基于重组酶聚合酶扩增技术建立实时荧光法快速检测鲍曼不动杆菌的研究

Rapid detection of Acinetobacter baumannii by real-time fluorescence method based on recombinase polymerase amplification

目的建立一种基于重组酶聚合酶扩增技术(recombinase polymerase amplification RPA)快速鉴定鲍曼不动杆菌的实时荧光检测方法。方法根据鲍曼不动杆菌16S-23S rRNA ITS基因特异保守序列设计RPA引物与探针,采用铜绿假单胞菌、白色念珠菌、金黄色葡萄球菌、大肠埃希菌4种非鲍曼不动杆菌作为对照进行鲍曼不动杆菌实时荧光RPA特异性验证;对菌液进行梯度稀释后提取模板,验证方法的灵敏性;采用RPA检测30株鲍曼不动杆菌临床分离株,以验证方法的临床应用性。结果建立的RPA扩增方法在42℃条件下18 min内完成对鲍曼不动杆菌的快速检测,与对照菌相比,仅鲍曼不动杆菌呈现典型的扩增曲线;RPA检测鲍曼不动杆菌最低检出限为2.86 cfu/ml菌液,与PCR及实时荧光PCR结果一致。30株鲍曼不动杆菌临床分离株RPA检测均出现扩增曲线,检出率为100%。结论建立的鲍曼不动杆菌实时荧光RPA方法具有高特异、灵敏、简便、迅速等优点,可用于鲍曼不动杆菌感染快速检测。

Objective To construct a real-time detection method of recombinase polymerase amplification(RPA) to rapid detection Acinetobacter baumannii.Methods A set of primer and probe were designed for RPA detection based on the specific conserved sequence of 16 s-23 s rRNA ITS gen of A.baumannii according to manufacturer guidelines(TwistDx Inc.Cambridge,UK),the four non-Acinetobacter isolates of Pseudomonas aeruginosa,Candida albicans,Staphylococcus aureus,and Escherichia coli as negative control were used to test the specificity of real-time fluorescent RPA method using the primers and probe designed.The bacterial fluid was serially diluted 10-fold with physiological saline and the template was extracted to verify the sensitivity of the RPA method.At the same time,30 clinical isolates of A.baumannii were detected respectively by RPA and PCR to verify the clinical application of RPA.Results The RPA method could achieve successfully rapid detection of A.baumannii within 18 minutes under a low-temperature condition of 42℃.Compare with negative control strains,the typical amplification curve was only detected in A.baumannii.The detection limit of the RPA method was 2.86 cfu/ml,which was consistent with the PCR and qPCR.Amplification curves of RPA were observed in all 30 clinical isolates of A.baumannii,and the detection rate was 100%.Conclusion This study established successfully a real-time RPA method for detection of A.baumannii,which has high specificity,high sensitivity,simple and fast operation,and provides a new method for rapid detection of A.baumannii.