Establishment Of A Method For Rapid Detection Of Escherichia Coli And Its Common Drug Resistance Phenotype By Microbial Culture Combined With Molecular Identification

The extensive use of antibiotics,especially the use without indication,improper selection of antibiotics,overtreatment,and frequent drug changes,lead to the serious antibiotic resistance.Drug-resistant bacteria,especially multidrug-resistant bacteria,have become a major threat to public safety.Rational use of antibiotics is one of the core factors to solve this dilemma,which needs the support of clinical examination.In clinic,the isolation and culture of bacteria,biochemical identification,drug sensitivity test,and mass spectrometry analysis were often used for species identification and drug sensitivity analysis of bacteria.However,these methods have some limitations.In recent years,molecular diagnostic techniques have been widely used in the identification of bacteria and the detection of antibiotic resistance genes.Molecular diagnosis based on the nucleic acid amplification has been widely used in the clinical examination since the invention of PCR.However,due to the variety of drug resistance mechanisms and the inconsistency between the drug resistance phenotypes and drug resistance genes,the discrimination of drug resistance phenotypes based on drug resistance genes was not the best.In this study,the whole genome of Escherichia coli was screened by the local Blast and online Blast,and the specific gene hypothetical protein gene（ID:13702648）was obtained.It has been confirmed that this specific gene exists widely in 240 strains of E.coli and can not be detected in the 150 non-E.coli strains,thus it can be used as a specific gene for bacterial species identification.Combined with the enrichment culture of E.coli under antibiotics,a rapid molecular drug sensitivity detection system for PCR and q PCR was established.Both PCR and q PCR rapid molecular drug sensitivity detection systems can detect the drug sensitivity of bacteria at the original concentration of 10^2-4CFU/m L within 30-60 minutes accurately,and the total detection time is no more than 2.5 hours.PCR can detect the drug sensitivity of the bacteria with the original concentration of 10²CFU/m L within 60 minutes,and the drug sensitivity of the bacteria with the original concentration of 10^3-4CFU/m L within30 minutes.q PCR can detect the drug sensitivity of the bacteria with the original concentration of 10²CFU/m L in the shortest enrichment time of 30 minutes real-time and accurately.The q PCR rapid molecular drug sensitivity test system was used to evaluate the drug resistance of the clinical samples to levofloxacin,aztreonam,ampicillin and amikacin,and the results were consistent with the results of hospital drug sensitivity.To sum up,this study organically combined the traditional drug sensitivity identification with molecular diagnosis,and successfully constructed a rapid molecular drug sensitivity identification method of PCR and q PCR based on bacterial specific genes.It has the characteristics of intuition,simplicity,rapidity,sensitivity,and specificity,and can identify the infection and phenotype of common multidrug resistant pathogens in the clinical samples within 2.5 hours.It can provide a rapid and accurate method for the detection of bacterial species and their antibiotic resistance,and provide a technical basis for the development of related diagnostic reagents.