多重PCR方法快速检测血流感染10种常见病原菌方法的建立与应用

Establishment and Application of multiplex PCR for rapid identification of ten common pathogenic bacteria in bloodstream infection

目的应用多重聚合酶链式反应(PCR)技术,建立肺炎克雷伯菌、鲍氏不动杆菌、肺炎链球菌、屎肠球菌、粪肠球菌、大肠埃希菌、表皮葡萄球菌、金黄色葡萄球菌、铜绿假单胞菌、奇异变形杆菌等临床常见病原菌的快速、高效检测方法,并应用于血培养后病原菌的检测。方法以肺炎克雷伯菌khe基因、鲍氏不动杆菌OXA基因、肺炎链球菌LytA基因、屎肠球菌和粪肠球菌ddl基因、大肠埃希菌phoA基因、表皮葡萄球菌2312基因、金黄色葡萄球菌nuc基因、铜绿假单胞菌ecfX基因、奇异变形杆菌ure基因序列为模板,分别设计特异性引物,建立多重PCR扩增体系并优化。收集临床血培养报阳标本200份,应用所建立的多重PCR法鉴定,并与传统血培养方法比较。结果多重PCR法可特异性扩增出10种病原菌目标DNA条带,200份报阳样本目标菌阳性预测值为60.5%,除传统血培养有1份漏检外,多重PCR与血培养法结果符合率为100%。结论多重PCR方法与传统鉴定方法相比,可大幅度缩短临床培养报阳后鉴定时间,其多种病原菌同时检测的优势,具有良好的临床应用前景。

OBJECTIVE To establish a rapid and efficient detection method for clinical common pathogenic bacteria including Klebsiella pneumonia,Acinetobacter baumannii,Streptococcus pneumonia,Enterococcus faecium,Enterococcus faecalis,Escherichia coli,Staphylococcus epidermidis,Staphylococcus aureus,Pseudomonas aeruginosa and Proteus species by application of multiplex polymerase chain reaction（PCR）technique,and apply to the detection of pathogenic bacteria after blood culture.METHODS According to K.pneumonia khe gene,A.baumannii OXA gene,S.pneumonia LytA gene,E.faecium ddl gene,E.coli phoA gene,S.epidermidis2312 gene,S.aureus nuc gene,P.aeruginosa ecfX gene and P.species ure gene,specific primers were designed to amplify the target gene of above mentioned pathogens by multiplex PCR.Totally 200 positive specimens in blood culture were collected to verify this reaction system.The established multiplex PCR method was used for identification,and the results were compared with the traditional blood culture method.RESULTS Ten target fragments were amplified by multiplex PCR specifically.The positive rate of multiplex PCR system was 60.5%（121/200）,except for one case.The coincidence rate of the multiple PCR and blood culture was 100%.CONCLUSION Compared with traditional identification methods,multiplex PCR method can significantly shorten the time of clinical identification,and simultaneously detect multiple pathogens,which has good clinical application prospects.