建立检测血液中EC、KP、SA和MRSA的多重PCR方法  

Development of a multiplex PCR method for the rapid detection of Escherichia coli,Klebsiella pneumoniae,Staphylococcus aureus and methicillin-resistant Staphylococcus aureus

作　　者：吴许文 温旺荣[1] 李沛樟 倪金良 简仕铭 WU Xuwen;WEN Wangrong;LI Peizhang;NI Jinliang;JIAN Shiming(Department of Clinical Laboratory,The First Affiliated Hospital of Jinan University,Guangzhou,Guangdong 510163,China;Department of Clinical Laboratory,Kiang Wu Hospital,Macao 999078,China)

机构地区：[1]暨南大学附属第一医院检验科,广东广州510163 [2]镜湖医院检验科,澳门999078

出　　处：《国际检验医学杂志》2019年第1期19-24,28,共7页International Journal of Laboratory Medicine

摘　　要：目的建立快速检测血液中常见致病菌包括大肠埃希菌(EC)、肺炎克雷伯菌(KP)、金黄色葡萄球菌(SA)及耐甲氧西林SA(MRSA)的多重聚合酶链反应(PCR)方法,有利于败血症的及时治疗。方法建立多重PCR方法对EC phoA基因、KP mdh基因、SA femA基因和MRSA mecA基因进行检测,并将16SrDNA作为致病菌感染对照。结果采用建立的多重PCR方法进行检测的特异性为100%,EC检测限为2.75×102CFU/mL,KP为2.43×103 CFU/mL,SA为3.13×102 CFU/mL,MRSA为3.03×102 CFU/mL;用多重PCR方法和传统血培养方法对300个血标本进行检测,传统血培养方法检测出187个血标本内阳性,多重PCR方法有4个血标本为假阴性,未能被检出。结论建立的多重PCR方法可简便、及时地检测血流感染中的EC、KP、SA和MRSA。Objective Septicemia is caused by a variety of pathogenic bacteria,including Escherichia coli,Klebsiella pneumoniae and Staphylococcus aureus(especially for methicillin-resistant S.aureus,MRSA),thus to establish a multiplex PCR method for the rapid detection of common pathogenic bacteria in the blood,is in favor of the timely treatment of septicemia.Methods The target genes of this assay were phoA for E.coli,mdh for K.pneumoniae,femA for S.aureus and mecA for MRSA,and 16S rDNA as pathogen infection control.Results A perfect concordance(100%)was observed when the specificity was validated with 20 strains belonging to 15 species pathogens containing target strains and other clinical pathogens.The detection limits of the multiplex PCR method were 2.75×10^2 CFU/mL for E.coli,2.43×10^3 CFU/mL for K.pneumoniae,3.13×10^2 CFU/mL for S.aureus and 3.03×10^2 CFU/mL for MRSA.Multiplex PCR method was compared with conventional blood culture method(BCM)by a practical sample test assay with 300 blood specimens.The multiplex PCR method provided nearly concordant results with BCM except a few false-negative results that 4 in total 187 BCM-positive samples could not be determined by PCR method.Conclusion We successfully developed a simple,timely multiplex PCR method for detection of E.coli,K.pneumoniae,S.aureus and MRSA in blood stream infection.

关 键 词：聚合酶链反应 脓毒症 大肠杆菌 克雷伯菌 肺炎 葡萄球菌 金黄色 甲氧西林抗药性 

分 类 号：R446.61[医药卫生—诊断学] R378[医药卫生—临床医学]