Study On Plasmid DNA Standard Reference Samples For Detection Of Mechanisms Of ?-lactam And Polymyxin Antibiotic Resistance

?-lactam antibiotics and polymyxin antibiotics,as extremely effective antibacterial drugs,are widely used in the treatment of food-borne diseases.But the emergence of drug-resistant microorganisms has an impact on the effectiveness of these two types of drugs,making the detection of drug resistance of microorganisms a major focus of work.With the continuous breakthrough of microbial antibiotic resistance mechanism at the genetic level,the detection of resistance genes has become a necessary means to screen antibiotic-resistant strains.In the testing process,in order to ensure that the test results are accurate,fair,and comparable,the reference material is particularly important as a quality control standard.At present,the research on nucleic acid reference materials at home and abroad is mostly limited to viruses and transgenics,leading to blanks in reference materials for the detection of drug resistance mechanisms,making the test results between institutions and institutions incomparable and unable to form a unified quantification.In this study,a total of 8 plasmid DNA standard samples for detection of genes encoding the resistance mechanism of?-lactam and polymyxin antibiotics were developed.The specific results are as follows:(1)5 kinds of?-lactam antibiotics resistance mechanism coding genes and 3 kinds of polymyxin antibiotic resistance mechanism coding genes were obtained by consulting the literature,the comparison verification results show that they are universal.(2)Synthesize 8 gene sequences by pure chemical method,insert into the p UC57 plasmid vector and introduce into the DH5?engineering strain.The results of the 15 generations of the recombinant strains showed that the 8 recombinant plasmids carrying genes encoding resistance mechanisms have good genetic stability.(3)Use the kit to extract the plasmid,and the purity of the solution meets the requirements.Dilute the plasmid solution stepwise,the result shows that the PCR detection limit of each plasmid DNA is between 1.67×10~4-2.25×10~6 copies/?L,and the detection limit of Real-Time q PCR is between 1.15-1850 copies/?L.The linear correlation coefficients of the standard curve of Ct value-template concentration logarithm of each plasmid DNA were all greater than0.99.(4)The uniformity of the plasmid DNA standard samples prepared by vacuum drying meets the requirements.After storage at 37?for 14 d,4?for 90 d,and-20?for 360 d,the stability test results meet the requirements.(5)The result of joint certificate of plasmid DNA standard samples of polymyxin antibiotic resistance gene mcr-1 is consistent with the characteristic value.