| Food safety problems caused by food-borne pathogens are becoming more and more serious.To treat foodborne illness,antibiotic usage was the most direct and effective method,while the problem of drug resistance was becoming more and more seriously,it is more difficult to prevent and control foodborne pathogenic bacteria because drug-resistant genes can be transmitted horizontally among microorganisms and superbacterias appeared.Therefore,it is very significant to research the mechanism of drug resistance,detect and control drug resistant strains.In order to ensure the accurate and rapid detection,and diagnosis of drug resistance mechanism of pathogenic bacterial in daily research,food safety monitoring institutions and hospitals,and ensure the accuracy and fairness of the result,the reference materials as"benchmarks"are essential.However,the microbial reference materials were seldom,certificated reference materials for microbial drug resistance encoding genes were almost blank,it is necessary and significant for rapid screening and detection of food-borne pathogens resistance mechanism to develop homogeneous and stable standard reference strains and reference DNA.Target genes were collected from the National Center for Biotechnology Information(NCBI),and target gene sequences were compared by Bio Edit to obtain,constructed recombinant plasmids and strains.All recombinant strains were subcultured for 15 generations,the genetic stability of the plasmid and the DNA was detected by PCR.The limit of detection of the plasmid DNA were detected using PCR and RT-qPCR.Plasmid DNA was extracted and reference materials were prepared by vacuum drying after subpackaged.According to the requirements of GB/T 15000.3-2008 for standard sample making guidelines,the homogeneity and stability of the plasmid DNA reference samples that stored at different temperature with different storage time were evaluated.The determination indexes including the concentration of plasmid DNA,resistance genes detection by PCR and the Ct value for RT-qPCR detection.The major results of this work are as follows:(1)The target fragments associated with fluoroquinolones resistance(qnr A,qnr B,qnr S,aac(6’)-Ib,oqx A,5 common mutations in gyr A and 1 common mutation in par C)were successfully synthesized,and constructed recombinant plasmids and strains.All genes in the recombinant strains can stably inherited within 15 generations.(2)The plasmids DNA in recombinant strains were extracted.The limit of detection of plasmids DNA concentrations measured by conventional PCR were range from 1.85×10~3 to1.77×10~5 copies/μL;The limit of detection of plasmids DNA concentrations measured by RT-qPCR were range from 1.74×10~1 to 3.26×10~4 copies/μL,and the Ct values based on DNA template amplification at different concentrations and log of concentration exhibited excellent linear relationships.the regression coefficients(R~2 value)exceed 0.99.The results showed that plasmid DNA reference materials could be used as the positive reference materials for PCR and RT-qPCR detection for quinolone antibiotic resistance genes.(3)Two hundred plasmid DNA reference materials were acquired by vacuum dried.The results of homogeneity test showed that all target genes can be detected and no mutation,and no significant difference was found in the quality of each tubes;The results of short-term stability of the plasmid DNA reference materials stored at 37℃for 13 d and 4℃for 3 months showed that all target genes can be detected and no mutation,and no significant difference was found in the quality of each tubes,RT-qPCR results showed that the Ct values of all genes under the limit of detection of each genes;The results of long-term stability of the plasmid DNA reference materials stored at-20℃for 12 months showed that all target genes can be detected and no mutation,the quality in each tubes decreased significantly,but there was no significant difference in the first 300 days,RT-qPCR results showed that the Ct values of all genes under the limit of detection of each genes.To sum up,the homogeneity and stability of the 11 plasmid DNA qualitative reference materials can up to the requirements of GB/T15000.3-2008.(4)The joint certification result of par C plasmid DNA qualitative reference materials are consistent with previous detection results acquired in our lab,and have been concluded by National Standards Committee.The joint evaluation of other declared samples is being carried out and the project is ready to be concluded.To summarize,11 plasmid DNA reference materials,which conformed to GB/T 15000.3-2008 requirements,for quinolone and fluoroquinolones antibiotic resistance mechanism detection were successfully developed in this study.the reference materials can be reliably used as reference materials to detect quinolone and fluoroquinolone resistance and mechanism in foodborne pathogens. |
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