三种食源性致病菌的多重LAMP检测方法研究

Development of multiplex loop-mediated isothermal amplification assays to detect three kinds of food-borne pathogens

针对沙门氏菌的invA基因、金黄色葡萄球菌的nuc基因和志贺氏菌的ipaH基因分别设计LAMP引物,通过对实验条件和反应体系的优化,分析鉴定扩增产物,验证了LAMP方法的灵敏度及特异性,初步建立了针对三种致病菌的多重LAMP(m-LAMP)检测方法。分别对沙门氏菌、金黄色葡萄球菌和志贺氏菌的基因组DNA提取物梯度浓度稀释后进行实时荧光检测,结果表明:多重LAMP反应体系检测沙门氏菌、金黄色葡萄球菌和志贺氏菌的敏感度均为10fg/反应。对目标菌和其他非目标菌的特异性检测均未出现假阳性和假阴性结果,并通过熔解温度曲线分析对目标菌的扩增产物进行初步鉴定。鉴定结果表明:该方法快速准确、简便高效、特异性强,可广泛应用于致病菌的筛选检测。

A multiplex loop-mediated isothermal amplification （m-LAMP） method to rapidly detect food-borne pathogens is developed. The primers are designed according to the sequences of invA gene of Salmonella, nuc gene of Staphylococcus aureus and ipaH gene of Shigella. The reaction system is optimized.The amplification products are analyzed by melting curves, electrophoresis and real-time flu-orescence observation as well.The specificity and sensitivity of this multiplex loop-mediated isothermal amplification （m-LAMP） method is evaluated and the method is established. No false positive and negative results emerge during tests of 5 target and 3 non-target bacteria strains, and the pathogens can be determined by melting temperature on melting curves. The detection limits are 10 fg DNA/ tube. This proposed method provides a potential and valuable means for detection of food-borne patho-gens, especially for its rapidity, simplicity and high specificity.