葡萄球菌耐药基因aph的LAMP检测方法研究

Application of Loop-mediated Isothermal Amplification to Detect the aph Gene in Staphylococcus

随着抗生素大量而广泛的使用,细菌的耐药性已成为世界范围内关注的问题,对细菌携带耐药基因的检测已成为生物安全调查评估的新途径。本实验应用PCR方法扩增新霉素耐药基因aph,将其克隆于p EASY-T1载体并测序。通过Genbank同源性比较,在aph基因保守区设计了一套环介导等温扩增(LAMP)特异性引物,通过对LAMP反应体系和反应条件的优化,建立了耐新霉素aph基因的LAMP检测方法,其最佳工作条件为:甜菜碱浓度为0.8 mol/L,Mg2+浓度为20 mmol/L,反应温度63℃,时间50 min。此方法可在60 min左右完成对aph基因的检测,特异性好。应用系列稀释的aph基因质粒进行敏感性检测比较,结果表明aph基因的LAMP检测灵敏度与PCR相当,检测限约20个拷贝。对38株葡萄球菌分离株的aph基因的LAMP检测表明,aph基因阳性携带率为84.2%。

Since the use of antibiotics in animal breeding, bacterial resistance has become a worldwide concern. Detection of drug-resistance genes is a new method for assessing biosecurity. The aph gene was cloned into the p EASY-T1 vector using PCR-based methods and sequenced. A set of loop-mediated isothermal amplification(LAMP) detection primers was designed against a highly conserved region of the aph gene based on homology with aph loci in Gen Bank. The reaction conditions were optimized and LAMP detection methods for the aph gene were established. The optimal detection conditions were as follows: 0.8 mol/L betaine and 20 mmol/L Mg2+, completed within 50 min in a 63°C isothermal water bath. Compared to PCR detection, the LAMP method can be completed in a shorter time period with higher specificity. The sensitivity threshold was approximately 20.4 copies for the aph gene and was similar to that of the PCR method. The positive detection rate of the aph gene was 84.2% for 38 Staphylococcus aureus isolates.