猪轮状病毒RT-LAMP检测方法的建立及应用Development and Application of Reverse Transcription Loop-mediated Isothermal Amplification Method for Detection of Porcine Rotavirus of Swine
胡兴义,张双翔,冯旭芳,周碧君,文明,程振涛,王伟,王开功
摘要(Abstract):
为了建立一种适用于猪轮状病毒(PoRV)的逆转录-环介导等温核酸扩增(RT-LAMP)的快速、灵敏检测方法。依据GenBank上登录的PoRV VP7基因保守序列,设计了6条特异性引物,通过对外引物与内引物浓度比、Bst DNA聚合酶浓度、Mg2+浓度、dNTP浓度和反应条件等进行优化。结果显示,当外引物与内引物浓度比为200nmol/L∶2 400nmol/L(1∶12)、Bst DNA聚合酶浓度为0.64 U/μL、Mg2+浓度为2.5 mmol/L、dNTP浓度为1.0mmol/L,在恒温(60℃)条件下作用60min,扩增效果出现明显"梯状"条带,同时对建立的RT-LAMP检测方法进行特异性和敏感性验证,其只有PoRV获得特异性扩增条带,与其他猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪瘟病毒等无交叉反应,具有良好的特异性,最低检测分子拷贝数为1.0×102拷贝/μL,具有极高的敏感性。反应结束后肉眼可见阳性扩增产物出现白色沉淀,加入SYBR GreenⅠ观察颜色变化可以判定结果。该方法适用于野外、基层部门和海关快速检测PoRV的新方法,在临床上有良好的推广意义。
关键词(KeyWords): 猪轮状病毒(PoRV);RT-LAMP;建立;应用
基金项目(Foundation): 贵州省科学技术厅农业攻关项目(黔科合NY[2015]3009-1号);; 贵州省生猪质量安全工程技术研究中心建设项目(黔科合农C字[2011]4022号);; “贵州省动物疫病防控与兽医公共卫生保障科技创新人才团队”(黔科合人才团队[2015]4016号)
作者(Author): 胡兴义,张双翔,冯旭芳,周碧君,文明,程振涛,王伟,王开功
DOI: 10.13242/j.cnki.bingduxuebao.003071
参考文献(References):
- [1]Estes M K,Cohen J.Rotavirus gene structure and function[J].Microbiol Rev,1989,53:410-449.
- [2]田冲,欧阳金旭,罗碧毅,熊涛,钱平.仔猪病毒性腹泻的流行病学研究进展[J].长江大学学报(自科版),2013,05:57-60+63.
- [3]Wakuda M,Ide T,Sasaki J,Komoto S,Ishii J.Porcine rotavirus closely related to novel group of human rotaviruses[J].Emerging Infectious Diseases,2011,17(8):1491.
- [4]Aianot P K,Chupin S A,Dorohenkova G N,Kudriavtsev V A.Use of PCR and Nueleotide sequenee an alysis Of the VP7gene for deteetion of bovine Rotaviruses and identifieation Of their G serotypes[J].Mol Gen Mikrobiol Virusol,2003,(2):25-32.
- [5]时洪艳,冯力,陈建飞,孙东波,吴波平.猪轮状病毒OSU强毒株VP7基因的克隆及其抗原表位原核表达和免疫学活性分析[J].中国预防兽医学报,2008,02:136-140.
- [6]陈晓春,王继文,吴华伟,蔡青秀,邓永,高金源,郎洪武.猪轮状病毒L1株VP7基因克隆及序列分析[J].中国兽药杂志,2014,01:6-10.
- [7]滑翔,胡中凯,黄小波,文心田,曹三杰,文翼平,伍锐,邓静,赵松,尹人杰,常晓霞,欧阳达,张仙.检测猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪轮状病毒的cDNA芯片的构建[J].畜牧兽医学报,2015,12:2235-2242.
- [8]Notomi T,Okayama H,Masubuchi H,Yonekawa T,Watanabe K.Loopmediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
- [9]Shirato K,Yano T,Senba S,Akachi S,Kobayashi T.Detection of Middle East respiratory syndrome coronavirus using Reverse Transcription Loop-mediated Isothermal Amplification(RT-LAMP)[J].Virology Journal,2014,11(1):1-11.
- [10]Mori Y,Nagamine K,Tomita,Notomi T.Detecting of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[J].Biochem Biophys Res Commun,2001,289:150-154.
- [11]Duan Y B,Ge C Y,Zhang X K,Wang J X,Zhou M G.Development and evaluation of a novel and rapid detection assay for botrytis cinerea base on loop-mediated isothermal amplification[J/OL].PLoS One,2014,9(10):e111094.
- [12]耿合员,汪圣强,谢晓谦,肖雨,张汀,谭文杰,苏川.基于颜色判定的RT-LAMP技术检测HCoV-NL63冠状病毒基因[J].病毒学报,2016,01:56-61.
- [13]Estes M K,Greenberg H B.Rotaviruses[A].In:Knipe D M,Howley P M(Eds),[J].Fields Virology[J].Lippincott williamas and Wilkins,Philadelphia,2013,1347-1401.
- [14]Iturriza-Gomara M,Dallman T,Banyai K,Bottiger B,Buesa J.Rotavirus genotypes Co-circulating in Europe between 2006and 2009as determined by EuroRotaNet,apan-European collaborative Strain surveillance network[J].Epidemiol Infect,2011,139(6):895-909.
- [15]Iturriza-Gomara M,Dallman T,Banyai K,Bottiger B,Buesa J.Rotavirus surveillance in Europe,2005-2008:web-enabled reporting and real-time analysis of genotyping and epidemiological data[J].J Infect Dis,2009,200suppl 1:S215-221.
- [16]Maeda H,Kokeguchi S,Fujimoto C,Tanimoto I,Yoshizumi W.Detection of periodontal pathogen Porphyromonas gingivalis by loop mediatedisothermal amplification method[J].Fems Immunol Med Microbiol,2005,43(2):233-239.
- [17]Ogura A.Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue[J].Bio Techniques,2009,46(3):167-172.
- [18]Tomita N,Mori Y,Kanda H,Notomi T.loop-mediated isothermal amplification(LAMP)of gene sequences and simple visual detection of products[J].Nature Products,2008,3(5):877-882.