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目的建立一种基于颜色判断结果的金黄色葡萄球菌(简称金葡菌)快速、敏感和特异的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)方法并应用于食源性疾病样品的金葡菌筛查。方法针对金葡菌耐热核酸酶(thermostable nuclease,nuc)基因特异性序列的6个区域设计2对引物,建立LAMP方法。利用LAMP和普通PCR方法同时检测金葡菌和对照组菌株来验证LAMP方法的特异性;将金葡菌菌液作10倍系列稀释后利用LAMP和PCR方法同时进行检测来评价两者的敏感度,利用LAMP和PCR方法分别对330份食源性疾病样品进行检测来比较两者的检出率。结果 LAMP方法可以利用肉眼进行结果判断,其特异性与普通PCR相当,但其敏感性比普通PCR高10倍,LAMP方法检测下限为10 cfu/ml,PCR方法检测下限为100 cfu/ml;LAMP和普通PCR方法对食源性疾病样品的检出率相符。结论可视化金葡菌LAMP方法是一个快速、敏感和特异的方法,可应用于食源性疾病样品中金葡菌的筛查。
Objective To establish a rapid, sensitive and specific Loop-mediated isothermal amplification (LAMP) method for Staphylococcus aureus (Staphylococcus aureus) based on color judgment and apply it to foodborne disease samples Staphylococcus aureus screening. Methods Two pairs of primers were designed for the six regions of the thermostable nuclease (nuc) gene-specific sequence and the LAMP method was established. The specificity of LAMP method was tested by simultaneous detection of Staphylococcus aureus and control strains by LAMP and ordinary PCR methods. The sensitivity of both LAMP and PCR methods was evaluated by using 10-fold serial dilutions of S. aureus bacteria The detection rate of 330 samples of food-borne diseases were compared by LAMP and PCR respectively. Results The LAMP method could be judged by the naked eye. The sensitivity was 10 times higher than that of ordinary PCR, the detection limit of LAMP was 10 cfu / ml, and the detection limit of PCR was 100 cfu / ml. LAMP The detection rate of foodborne disease samples was in accordance with the common PCR method. Conclusion The Staphylococcus aureus LAMP method is a rapid, sensitive and specific method for the screening of Staphylococcus aureus in foodborne disease samples.