用环介导等温扩增技术快速检测粪便样本中的沙门菌

目的:建立快速检测粪便样本中的沙门菌的环介导等温扩增技术(LAMP),并着重在灵敏度和特异性方面对此方法进行评价。方法:利用LAMP针对沙门菌特定基因invA(靶基因)设计的6条特异引物,通过引物特异性识别特定基因invA上的8个独立区域来快速检测沙门菌;LAMP反应过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果。结果:实时浊度仪监测反应结果表明,LAMP反应在60~65℃等温条件下50 min内完成;如果在反应前添加羟基萘酚兰,蓝色阳性结果很明显区别于紫色阴性结果;LAMP的最低检出限为6.97 pg/μL,PCR为69.7pg/μL,LAMP方法的检测灵敏度是PCR的10倍,且具有良好的特异性。结论:LAMP方法用于快速检测沙门菌,具有检测过程简单、实验装置简便、反应结果肉眼可辨、灵敏度高、特异性强的特点,对非沙门菌菌株的结果呈阴性,表明引物设计有很好的特异性。对粪便样本进行检测,发现具有同样的敏感性和特异性。这表明LAMP法是潜在的和有价值的在粪便样本中直接检测沙门菌的方法,具有快速、简便、低成本的特点。LAMP法适用于快速临床诊断。

Sensitive and Rapid Detection of Salmonella in Stool Samples by Loop-Mediated Isothermal Amplification

Objective： A loop-mediated isothermal amplification（LAMP） assay was developed and validated for the specific detection of Salmonella in stool samples. Methods： One set of primers were designed for recognizing 8 distinct sequences on Salmonella-specific gene invA. Specificity and sensitivity of the primers in the LAMP reac tions for Salmonella detection were determined. To compare the sensitivity and specificity of the LAMP assay, PCR was performed as control. The sensitive of LAMP assay for Salmonella detection in spiked stool samples was tested too. Two methods including monitoring turbidity and adding hydroxy naphthol blue before reaction to reac tion tube were used to determine the negative and positive results. Results： The results showed that target DNA was amplified and visualized by the two detection methods within 50 rain at isothermal temperature 63℃. The sen sitivity of LAMP with detection limits of 6.97 pg/μL is 10 times than that of PCR. Non-Salmonella strains were selected for specificity and the resuhs of the amplification were negative, showed the primers designed had a good specificity. Conclusion： For stool samples, the same sensitivity and specificity were observed. The LAMP method reported here demonstrates a potential and valuable means for directly detection of Salmonella in stool samples, es pecially for its rapidity, simplicity and low cost. The LAMP assay is suitable for rapid clinical diagnosis.