沙门菌耐药基因多重PCR检测方法的建立

Establishment of multiplex PCR detective methods for the resistance genes of Salmonella

目的建立检测沙门菌耐药基因的多重PCR方法。方法在已有的单一PCR检测沙门菌耐药基因的基础上,采用正交试验设计法,对Mg2+、dNTPs和引物浓度比例以及退火温度进行优化,确定沙门菌耐药基因多重PCR最佳反应体系和条件,并运用多重和单一PCR同步比较检测细菌耐药基因。结果成功建立了氨基糖苷类药物耐药基因aph(3)-IIa、aadA2、aacC4三重PCR方法、氯霉素类药物耐药基因flor、cmlA和catI三重PCR方法、四环素类药物耐药基因tetA和tetB二重PCR方法,且多重和单一PCR同步检测相关耐药基因结果符合率达100%。结论建立的多重PCR具有特异、快速、简便的特点,可为沙门菌耐药基因的检测以及耐药性的监控提供技术支撑。

Objective To establish multiplex PCR methods to detect the resistance genes of Salmonella. Methods The multiplex PCR reaction system and condition was optimized to find out the best concentration of Mg2＋, dNTPs, and primers, and the best annealing temperature by orthogonal experimental design, and the multiplex PCR methods were established based on the single PCR methods established already to detective the resistance genes of Salmonella. The resistance genes of Salmonella were detected simultaneously by multiplex PCR and single PCR. Results There were established that 3 multiplex PCR methods to simultaneously amplify three aminoglycoside resistance genes aph（3）-IIa, aadA2 and aacC4, three choramphenicols resistance genes flor, cmlA and catI, two tetracyclines resistance genes tetA and tetB, respectively. Besides, the consistency rate of the results in multiplex PCR with that in single PCR was 100%. Conclusion Multiplex PCR established in the study is specific, rapid, simple and convenient. It provides the technical support to detect resistance genes monitor drug resistance of Salmonella.