Establishment And Application Of Multiple PCR Method For Rapid Detection Of Common Pathogenic Bacteria

The traditional culture method has always been the "gold standard" for the identification of common clinical pathogens.It includes identification of bacteria separation and a series of biochemical reactions,but it takes a long time,complex operation and low positive rate,which can not meet the clinical large-scale detection.Gram negative bacteria and gram positive bacteria accounted for most of the current infections in large hospitals in China,followed by some common fungal infections,which accounted for a small proportion of bloodstream infections.In bacterial infection,Escherichia coli and Staphylococcus aureus accounted for the vast majority,in recent years,Klebsiella pneumoniae,Acinetobacter Bauman and Enterococcus infection ratio also increased year by year,Pseudomonas aeruginosa,Streptococcus pneumoniae,proteus occupies a small proportion.The application of multiplex polymerase chain reaction technology,in view of the above clinical common bacterial infection of Klebsiella pneumoniae,Bauman Acinetobacter,Streptococcus pneumoniae,Enterococcus faecium,Enterococcus faecalis,Escherichia coli,Staphylococcus epidermidis,Staphylococcus aureus,Pseudomonas aeruginosa,Proteus and other ten kinds of clinical common pathogens bacteria fast and efficient detection methods,and to detect the pathogenic bacteriaThe virulence factor sequences of ten pathogenic bacterias were searched on NCBI,and the specific gene sequences of each pathogen were selected According to Klebsiella pneumonia khe gene,Acinetobacter baumannii OXA gene,Streptococcus pneumonia Lyt A gene,Enterococcus faecium ddl gene,Enterococcus faecalis ddl gene,Escherichia coli pho A gene and O83H1 gene,Staphylococcus epidermidis 2312 gene and gse A gene,Staphylococcus aureus nuc gene,Pseudomonas aeruginosa ecf X gene and Proteus ure gene.Specific primers were designed to amplify the target gene of above mentioned pathogens by Multiplex PCR.Primers were designed with the primer 5.0 software in the conserved regions of these bacterial sequences.The Tm value of each primer of bacteria was designed at the same temperature,and the amplified fragment was designed as a phase difference of about 50 bp,The specificity and sensitivity of this method were evaluated after optimizing the Multiplex PCR reaction system.which is designed and used to establish Multiplex PCR amplification system respectively on blood culture and culture fluid were detected.500 cases of clinical blood culture and humoral culture were collected and tested with multiple PCR,and the results were compared with those of traditional culture methods.The proposed method can amplify the target DNA band of specific pathogenic bacteria,with clear strip and uniform brightness,no specific bands.500 cases of blood and body fluids at multiple PCR system Yang sample application target bacteria detection rate was 71.2%(356/500),which the single bacterial infection in 310 cases,46 cases of mixed infection,cultivation method of target bacteria detection rate was 70.2%(351/500),which the single bacterial infection in 318 cases,mixed infection in 33 cases,the major infection were fungal infection,followed by some bacteria,such as staphylococcus hominis,Enterobacter cloacae,staphylococcus haemolyticus and Morgan infection.Compared with the traditional identification method,Multiplex PCR can detect a variety of pathogenic bacteria at the same time.Greatly improve the efficiency of clinical blood culture detection.So it has unique advantage in terms of identifying clinic common pathogenic bacteria.