实时逆转录聚合酶链式反应检测活的金黄色葡萄球菌

Detection of Live Staphylococcus aureus Using Real-time Reverse Transcription-polymerase Chain Reaction

针对基于DNA水平的聚合酶链式反应(DNA-PCR)检测食品中金黄色葡萄球菌时会出现假阳性结果的缺点,建立一种能够区分死、活金黄色葡萄球菌的检测方法。根据金黄色葡萄球菌的femA基因,自设计Taqman探针、引物,采用一步法逆转录聚合酶链式反应(RT-PCR),以femA mRNA为检测对象,发现只有活的金黄色葡萄球菌显阳性,死亡的则呈阴性;纯培养时,重复检测变异系数为0.15,灵敏度为9×102CFU/mL,检出限可达1/3 CFU/3 mL。实验表明,该研究所建立的一步法逆转录聚合酶链式反应检测法,不仅灵敏度高、特异性强,而且能够有效地区分死、活金黄色葡萄球菌。

This research focuses on establishment of a way that distinguish dead Staphylococcus aureus from the via-ble to avoid false-positive results caused by the ordinary DNA-based polymerase chain reaction in food detection. Primer and probe are designed by ourselves. One-step reverse transcription polymerase chain reaction （RT-PCR） is applied to detect mRNA of Staphylococcus aureus femA. Results show that only living Staphylococcus aureus are positive and deaths are negative. In pure culture, the coefficient of variation is 0.15 by repeat detection, and the analytical sensi-tivity and detection limit of the assay are 9×1062 CFU/mL and 1/3 CFU/3mL respectively. Experiments have shown that the one-step reverse transcription-polymerase chain reaction assay established in this study is not only a rapid, sensitive and specific method but also can effectively distinguish the dead from the viable Staphylococcus aureus.