RT-PCR检测金黄色葡萄球菌

Reverse transcription-PCR assay for detection of viable Staphylococcus aureus

目的探讨检测金黄色葡萄球菌及其活菌的RT-PCR方法。方法用RT-PCR方法对金黄色葡萄球菌的spa基因进行检测,并做灵敏度和特异性测定,用RT-PCR检测细菌灭活前后的spa基因。结果用spa基因检测金黄色葡萄球菌灵敏度为1.5×104CFU/mL;Spa引物能特异性扩增出金黄色葡萄球菌的标准株和14株临床株的目的片段,对大肠埃希菌、铜绿假单胞菌、表皮葡萄球菌和化脓性链球菌则无特异性扩增条带,而对白色念珠菌有较弱条带扩出;细菌灭活前可以检测出目的基因,灭活后4℃放置24、48和72 h均无目的基因片段扩出。结论可以用spa基因对金黄色葡萄球菌进行活菌检测。

Objective To observe the effect of reverse transcription-polymerase chain reaction（RT-PCR） in detecting viable Staphylococcus aureus using.Method The spa gene of Staphylococcus aureus was detected by mRNA-based RT-PCR both before and after inactivation.The sensitivity and specificity of the RT-PCR method were determined.Result The Special fragment of Staphylococcus aureus was extended by the pair of primers.There was no crossreaction with E.coli,Pseudomonas aeruginosa,Staphylococcus epidermidis,Pyogenic streptococcus.The sensitivity of detection was 1.5×10^4 CFU/mL.mRNA from uninactivated cells was detected,while in inactivated cells,.mRNA became undetectable when dead cells were held at 4 ℃ temperature for over 24 h.Conclusion This gene can be used to detect viable Staphylococcus aureus.