EMA-PCR检测金黄色葡萄球菌活菌的方法

Method to Detect Only Live Staphylococcus Aureus by EMA-PCR

根据金黄色葡萄球菌(SA)耐热核酸酶编码基因(nuc基因)设计特异性引物,对试验菌株进行PCR检测,结果显示,金黄色葡萄球菌扩增出大小为480bp的特异性条带,而其他对照菌株未扩增出条带。由于传统PCR技术无法区分样品中的死细菌与活细菌,从而使检测结果往往出现很高的假阳性。本试验利用EMA能穿过死细菌的细胞膜并在光激活的作用下能与基因组DNA共价结合,从而能抑制死菌DNA进行PCR扩增的特性,建立了一种快速、有效的检测金黄色葡萄球菌活菌的EMA-PCR方法,较传统PCR大大提高了检测的准确性和可靠性,该方法检测灵敏度可达15CFU/mL。

A primer was designed according to heat resistant nuclease （nuc） encoding gene of Staphylococcus aureus （SA） and the 480 bp amplicon was specifically detected in SA but not in control strains. Traditional PCRtechnique can not distinguish dead and live bacteria in samples, which results in relatively high false positives. In this study, EMA can pass through the cell membrane of dead bacteria and conjugate with genomic DNA throughactivation by light, which can inhibit PCR amplification of the dead bacteria. Based on the EMA-PCR, a rapid and effective detection method for SA was established. Compared with the conventional PCR, the sensitivity of thismethod is up to 15 CFU/mL, which greatly improves the accuracy and validity of the detection.