摘要
根据对虾白斑综合征病毒(WSSV)囊膜蛋白VP28基因保守序列,利用Primer Explorerv 4.0软件设计了4条引物,建立了白斑综合征病毒环介导等温扩增快速检测方法,对反应温度和反应时间等参数进行了优化,同时将建立的LAMP检测方法与巢式PCR进行了比较分析。结果表明,LAMP最适反应在64℃恒温条件60min内完成,凝胶电泳呈现梯型条带;反应体系中添加SYBR Green I荧光染料后,绿色的阳性结果明显区别于橙色阴性结果。LAMP方法的最低检出限为100拷贝/μL,灵敏度较巢式PCR高100倍,而且LAMP方法在1h内即可完成检测,操作简单,无需复杂仪器,肉眼可直接观察检测结果。用建立的LAMP方法对临床发病南美白对虾样品进行了检测,结果表明,LAMP方法适合对虾白斑综合征病毒的现场快速检测。
A rapid,sensitive and simple method for white spot syndrome virus（WSSV） detection was established.Loop-mediated isothermal amplification（LAMP） assay is a novel method of gene amplification with high specificity,sensitivity and rapidity,which can be applied for disease diagnosis in shrimp aquaculture.The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target.In the present study,a set of four specific primers were designed based on the conservative sequence of envelope protein gene VP28 using Primer Explorer v4 software,and a rapid detection of WSSV was established by using LAMP assay.The parameters of reaction time and temperature were optimized,and its specificity and sensitivity were assessed.The reactions were carried out at 58,60,62,63,64,65,66,67,68 ℃ for different time（15 -75 min）.A plasmid pMDWSSV carrying target sequence of LAMP detection was constructed.Ten-fold diluted pMDWSSV（107-100 copies/ μL） was used as template for LAMP assay to investigate the detection limit.To determine the specificity,LAMP assays were carried out with DNA templates from other pathogens（Infectious Hypodermal and Hematopoietic Necrosis Virus IHHNV,Taura Syndrome Virus TSV,A.hydrophila,V.alginolyticus,V.parahaemolytious,E.coli）.The results showed the optimum LAMP assay for the rapid detection of WSSV was performed at 64 ℃ for 60 min.The LAMP assay had an unequivocal detection limit of 100 copies/μL,and it was 100 times lower than that of the nested PCR.The nucleic acids of other pathogens were not amplified by this LAMP system with the specific primers,which showed a good specificity.The resulting amplicons were detected using visual observation after the addition of SYBR Green I and gel electrophoresis.We investigated the efficacy of uracil-N-glycosylase（UNG） in preventing contamination in the LAMP assay procedure and explored its effect on the amplification efficiency.Products of LAMP with dUTP adding could b
出处
《水产学报》
CAS
CSCD
北大核心
2010年第4期598-603,共6页
Journal of Fisheries of China
基金
浙江省科技计划项目(2005C33053)
宁波市农业科技攻关项目(2008C10041)
宁波市海洋与渔业专项资金项目(7-3)
关键词
对虾
白斑综合征病毒
环介导等温扩增
快速检测
shrimp
white spot syndrome virus
loop-mediated isothermal amplification
rapid detection
