基于12S序列的蛤蚧LAMP快速鉴定方法

Rapid identification of Gekko gecko by loop-mediated isothermal amplification based on 12S rRNA sequence

蛤蚧为我国名贵动物中药材,为建立蛤蚧的LAMP可视化检测方法,实现其与混伪品的高效特异鉴别,该研究通过MEGA 6.0分析蛤蚧与其13种混伪品共65条12S rRNA序列,在线设计LAMP引物,并优化扩增条件,考察反应灵敏度,对蛤蚧及其混伪品进行鉴别验证。结果表明,基于12S rRNA序列的蛤蚧及其13种混伪品均可通过BLAST比对准确区分。蛤蚧12S rRNA序列较为保守,10批次30条序列在引物设计区仅含2条单倍型和3个变异位点,成功设计了6条LAMP特异引物。64℃反应1 h,80℃灭活5 min,即可完成蛤蚧的可视化检测,最低检测浓度为5μg·L-1,高于传统PCR 2个数量级。该方法特异性强、灵敏度高、检测方便、反应时间短,且闭管操作,避免了污染,可实现蛤蚧与混伪品的快速可视化鉴别。

Gekko gecko（ Tokay Gecko） is a valuable traditional Chinese medicine. In this study,the loop-mediated isothermal amplification（ LAMP） technique was introduced for visual rapid identification of G. gecko from adulterants. A total of sixty-five 12 S rRNA sequences of fourteen species of G. gecko and its adulterants were obtained. The results showed that G. gecko could be identified from its adulterants through BLAST analysis based on 12 S rRNA regions. The 12 S rRNA sequences of ten batches of G. gecko were conserved. There were only two haplotypes and three variation sites in the available regions for primers design. Six specific LAMP primers were successfully designed online based on 12 S rRNA sequences. The visual rapid detection of G. gecko could be achieved with the optimized conditions（ 64 ℃ for 1 h and 80 ℃ for 5 min）. And the required minimal template concentration was 5 μg·L-1 while conventional PCR with 0. 5 mg·L-1. Consequently,the LAMP method established from this study was rapid,specific,highly sensitive,and simple. It could be applied to detect G. gecko from its adulterants efficiently.