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

WU Xuwen;WEN Wangrong;LI Peizhang;NI Jinliang;JIAN Shiming;Department of Clinical Laboratory,The First Affiliated Hospital of Jinan University;Department of Clinical Laboratory,Kiang Wu Hospital;  
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 16 SrDNA 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 2.75×102 CFU/mL for E.coli,2.43×103 CFU/mL for K.pneumoniae,3.13×102 CFU/mL for S.aureus and 3.03×102 CFU/mL for MRSA.Multiplex PCR method was compared with conventional blood culture method(BCM)by apractical 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.
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