反转录-环介导等温扩增(reverse transcriptase loop-mediated isothermal amplification,RT-LAMP)技术是以RNA为检测模板的扩增反应.针对金黄色葡萄球菌特异保守nuc基因设计多组引物,通过对引物筛选和反应条件优化,建立了检测金黄色葡萄球菌的实时荧光RT-LAMP方法.利用该方法对菌株特异性、纯培养金黄色葡萄球菌灵敏度和人工污染脱脂乳样品灵敏度进行检测.等温65℃条件下,30 min内完成RT-LAMP反应.该方法具有较强的菌株特异性,对纯培养金黄色葡萄球菌灵敏度检测限为20 fg/μL,在人工污染脱脂乳样品的检测中,灵敏度达到230 CFU/g.建立的实时荧光RT-LAMP方法可以为金黄色葡萄球菌现场快速检测提供有力手段.%In this paper,a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) method has been developed to detect Staphylococcus aureus.Series of primers were designed according to the specific conservative nuc gene,The optimized primer usage and reaction conditions for Staphylococcus aureus detection using RT-LAMP was established.The specificity and sensitivity of the method was studied using Staphylococcus aureus-contaminated skim milk,and was compared to the reverse transcription polymerase chain (RT-PCR) method in terms of sensitivity.The RT-LAMP assay successfully detected Staphylococcus aureus within 30 minutes at 65 ℃.There were 5 out of 26 strains of Staphylococcus aureus detected.The sensitivity of detecting the nucleotide extract of the contaminated skim milk samples was 230 CFU/g,which was 100 fold higher than that of RT-PCR.This RT-LAMP method is a powerful way for rapid detection for Staphylococcus aureus.
展开▼
