龟鲍曼不动杆菌和摩氏摩根菌双重PCR诊断方法建立

Establishment of Duplex PCR Diagnostic Method for Acinetobacter baumannii and Morganella morganii from Turtle

为建立龟源致病性鲍曼不动杆菌和摩氏摩根菌双重PCR诊断方法,应用鲍曼不动杆菌rpoB基因和摩氏摩根菌gyrB基因的特异性引物,对单重和双重PCR反应的退火温度、引物浓度、MgCl2浓度、dNTP浓度等进行优化。试验结果显示,鲍曼不动杆菌rpoB基因和摩氏摩根菌gyrB基因引物分别进行PCR扩增后,均获得特异性条带,与预期结果一致;rpoB和gyrB基因的上、下游引物各为0.3μmol/L,退火温度为59.3℃,dNTP浓度为0.5 mmol/L,MgCl2浓度为5 mmol/L时,PCR扩增效果最佳;特异性试验结果显示,本方法仅对摩氏摩根菌和鲍曼不动杆菌有扩增,对其他病原菌如肺炎克雷伯氏菌、维氏气单胞菌及恶臭假单胞菌等无扩增;灵敏性结果表明,本方法对鲍曼不动杆菌和摩氏摩根菌最低检测质量浓度分别为4.12×10^-3 ng/μL和1.257×10^-3 ng/μL。通过对临床病料样本的检测分析证明,本方法与单重PCR检测结果一致。本试验所建立的针对两种病原菌检测的双重PCR方法特异性强、灵敏度高,可为龟鳖类鲍曼不动杆菌和摩氏摩根菌的鉴别诊断和临床调查提供帮助。

To establish duplex PCR diagnostic methods for pathogenic Acinetobacter baumannii and Morganella morganii from turtles,the specific primers of rpoB gene of A.baumannii and gyrB gene of M.morganii were used to optimize the reaction system and reaction conditions of single and duplex PCR reaction by annealing temperature,primer concentration,MgCl2 concentration and dNTP concentration.The results showed that the specific bands of the primers of rpoB gene of A.baumannii and gyrB gene of M.morganii were obtained by PCR amplification,which were consistent with the expected results.The PCR amplification effect was the best when the forward and reverse primers of rpoB gene and gyrB gene were 0.3μmol/L,annealing temperature of 59.3℃,dNTP concentration of 0.5 mmol/L and MgCl2 concentration of 5 mmol/L,respectively.It was found that the method was only used for the amplification of A.baumannii and M.morganii,not for other pathogens such as Klebsiella pneumoniae,Aeromonas veronii and Pseudomonas putida.The sensitivity results showed that the minimum detection concentrations was 4.12×10^-3 ng/μL in A.baumannii and 1.257×10^-3 ng/μL in M.morganii.The detection of clinical samples showed that the results were consistent with those of single PCR.The findings indicated that the duplex PCR method for the detection of the two pathogens was highly specific and sensitive,which provides references for the differential diagnosis and clinical investigation of A.baumannii and M.morganii.