复合探针实时荧光PCR检测细菌耐药基因blaNDM-1方法的建立

目的:建立一种检测编码新德里金属β内酰胺酶1(NDM-1)的细菌耐药基因blaNDM-1的复合探针实时荧光PCR方法。方法:基于复合探针技术原理,以blaNDM-1基因作为待检靶基因建立检测方法,对PCR扩增体系中镁离子浓度、PCR退火温度等进行优化,并对检测的灵敏性、特异性、重复性等进行评价。结果:优化了最佳反应体系和扩增条件;以灵敏度质控品进行灵敏度实验,最低检测限可达2拷贝/体系;非耐药性菌株的检测结果均为阴性;批间批内变异系数均小于5%;只有产NDM-1的鲍曼不动杆菌检测为阳性,其他377株临床分离菌和阴性对照均无响应。结论:建立了检测含blaNDM-1基因的菌株的方法,具有很好的灵敏性、特异性和重复性。

Development of Complex Probe Real-Time Fluorescent PCR for Detection of the Antibiotic Resistance blaNDM-1 Gene

Objective: To establish a complex probe real-time fluorescent PCR method to detect the antibiotic resistance blaNDM-1 gene.Methods: This detection method based on the principles of complex probe,and use blaNDM-1 gene sequences coding New Delhi metallo-β-lactamase 1(NDM-1) as the target gene.The PCR amplification system parameters such as the concentration of magnesium ions and PCR annealing temperature were opti mized,and the specificity,precision and reproducibility of detection were evaluated.Results: Optimizing the PCR reaction system and the best conditions to test the sensitivity of sensitivity control samples amplified with satisfacto ry results.The sensitivity of the assay was about 2 copies/system,all of the blaNDM-1 gene containing strains were positively detected and none of the other bacteria was detected by the assay.The coefficient of variation of in tra-assay and inter-assay was less than 5%.Only Acinetobacter baumannii strain containing blaNDM-1 gene isolated from clinical samples tested positive,other 377 clinical specimens and negative control strains had no response.Conclusion: The established assay detects blaNDM-1 gene containing strains with excellent sensitivity,specificity and reproducibility.