| Objective:1.To explore a simple,rapid,effective and economical method for the preparation of bacterial DNA.2.To establish a real-time fluorescent quantitative PCR technique for rapid detection of bacteria and preliminary bacterial gram-classification.3.Taking the results of the bacterial susceptibility test obtained by BioMerieux VITEK-2 Com-pact bacterial analysis and drug susceptibility test system as the gold standard.Clinically common bacteria growing situation was detected in the M-H broth culture medium with antibiotics by the established real-time fluorescence quantitative PCR technique,finding the appropriate susceptibility and resistance cutoff value to determine the susceptibility and resistance phenotype of bacteria to antibiotics.Methods:1.The Escherichia coli and Staphylococcus aureus DNA were prepared by 8simple bacteria DNA preparation methods,and compared the effect of different boil time on the DNA quality and quantity and observed the DNA integrity,content,purity,as well as the effect of Real-time fluorescence quantitative PCR amplification reaction which use it as a template.2.Universal primer,universal probe and gram-typing probe were designed to target bacteria 16S rRNA gene,A variety of bacterial DNA was detected by real-time fluorescence quantitative PCR with universal primers and universal probes.The preliminary gram-type of bacteria was carried out by real-time fluorescence quantitative PCR with universal primers and typing probes.3.The shortest culture time for the growth of bacteria was looked for by the way of analyzing the growth curve of Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa.And then the relative growth rate of clinical isolates with known drug-susceptibility test results were detected in antibiotic-containing M-H broth by the real-time fluorescence quantitative PCR technique established in this study,and to explore optimal drug-resistance and susceptibility cutoff value(Intermediaries as resistant)to determine the susceptibility and resistance phenotype of bacteria to antibiotics.4.The cutoff value of susceptibility and resistance phenotype of bacteria to antibioticswasverifiedusingotherbacteriaexceptforEscherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa isolated clinically,and calculate the coincidence rate.Results:1.There was no significant difference in the amount of DNA after boiling for 5minutes or 10 minutes(P>0.05).There was no significant difference in the PCR amplification reaction effect which use DNA after boiling for 5 minutes or 10 minutes as a template.Boiling for 5 minutes was able to meet the PCR test requirements.The integrity,content and PCR amplification effect of DNA prepared by alkaline-SDS boiling method were obviously higher.2.The universal probe could correctly distinguish the bacterial DNA and non-bacterial DNA.The detection limit was 1×10~2cfu/ml for Escherichia coli,1×10~3cfu/ml for Staphylococcus aureus,and 1×10~2cfu/ml for Pseudomonas aeruginosa.Through the optimization of the experimental conditions,the typing probe can distinguish gram-negative and gram-positive bacteria,and the detection limit of Escherichia coli and Pseudomonas aeruginosa can reach 1×10~1cfu/ml.3.The shortest culture time of three standard strains was 3h.The research on 30strains of Escherichia coli isolated clinically showed that the critical relative growth rate of bacteria was 0.4416(the relative growth rate of bacteria?0.4416 was susceptibility,relative growth rate>0.4416 was resistance.),the sensitivity of bacteria susceptibility phenotype to antibiotics was 98.4%,the specificity was 100%.The experiments on 30 strains of Staphylococcus aureus isolated clinically showed that the critical relative growth rate of bacteria was 0.4010,the sensitivity and specificity of bacteria susceptibility phenotype to antibiotics was 100%.The study on 30 strains isolates of Pseudomonas aeruginosa isolated clinically showed that the relative growth rate of bacteria 0.4830 as the cutoff value,the sensitivity of bacteria susceptibility phenotype to antibiotics was 60%,and the specificity was 64%.4.For 20 strains Gram-negative bacilli,with 0.4416 as the cutoff value of susceptibility and resistance,the total concordance was 92%with BioMerieux VITEK-2 Com-pact bacterial analysis and drug susceptibility test system,there are 5major errors(judging susceptibility as the resistance is the major error,judging resistance as the susceptibility is the great error),which was 8%of the total number of test results.For 10 strains Gram-positive cocci,with 0.4010 as the cutoff value of susceptibility and resistance,the total concordance was 93%,there are 2 major errors,accounting for 7%of the total number of test results.Conclusions:1.The alkaline-SDS-boiling method for bacterial DNA preparation is not only simple and rapid,but also the obtained DNA is most suitable for Real-time fluorescent quantitative PCR experiments.2.The established real-time fluorescence quantitative PCR technique has a low detection limit in the detection of bacteria,and can be applied to detection and gram-type of bacteria in clinical aseptic body fluid.3.Using real-time fluorescence quantitative PCR method to detect the relative growth rate of bacteria,which can help us to judge the susceptibility and resistance phenotype of bacteria to antibiotics.High sensitivity and specificity could be ebtained by this method for Escherichia coli and Staphylococcus aureus,For Pseudomonas aeruginosa,the cutoff value obtained by this method is undesirability for judging antimicrobial susceptibility and resistance.4.Applying the cutoff value of susceptibility and resistance obtained through representative bacteria to the same kind of bacteria,the total concordance was highly consistent with BioMerieux VITEK-2 Com-pact bacterial analysis and drug susceptibility test system,but it influenced greatly by the percentage of different bacteria,therefor the universality of the cutoff value has some boundedness,maybe need increase and subdivide the bacteria species to complete the experiment. |
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