 2002, 59(1): 97-104.
[11] 谢金鑫, 段招军, 李丹地, 等. 中国大庆奶牛中发现轮状病毒G10P
[11] [J]. 病毒学报, 2010, 26(5): 407-409.
XIE J X, DUAN Z J, LI D D, et al. Detection of bovine rotavirus G10P
[11] in a diary farm in Daqing, China[J]. Chinese Journal of Virology, 2010, 26(5): 407-409. (in Chinese)
[12] 甘瑜萍, 余祖琼, 黄恒, 等. 我国牛传染性鼻气管炎的流行与防控现状[J]. 黑龙江畜牧兽医, 2016(2): 113-114.
GAN Y P, YU Z Q, HUANG H, et al. Epidemic and control of infectious bovine rhinotracheitis[J]. Heilongjiang Animal Science and Veterinary Medicine, 2016(2): 113-114. (in Chinese)
[13] WU X, LI L, LI J, et al. Peste des petits ruminants viruses re-emerging in China, 2013-2014[J]. Transbound Emerg Dis, 2016, 63(5): e441-e446.
[14] 胡长敏, 郭爱珍, 潘志明, 等. 发达国家重大牛病防控历史和经验[J]. 中国兽医学报, 2011, 31(3): 453-456.
HU C M, GUO A Z, PAN Z M, et al. Lessons from the history of cattle major epidemic disease control in developed countries[J]. Chinese Journal of Veterinary Science, 2011, 31(3): 453-456. (in Chinese)
[15] DREW J E, MAYER C D, FARQUHARSON A J, et al. Custom design of a GeXP multiplexed assay used to assess expression profiles of inflammatory gene targets in normal colon, polyp, and tumor tissue[J]. J Mol Diagn, 2011, 13(2): 233-242.
[16] YANG M J, 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.
[17] HU X M, ZHANG Y, ZHOU X M, 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.
[18] RAI A J, KAMATH R M, GERALD W, et al. Analytical validation of the GeXP analyzer and design of a workflow for cancer-biomarker discovery using multiplexed gene-expression profiling[J]. Anal Bioanal Chem, 2009, 393(5): 1505-1511.
[19] 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.
[20] XIE Z X, LUO S S, XIE L J, 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.
[21] ZHANG Y F, XIE Z X, XIE L J, et al. GeXP analyzer-based multiplex reverse-transcription PCR assay for the simultaneous detection and differentiation of eleven duck viruses[J]. BMC Microbiol, 2015, 15: 247.
[22] ZHANG M X, XIE Z X, XIE L J, et al. Simultaneous detection of eight reproductive and respiratory swine pathogens using a novel GeXP analyser-based multiplex PCR Assay[J]. J Virol Methods, 2015, 224: 9-15.
[23] ZENG T T, XIE Z X, XIE L J, et al. Simultaneous detection of eight immunosuppressive chicken viruses using a GeXP analyser-based multiplex PCR assay[J]. Virol J, 2015, 12: 226.
[24] LI M, XIE Z X, XIE Z Q, et al. Simultaneous detection of four different neuraminidase types of avian influenza A H5 viruses by multiplex reverse transcription-PCR using a GeXP analyser[J]. Influenza Other Respir Viruses, 2016, 10(2): 141-149.
[25] 朱晓光, 李斌, 刘冰, 等. 麻疹病毒属病毒检测基因芯片的建立[J]. 中国病原生物学杂志, 2010, 5(5): 329-331, 405.
ZHU X G, LI B, LIU B, et al. Microarray for species-specific detection of Morbillivirus[J]. Journal of Pathogen Biology, 2010, 5(5): 329-331, 405. (in Chinese)
[26] 史茜, 唐慧芬, 牛国辉, 等. 牛新孢子虫基因芯片检测方法的建立[J]. 中国预防兽医学报, 2014, 36(9): 715-719.
SHI Q, TANG H F, NIU G H, et al. Development gene microarray method for the detection of bovine Neospora caninum[J]. Chinese Journal of Preventive Veterinary Medicine, 2014, 36(9): 715-719. (in Chinese)

同时监测8种牛常见病原体的GeXP高通量快速检测技术

Development of a GeXP Assay for Simultaneous Differentiation of 8 Pathogens of Bovine Infectious Diseases

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 4

编辑推荐

Metrics

本文评价

[1]	李甜, 杨洋, 谢黎卿, 王远兰, 李攀, 彭远义, 李能章. 牛溶血性曼氏杆菌及牛荚膜A型多杀性巴氏杆菌灭活疫苗对小鼠的保护性研究[J]. 畜牧兽医学报, 2021, 52(9): 2579-2588.
[2]	刘梦瑶, 王展慧, 吴浩, 谷月, 吴文学. 牛星状病毒、牛病毒性腹泻病毒1型、牛冠状病毒和牛轮状病毒四重实时荧光定量RT-PCR检测方法的建立[J]. 畜牧兽医学报, 2021, 52(7): 1942-1952.
[3]	马小静, 李梦莹, 张淮瑜, 宋阿北, 许立华, 吴心华, 张巧娥, 李继东. 共表达牛病毒性腹泻病毒E0基因和牛传染性鼻气管炎病毒gD基因的重组质粒DNA构建及其对小鼠的免疫效应[J]. 畜牧兽医学报, 2021, 52(1): 154-165.
[4]	米思远, 师科荣, 张瑞强, 张海亮, 徐庆磊, 俞英. 牛病毒性腹泻病毒的RT-PCR检测及感染犊牛相关基因差异表达分析[J]. 畜牧兽医学报, 2018, 49(7): 1432-1439.