实时荧光定量PCR检测ESBLs基因型方法的研究

Real-time Fluorescence Quantitative PCR for Detecting Extended Spectrum-β-Lactamases Genotype

目的:建立一种快速检测革兰阴性杆菌产超广谱β内酰胺酶(ESBLs)耐药基因分型的SYBRGREENⅠ实时荧光定量PCR方法。方法:针对临床常见ESBLs的耐药基因SHV、TEM、CTX-M、OXA及其同源性分析,设计了SHV、TEM、CTX-M-1、CTX-M-2、CTX-M-8、CTX-M-9、OXA-1、OXA-2及OXA-10共9对特异性引物,煮沸法提取DNA模板,建立并优化SYBRGREENI实时荧光定量PCR反应体系,并对其精密度、线性范围进行测定。利用建立方法对51株表型阴性的多重耐药的大肠埃希菌、肺炎克雷伯菌进行ESBLs耐药基因检测,并与改良三维实验进行对比。结果:从39株ESBLs表型阳性菌株及51株ESBLs表型阴性多重耐药菌株中扩增出除OXA-2外共8种耐药基因型并经测序证实。线性检测范围3×103～3×108拷贝/mL,r=-0.9947;批间重复性试验变异系数(CV)为9.6%。荧光定量PCR方法与改良三维实验方法比较差异无统计学意义(χ2=1.125,P>0.05)。结论:SYBRGREENⅠ实时荧光定量PCR检测ESBLs的耐药基因具有特异性强、灵敏度高、快速、简便的特点,适于临床监测革兰阴性杆菌产ESBLs基因型。

Objective：To establish a rapid method to detect drug-resistance genotypes of extended spectrum-β- Lactamases （ESBLs） produced by gram negative bacillus using the real-time fluorescence quantitative PCR. Methods： According to clinical common genotypes of ESBLs, SHV, TEM, CTX-M, OXA and their homology, 9 pairs of specific primers were designed including SHV, TEM, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, OXA-1, OXA-2 and OXA-10. To extract DNA template by boiling assay, and then establish and grade up SYBR GREEN Ⅰ real-time fluorescence quantitative PCR reaction system, finally definite real-time fluorescence quantitative PCR method. Its precision and range of linearity were tested. With established assay 51 multidrug resistant ESBLs- E. coli K. pneumoniae were detected and compared with improved three dimensional extract tests. Results： Except OXA-2, 8 genotypes SHV, TEM, CTX-M-1, CTX-M-2, CTX-M- 8, CTX-M-9, OXA-1 and OXA-10 were amplified by quantitative PCR from 39 ESBLs＋ and 51 multi-drug resistant ESBLs- E. coli K. pneumoniae and confirmed by sequence testing. The range of linearity was 3×10^3～3×10^8 copies/mL, r = -0.994 7. Repetitive experiments showed that the average coefficient of variation between-runs was 9.6%. Comparing with three dimensional extract test, there was no significant difference （χ^2 = 1.125,P 〉 0.05）. Conclusion： Testing drug-resistance genotypes of ESBLs with SYBR GREEN Ⅰ real-time fluorescence quantitative PCR is a rapid, specific and sensitive method, which is capable of inspecting genotypes of ESBLs from clinical strains.