多重聚合酶链反应快速鉴定三种常见致病菌的方法学研究及应用

Methodology Research and Application on Rapid Identifying the Common Pathogenic Bacteria by Multiplex Polymerase Chain Reaction

目的应用多重聚合酶链反应(MPCR)技术,建立铜绿假单胞菌、金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌等常见致病菌的快速、高效鉴定方法,并用于气管切开患者气道分泌物和痰液标本检测。方法根据铜绿假单胞菌opr L基因、金黄色葡萄球菌nuc基因和耐甲氧西林金黄色葡萄球菌mec A基因分别设计特异性引物,通过MPCR方法扩增上述病原菌的目的基因,同时优化MPCR反应体系,检测该方法的特异性、灵敏度和稳定性。收集气管切开患者的气道分泌物和痰液标本,分别采用传统微生物鉴定方法和MPCR法进行鉴定,记录鉴定结果和检测周期。结果 MPCR法在各引物比例条件下均可特异地扩增出3条目的片段,58℃为最佳退火温度,50ng·μL^(-1)DNA模板可被稀释至100倍,重复试验结果与上述结果一致。MPCR法与传统检测方法对临床标本的鉴定结果一致,但是前者相对于后者操作简单,耗时少。结论这种基于MPCR的检测方法操作简单,特异性、灵敏度和稳定性高,检测周期短,成本低,能同时对多种病原菌进行检测,在鉴定气管切开术后的复合菌感染中具有较大优势。

Objective To establish an efficient and accurate method based on multiplex polymerase chain reaction( MPCR) technology and identify common pathogen of tracheotomy infection including Pseudomonas aeruginosa and Staphylococcus aureus. Methods According to Pseudomonas aeruginosa opr L gene,Staphylococcus aureus nuc gene and methicillin- resistant Staphylococcus aureus mec A gene,specific primers were designed to amplify the target gene of above- mentioned pathogens by MPCR. The specificity,sensitivity and stability of this method was evaluated after optimizing the MPCR reaction system. Secretions and sputum specimens were collected from tracheostomy infection patients of ICU and neurosurgery. Specimens were identified by the traditional microbial identification method and MPCR method respectively. Identification results and testing cycle were recorded. Results Three target fragments were detected by MPCR method under the condition of each primer proportion specificly. As well as,58℃ was the best annealing temperature. 50ng·μL^(-1) DNA template could be diluted to 100 times. Results of repeat test were consistent with above results. The clinical specimens identification results of two methods are consistent. MPCR method was simple and timesaving compared to traditional identification method. Conclusion This method based on MPCR technology has some characteristics of simple operation,high specificity,sensitivity and stability. Compared to traditional identification method,MPCR can detect a variety of pathogenic bacteria at the same time with short testing cycle and low cost. So it has high efficiency and unique advantage in terms of identifying compound bacteria of tracheostomy infection.