β-内酰胺类抗生素耐药编码基因检测用质粒标准样品研制

Development of plasmid standard sample for detection of β-lactam antibiotics resistance coding genes

目的研制可作为β-内酰胺类抗生素耐药编码基因检测质控用标准样品。方法通过检索美国国家生物技术信息中心基因库,获得β-内酰胺类抗生素耐药性编码基因bla_(TEM)、bla_(PSE)、bla_(CTX-M)、bla_(CMY)和bla_(OXA)的DNA序列,构建耐药基因重组质粒和工程菌。将工程菌传代培养15代,同时使用聚合酶链式反应(polymerase chain reaction,PCR)扩增和DNA测序测定确认目标基因的遗传稳定性。大量制备菌悬液,提取重组质粒,真空干燥得到质粒标准样品。使用PCR和定量逆转录PCR(quantitative reverse transcription PCR,RT-qPCR)检测质粒标准样品的最低检出限。使用超微量分光光度计测定质粒标准样品的质量,RT-qPCR测定质粒作为模板扩增时的循环数(cycle threshold,Ct),以此评价标准样品的均匀性及贮藏稳定性。结果5种重组质粒DNA可在工程菌中稳定遗传且无突变发生,5种质粒DNA标准样品均匀性良好。各质粒DNA经梯度稀释后,PCR最低检出限在2.33×10^(4)~2.25×10^(6)拷贝数/μL之间,RT-qPCR最低检出限在1.93~1850拷贝数/μL之间。样品在37℃贮存14 d、4℃贮存3个月、-20℃贮存12个月稳定性良好。结论研究制备得到的β-内酰胺类抗生素耐药编码基因检测用质粒标准样品的目的基因遗传稳定性、标准样品的均匀性和贮存稳定性均良好。

Objective To develop a standard sample which can be used as the quality control for the detection of β-lactam antibiotics resistance coding genes.Methods By searching the gene bank of the National Biotechnology Information Center of the United States,the DNA sequences of the drug resistance coding genes bla_(TEM),bla_(PSE),bla_(CTX-M),bla_(CMY) and bla_(OXA) of β-lactam antibiotics were obtained,and the recombinant plasmid and engineering bacteria of drug resistance genes were constructed.The engineered bacteria were subcultured for 15 generations,and the genetic stability of the target gene was confirmed by polymerase chain reaction(PCR)amplification and DNA sequencing.A large amount of bacterial suspensions were prepared,the recombinant plasmids were extracted,and the standard plasmid DNA reference sample was obtained after vacuum drying.The minimum detection limit for the standard plasmid DNA reference sample were determined by PCR and quantitative reverse transcription PCR(RT-qPCR).The ultra-micro spectrophotometer was used to determine the mass of the standard plasmid DNA reference sample,and RT-qPCR was used to determine the cycle threshold(Ct)of plasmid amplification as a template,in order to evaluate the homogeneity and storage stability of the standard samples.Results The 5 kinds of recombinant plasmid DNA could be stably inherited in the engineering bacteria without mutation,and the homogeneity of the 5 kinds of plasmid DNA standard samples were good.After gradient dilution of each plasmid DNA,the minimum detection limit of PCR was 2.33×10^(4)-2.25×10^(6) copies/μL,and the minimum detection limit of RT-qPCR was 1.93-1850 copies/μL.The stability of samples stored at 37℃ for 14 d,4℃ for 3 months and -20℃ for 12 months was good.Conclusion The prepared standard plasmid DNA reference sample for detecting the gene encoding β-lactam antibiotic resistance has good genetic stability,uniformity and storage stability of the target gene.