Objective:To establish an authentication method for Eupolyphaga sinensis based on loop-mediated i-sothermal amplification(LAMP).Methods:Two pairs of primers(outer primer DB-F3,DB-B3 and inner primer DB-FIP,DB-BIP)were designed according to Eupolyphaga sinensis CO Ⅰ specific loci.The reaction system containing template,primers,Bst DNA polymerase and methyl red-phenol red indicator amplified at 63℃ for 90 min.The LAMP results were determined by visual observation and compared with DNA barcoding and pol-ymerase chain reaction(PCR)methods to investigate the specificity and sensitivity.Results:All 11 batches of the authentic Eupolyphaga sinensis sample tubes colour changed from purple red to orange yellow,while sample tubes of 1 batch of Steleophaga plancyi,9 batches Opisthoplatia orientalis and 1 batch of the adulterant,Cybister tripunctatus orientalis,remained purple red after the reaction.The LAMP results were consistent with those of DNA barcoding and PCR.The detection limit of LAMP was 0.984 ng·mL-1.Conclusion:The established LAMP method is ac-curate,sensitive and easy to operate,low equipment required,and can be applied to the rapid screening of the authentication of Eupolyphaga sinensis.
关键词
地鳖/环介导等温扩增技术/DNA条形码/聚合酶链式反应/中药材鉴别/目视检查/快速鉴别
Key words
Eupolyphaga sinensis/loop-mediated isothermal amplification/DNA barcoding/polymerase chain reaction/authentication of Chinese medicinal materials/visual check/rapid identification
维普
万方数据