【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。更多还原

【Abstract】 Pythium splendens is one of entry plant quarantine pests in China.This study established a rapid and sensitive 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·μL-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 naked 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.更多还原