逆转录荧光PCR鉴定活性铜绿假单胞菌方法的建立

Reverse transcription real time PCR assay for detection viable Pseudomonas aeruginosa

目的:建立快速检测和甄别铜绿假单胞菌死活状态的定量方法。方法:根据铜绿假单胞菌gyrB基因序列设计引物和探针;提取铜绿假单胞菌菌体总RNA,采用一步法逆转录实时荧光定量PCR反应液对其进行检测。评价逆转录实时荧光定量PCR方法的特异性和敏感性,对收集的临床病人样本和模拟样本进行检测。结果:本试验建立的逆转录实时荧光定量PCR方法能特异、准确、快速的鉴定铜绿假单胞菌菌体死活状态,敏感度达102cfu/m l。临床标本检测结果与细菌培养结果具有较高一致性。10份模拟样本鉴定结果也完全一致。结论:一步法逆转录实时荧光PCR方法能够有效快速检测铜绿假单胞菌,并能甄别死活状态。

Objective：To develop a rapid and reliable technique for detection of Viable Pseudomonas aeruginosa.Methods：The unique gyrB gene of Pseudomonas aeruginosa was chosen as target of detection,the primers and TaqMan probe were designed to specifically amplify the target gene.The RNA was extracted for analysis.The reverse transcription and real time PCR were conducted simultaneously in one step.The specificity and sensitivity were evaluated,and the clinical specimens and mocked double-blind samples were detected through the assay.Results：The assay was able to detect and identify as few as 100 cfu/ml of viable Pseudomonas aeruginosa.There is more consistency between the one step reverse transcription real time PCR and culture for detecting viable Pseudomonas aeruginosa in clinical samples and mocked double-blind samples.Conclusion：An assay combining one-step reverse transcription with real time PCR was successfully developed and validated for detection of viable Pseudomonas aeruginosa.