环介导等温扩增技术快速检测产气荚膜梭菌的研究

目的:建立环介导等温扩增技术(LAMP)快速检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌(ATCC13124)的α毒素全基因序列为保守序列,设计内、外、环引物,肉眼观察白色沉淀并判断检测结果。结果:LAMP检测产气荚膜梭菌的灵敏度为2.92×102CFU/mL,人工污染产气荚膜梭菌的全脂乳的检测限为15.7CFU/mL,耗时仅1h。对照PCR检测产气荚膜梭菌的灵敏度为2.92×105CFU/mL,人工污染产气荚膜梭菌的全脂乳的检出限1.57×105CFU/mL。采用同样的方法提取DNA,耗时3h。结论:建立LAMP快速、特异检测产气荚膜梭菌的方法,该方法灵敏度是PCR的1000倍,为食品中产气荚膜梭菌的检测构建了一个技术平台。

The Study on the Detection of Clostridium Perfringens by Loop-Mediated Isothermal Amplification

Objective:An assay using loop-mediated isothermal amplification was developed for rapidly detection of Clostridium perfringens.Method:Sequences of alpha-toxin of Clostridium perfrigens(ATCC13124) were used as target sequences to design outer primers,inner primers and loop primers.We judge the results through the visible by naked eye of the white precipitate.Results:The sensitivity of the LAMP assay was 2.92×102 CFU/mL and the detection limit of artificial contamination was 15.7 CFU/mL,the detection could be finished in an hour.Compared with LAMP,the sensitivity of the PCR assay was 2.92×105 CFU/mL and the detection limit of artificial contamination was 1.57×105 CFU/mL.When we used the same method to extract DNA,it would be spend three hours.Conclusion:A rapid and special method was established for detection of Clostridium perfrigens,the sensitivity of the LAMP were 1 000 times as high as that of PCR.This result indicates that LAMP can provide a technical platform for rapid detection of Clostridium perfrigens in food.