环介导等温扩增技术检测油棕猝倒病菌的研究  

Study on detection of Pythium splendensby LAMP

作　　者：张慧丽[1] 段维军[1] 张吉红[1] 王江岭[1] 徐瑛[1] 陈先锋[1] 

机构地区：[1]宁波出入境检验检疫局,浙江宁波315012

出　　处：《浙江农业学报》2013年第2期303-308,共6页Acta Agriculturae Zhejiangensis

基　　金：质检公益性行业科研专项项目(201010256);国家质检总局科研项目(2011IK169);国家质检总局科研项目(2013IK279);国家科技支撑计划(2012BAK11B02);浙江省重点科技创新团队(2010R50028);宁波市科技项目(2011C50080);宁波市社会发展项目(2012C50041);宁波出入境检验检疫局科研项目(甬K10-2013)

摘　　要：油棕猝倒病菌是我国进境植物检疫性有害生物。试验应用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术建立了一种快速敏感的油棕猝倒病菌检测方法。根据ITS区基因上的8个位点,共设计了6条引物,并对反应条件和反应体系进行优化。特异性试验结果表明,引物对油棕猝倒病菌的检测具有良好的特异性;灵敏度试验结果表明引物最低检出限为0.12 ng.μL-1比普通PCR高100倍;检测结果经SYBR GreenⅠ染色后,肉眼即可观察结果。该方法能够快速、灵敏、特异地检测油棕猝倒病菌,只需一台水浴锅就可以开展检测,非常适合基层现场检测。Pythium splendens is one of entry plant quarantine pests in China. This study established a rapid and sen- sitive method to detect P. splendens by using loop-mediated isothermal amplification （LAMP）. A set of 6 primers was designed specifically to recognize the ITS region gene of P. splendens. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay were also optimized at 63℃ for 60 min. Specificity test results showed that these primers were specific for detection of P. splendens. Sensitivity test results showed that the detection limit of this LAMP assay was 0. 12 ng. p^L-1, which was 100 times higher than conventional PCR. The results of the LAMP reactions were visualized by adding SYBR Green I to the reaction tube that can simply be judged by the na- ked eye. This LAMP-based assay is rapid, sensitive and specific detection of P. splendens, and the only equipment needed for the amplification reaction is a regular laboratory water bath. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in field conditions.

关 键 词：环介导等温扩增 油棕猝倒病菌 植物检疫性有害生物 

分 类 号：S435.659[农业科学—农业昆虫与害虫防治]