兔出血症病毒2型的VP60基因保守区人工合成及RT-PCR检测方法初探

doi:10.3969/j.issn.1004-1524.2016.10.04

浙江农业学报 ›› 2016, Vol. 28 ›› Issue (10): 1650-1656.DOI: 10.3969/j.issn.1004-1524.2016.10.04

兔出血症病毒2型的VP60基因保守区人工合成及RT-PCR检测方法初探

杨泽晓¹, 赵希仑¹, 李岩², 王波¹, 姚学萍¹, 王印^{1, 3, *}, 耿毅¹, 蒙正群¹, 白瑜¹

1.四川农业大学动物医学院, 四川温江 611130;
2.四川农业大学动物科技学院, 四川温江 611130;
3.动物疫病与人类健康四川省重点实验室,四川温江 611130

收稿日期:2016-01-11 出版日期:2016-10-15 发布日期:2016-11-20
通讯作者: 王印,E-mail:yaanwangyin@tom.com
作者简介:杨泽晓(1980—),男,河南南阳人,博士,副教授,研究方向为动物疫病防治与兽医公共卫生。E-mail:yzxyang2003@126.com
基金资助:
国家自然科学基金项目(31402222); “十二五”国家科技支撑计划(2013BAD12B04); 教育部《“长江学者和创新团队发展计划”》创新团队项目(IRT0848); 四川农业大学双支计划; 四川省科技支撑计划(2016N20002)

Artificial synthesis of VP60 conserved gene fragment of rabbit hemorrhagic disease virus type 2 and preliminary study of its RT-PCR detection method

YANG Ze-xiao¹, ZHAO Xi-lun¹, LI Yan², WANG Bo¹, YAO Xue-ping¹, WANG Yin^{1, 3, *}, GENG Yi¹, MENG Zheng-qun¹, BAI Yu¹

1. College of Veterinary Medicine,Sichuan Agricultural University, Wenjiang 611130, China;
2. College of Animal Science and Technology, Sichuan Agricultural University, Wenjiang 611130, China;
3. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Wenjiang 611130, China

Received:2016-01-11 Online:2016-10-15 Published:2016-11-20

摘要/Abstract

摘要： 为建立用于兔出血症病毒2型(RHDV2)的快速检测方法,根据GenBank已公布的RHDV和RHDV2 VP60基因,设计合成2对特异性引物和10条末端重叠引物,通过重叠PCR 人工合成RHDV2 VP60基因中保守区片段,构建重组质粒,并以其为模板,经过反应条件优化、敏感性试验、特异性试验和35份样品检测应用,初步建立RHDV2 RT-PCR检测方法。实验成功合成RHDV2 VP60基因的435 bp保守片段并构建了pMD-19T-RHDV2重组质粒, 初步建立的RHDV2 RT-PCR方法具有良好的特异性和敏感性,RHDV2的检测限度可达到230个拷贝的靶基因片段,检测RHDV、pGM-T-EBHSV(已构建)、兔巴氏杆菌、兔源大肠埃希菌和沙门氏菌均无特异性扩增,样品检测结果显示样品中RHDV2核酸阴性。该研究为中国及时进行RHDV2的防控提供技术储备。

关键词: 兔病毒性出血症, RHDV2, RT-PCR, 重叠PCR

Abstract: In order to develop a rapid detection method for RHDV2, 4 specific primers and 10 overlapping oligo primers were designed to amplify the conserved RHDV2 VP60 gene specific DNA fragment according to the genome sequences of RHDV and RHDV2 published in GenBank. Based on the synthesis of a conserved part of RHDV2 sequence using overlap extension PCR and the construction of recombinant plasmid, RT-PCR assay was firstly established and evaluated in China after a series of tests, including optimization of reaction conditions, sensitivity and specificity tests, and the application tests of 35 samples. It was shown that a 435 bp specific DNA fragment of the RHDV2 capsid protein (VP60) gene was synthesized in vitro and the recombinant plasmid pMD-19T-RHDV2 was constructed. The sensitivity of the established RT-PCR detection method for RHDV2 could reach about 230 copies of cloned viral genomic fragments of RHDV2, and there was no amplification for RHDV, pGM-T-EBHSV, Pasteurella multocida, Escherichia coli and Salmonella from rabbits by this method. Besides, the application results showed that there were not RHDV2 in the experimental infection RHDV samples and clinical samples. The present research set basis for RHDV2 control study and supplied a back-up method for the rapid detection and prevention of RHDV2.

Key words: rabbit hemorrhagic disease, RHDV2, RT-PCR, overlap extension PCR

中图分类号:

S855.3

杨泽晓, 赵希仑, 李岩, 王波, 姚学萍, 王印, 耿毅, 蒙正群, 白瑜. 兔出血症病毒2型的VP60基因保守区人工合成及RT-PCR检测方法初探[J]. 浙江农业学报, 2016, 28(10): 1650-1656.

YANG Ze-xiao, ZHAO Xi-lun, LI Yan, WANG Bo, YAO Xue-ping, WANG Yin, GENG Yi, MENG Zheng-qun, BAI Yu. Artificial synthesis of VP60 conserved gene fragment of rabbit hemorrhagic disease virus type 2 and preliminary study of its RT-PCR detection method[J]. , 2016, 28(10): 1650-1656.

参考文献

[1] 陈溥言.兽医传染病学[M].5版,北京:中国农业出版社,2008: 410-412.
[2] JOANA A, WESSEL V L, JACQUES L P, et al. Rabbit haemorrhagic disease (RHD) and rabbit haemorhhagic disease virus (RHDV): A review[J]. Veterinary Research , 2012, 43(6):12-31.
[3] 陈柳,云涛,刘光清,等. 兔出血症病毒衣壳蛋白VP60在杆状病毒表达系统中的表达及细胞内定位[J]. 浙江农业学报,2010, 22(2):135-139.
CHEN L, YUN T, LIU G Q, et al. Expression and localization analysis of VP 60 protein of Rabbit hemorrhagic disease virus in insect cells-baculovirus expression system[J]. Acta Agriculturae Zhejiangensis , 2010, 22(2):135-139. (in Chinese with English abstract)
[4] LE GALL-RECULÉ G, ZWINGELSTEIN F, BOUCHER S, et al. Detection of a new variant of rabbit haemorrhagic disease virus in France[J]. Veterinary Research ,2011,168(5):137-138.
[5] LE GALL-RECULÉ G, LAVAZZA A, MARCHANDEAU S, et al. Emergence of a new lagovirus related to rabbit haemorrhagic disease virus[J]. Veterinary Research , 2013,44(1):81-94.
[6] LE GALL-RECULÉ G, ZWINGELSTEIN F, LAURENT S, et al. Phylogenetic analysis of rabbit haemorrhagic disease virus in France between 1993 and 2000, and the characterisation of RHDV antigenic variants [J]. Archives of Virology , 2003, 148(1):65-81
[7] DUARTE M, CARVALHO C,BERNARDO S, et al, Rabbit haemorrhagic disease virus 2 (RHDV2) outbreak in Azores: Disclosure of common genetic markers and phylogenetic segregation within the European strains[J]. Infection , Genetics and Evolution , 2015, 35(10):163-171.
[8] PUGGIONI G, CAVADINI P, MAESTRALE C, et al. The new French 2010 Rabbit Hemorrhagic Disease Virus causes an RHD-like disease in the Sardinian Cape hare ( Lepus capensis mediterraneus )[J]. Veterinary Research , 2013, 44(1):96-103.
[9] CAMARDA A, PUGLIESE N, CAVADINI P, et al. Detection of the new emerging rabbit haemorrhagic disease type 2 virus (RHDV2) in Sicily from rabbit ( Oryctolagus cuniculus ) and Italian hare ( Lepus corsicanus )[J]. Research in Veterinary Science , 2014, 97(3):642-645.
[10] WESTCOOT D G, FROSSARD J P, EVEREST D, et al. Incursion of RHDV2-like variant in Great Britain[J]. Veterinary Record , 2014, 174(13):333.
[11] DALTON K P, NICIEZA I, ABRANTES J, et al. Spread of new variant RHDV in domestic rabbits on the Iberian Peninsula[J]. Veterinary Microbiology , 2014, 169(1/2):67-73.
[12] LOPES A M, CORREIA J, ABRANTES J, et al. Is the new variant RHDV replacing genogroup 1 in Portuguese wild rabbit populations? [J]. Viruses , 2015, 7(1):27-36.
[13] DALTON K P, ABRANTES J, LOPES A M, et al. Complete genome sequence of two rabbit hemorrhagic disease virus variant b isolates detected on the Iberian Peninsula[J]. Archives of Virology , 2015, 160(3):877-881.
[14] DUARTE M, HENRIQUES M, BARROS S C, et al. Detection of RHDV variant 2 in the Azores[J]. Veterinary Record ,2015,176(5):130.
[15] YANG Z, WANG B, XU Q, et al. Designment and evaluation of the primers for Rift Valley Fever (RVF) virus RT-PCR detection[J]. Advanced Materials Research , 2014, 989-994: 1115-1119.
[16] WANG X, HAO H, QIU L, et al. Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains[J]. Archives of Virology , 2012, 157(8):1523-1530.
[17] DUARTE M D, CARVALHO C L, BARROS S C, et al. A real time Taqman RT-PCR for the detection of rabbit hemorrhagicdisease virus 2 (RHDV2) [J]. Journal of Virological Methods ,2015, 219: 90-95.
[18] 朱晓文,李林,桑晓宇,等. 裂谷热病毒TaqMan荧光定量PCR检测方法的建立[J]. 中国人兽共患病学报,2012,28(4):315-318.
ZHU X W, LI L, SANG X Y, et al. Development of a TaqMan real-time fluorescent quantitative PCR assay for detection of Rift Valley Fever virus[J]. Chinese Journal of Zoonoses , 2012, 28(4):315-318. (in Chinese with English abstract)
[19] 王印,杨泽晓,韩雪清,等. 兔出血症病毒与欧洲野兔综合征病毒复合RT-PCR检测方法的建立[J]. 中国兽医科学, 2011, 41(11):1165-1170.
WANG Y, YANG Z X, HAN X Q, et al. Development of a complex RT-PCR assay for the detection and differentiation of rabbit hemorrhagic disease virus and European brown hare syndrome virus[J]. China Animal Husbandry and Veterinary Medicine , 2011, 41(11):1165-1170. (in Chinese with English abstract)

兔出血症病毒2型的VP60基因保守区人工合成及RT-PCR检测方法初探

Artificial synthesis of VP60 conserved gene fragment of rabbit hemorrhagic disease virus type 2 and preliminary study of its RT-PCR detection method

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