摘要
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。
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-1actamase I（NDM-1） as the target gene. The PCR amplification system parameters such as the concentration of magnesium ions and PCR annealing temperature were optimized, 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-i 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.
出处
《生物技术通讯》
CAS
2012年第3期411-414,共4页
Letters in Biotechnology
