沙门菌属stn基因LAMP快速检测方法的建立及应用

Establishment and application of LAMP method based on stn gene for rapid detection of Salmonella

目的本研究以沙门菌属肠毒素基因(stn)为靶基因,运用环介导恒温扩增技术(LAMP),建立LAMP快速检测方法。方法对沙门菌属stn基因的6个特征区域设计4条引物,利用具有链置换活性的Bst DNA聚合酶在恒温条件下催化新链合成,使目的基因高效扩增;对LAMP反应体系和反应条件进行优化,用沙门菌标准株和非沙门菌标准株检测方法的特异性和敏感性。与显色培养基法同时检测肉、蛋、奶、等样品共计100份,检测方法的实用性。结果以沙门菌属stn基因为靶基因建立的LAMP快速检测方法,沙门菌株阳性检出率100%,非沙门菌株检测均为阴性。100份检验样品检测结果与显色培养基检测结果对比,一致率100%。结论沙门菌属stn基因LAMP检测技术敏感性、特异性高,简便快速,是一种适合口岸及基层检验、检疫部门使用的沙门菌快速检测方法。

Objective To establish LAMP method for rapid detection of Salmonella with Salmonella enterotoxin gene（ stn） as target gene. Methods Four primers were designed based on 6 characteristic regions of stn gene of Salmonella,and Bst DNA polymerase was used to catalyze new chains synthesis under the constant temperature. Then the targeted gene was amplificated efficiently. The LAMP reaction system and conditions were optimized,then the specificity and sensitivity of this method were detected with Salmonella standard strains and non- Salmonella standard strains. One hundred samples including meat,milk and egg,were detected by the LAMP and color culture medium method respectively to validate the practicability of LAMP. Results The positive rate of Salmonella strains and the negative rate of non Salmonella strains were all 100%. The consistent rate of LAMP and color culture medium method was 100% in the detection of 100 samples. Conclusion The stn gene- based LAMP method is highly sensitive,specific,simple and rapid,without expensive instrument and equipment,so it is suitable for rapid detection of Salmonella in ports,primary inspection and quarantine departments.