环介导恒温扩增技术可视化检测携带tdh基因的致病性副溶血性弧菌
Rapid method for visual detecting pathogenic Vibrio parahaemolyticus in food by loop-mediated isothermal amplification
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摘要: 建立了环介导恒温扩增技术检测携带tdh基因的致病性副溶血性弧菌。基于副溶血性弧菌高度保守的tdh基因序列,设计了6条特异性引物,两条外引物F3、B3,两条内引物FIP、BIP及两条环引物LF、LB。在Bst DNA Polymerase作用下,60℃恒温水浴进行扩增。对10种细菌共21株菌进行LAMP扩增,所试6株副溶血性弧菌均为阳性,说明引物具有高度特异性。本LAMP方法对纯培养物的灵敏度可达到9.42cfu/m L。对污染食品中副溶血性弧菌的灵敏度为25.3cfu/25g,40~60min内即可完成检测。本方法操作简便、特异性强、灵敏度高,可以为临床提供简单、快速、高灵敏度和高特异性的检测应用。Abstract: A loop-mediated isothermal amplification technique was established to detect pathogenic Vibrio parahaemolyticus by using tdh gene in this paper. Six primers including two outer primers F3/B3, two inner primers FIP/BIP and two loop primers LF/LB were designed according to the highly conserved tdh gene of Vibrio parahaemolyticus. All bacteria strains including 6 pathogenic Vibrio parahaemolyticus strains and 15 other bacterial strains were amplified by using the primers with Bst DNA polymerase at 60℃. Among the 21 bacteria strains, only pathogenic Vibrio parahaemolyticus strains were LAMP positive result, which indicated that the LAMP primers designed were highly specific for target bacteria. The sensitivity of LAMP assay for Vibrio parahaemolyticus reached to 9.42 cfu/m L for the pure culture and 25.3cfu/25 g for the tested contaminated food. The test could complete in 40 ~60min. The LAMP assay established in this study was a sensitive, rapid and simple tool for detecting Vibrio parahaemolyticus.
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