基于可视化环介导等温扩增技术快速检测金黄色葡萄球菌

Rapid Visual Detection of Staphylococcus aureus Using Loop-Mediated Isothermal Amplification

应用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)建立基于颜色判定的快速、灵敏的金黄色葡萄球菌检测方法.根据金黄色葡萄球菌特异性靶基因SAR0395设计LAMP引物,建立可视化LAMP检测方法.优化的反应体系为:1.6μmol/L内引物(FIP和BIP),0.2μmol/L外引物(F3和B3),4.0 mmol/L MgSO4,1.0 mmol/L dNTPs,0.4 U/μL Bst 2.0 WarmStartTMDNA聚合酶,120μmol/L羟基萘酚蓝,扩增温度60℃,扩增时间60 min.在可视化LAMP体系中添加两条环引物(LF和LB)反应时间可缩短至20 min.该可视化LAMP反应60 min,其基因组灵敏度可以达到0.0103 fg/μL,菌落灵敏度可以达到1.9 CFU/mL;利用46株金黄色葡萄球菌和24株非金黄色葡萄球菌证实该可视化LAMP具有良好的特异性和可靠性.本研究中建立的可视化LAMP可为金黄色葡萄球菌的快速检测提供一种新的技术.

In this paper, a simple, rapid and sensitive method for the detection of Staphylococcus aureus based on color determination was established by using loop-mediated isothermal amplification (LAMP). LAMP primers were designed based on the specific target gene SAR0395 of S. aureus, and a visualized LAMP reaction system was built. The optimized conditions were determined as follows: inner primers (FIP and BIP), 1.6 μmol/L;outer primers (F3 and B3), 0.2 μmol/L;MgSO4, 4.0 mmol/L;dNTPs, 1.0 mmol/L, Bst 2.0 WarmStart. DNA polymerase, 0.4 U/μL;hydroxynaphthol blue, 120 μmol/L;and amplification at 60 ℃ for 60 min. Two loop primers (LF and LB) added to the LAMP system could reduce the reaction time to 20 min. After 60 minutes of reaction, the sensitivity of the LAMP assay was 0.010 3 fg/μL for genomic DNA and 1.9 CFU/mL for colonies. The specificity and reliability were confirmed by using 46 S. aureus and 24 non-S. aureus strains. The LAMP assay established in this study provides a new technique for rapid and visual detection of S. aureus.