Fluorescence Quantitative PCR For Rapid Detection Of Clinical Common Bacteria

Objectives:The objective is to establish a method of dual-color fluorescence quantitative PCR for rapidly detecting of clinical common bacteria and discriminating their Gram typeMethods:Universal primers for all bacteria and probes for gram-negative and gram-positive bacteria were designed in the conservative region of the bacterium 16sRNA gene.Probes were marked with MGB,FMN and HEX.The specificity and universal of primers and probes for bacteria were validated with conventional PCR.The conditions of dual-color fluorescence quantitative PCR were optimized.11 species of standard bacteria,19 species of clinical isolated bacteria,Hepatitis B virus,Cryptococcus neoformans,Candida albicans and human genome DNA were analyzed by the dual-color FQ-PCR for the specificity of the method.Different concentration bacteria were analyzed by the method for its sensitivity.Results:The amplified products of conventional PCR from 11 species of standard bacteria and 19 species of clinical isolated bacteria were detected.There was no amplified products were detected from Hepatitis B virus,Cryptococcus neoformans,Candida albicans and human genome DNA.With the G+probe,fluorescence quantitative PCR displayed positive results for 14 gram-positive bacteria but negative for 16 Gram-negative bacteria.There were vice versa with the G-probe.Ct values from the G+ probe were the 17.0-23.0 and 11.0-23.from G-probe.There were no positive results for Hepatitis B virus, Cryptococcus neoformans,Candida albicans and human genome DNA with the FQ PCR.The low limit of the dual-color fluorescence quantitative PCR were 35 CFU/ml for Escherichia coli and 100 CFU/ml for Staphylococcus aureus.Conclusions:(1) The primers and probes are special and universal all bacteria and can be used for detecting of clinical common bacteria and discriminating their Gram type.(2) The dual-color fluorescence quantitative PCR established has sufficient specificity and sensitivity for detecting of clinical common bacteria.(3)The method established is simple,rapid and cheap for detecting of clinical common bacteria and will be useful in clinical microbiology laboratory.