CAJ | 학술논문

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SYBR GREEN Ⅰ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SYBR GREEN I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3×10~3～3×10~8拷贝/mL , r ＝ -0.994 7 ;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:SYBR GREEN Ⅰ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.
목적:건립일충쾌속검측혁란음성간균산초엄보β내선알매(ESBLs )내약기인분형적SYBR GREEN Ⅰ실시형광정량PCR방법.방법:침대림상상견ESBLs적내약기인SHV、TEM、 CTX-M、OXA급기동원성분석,설계료SHV、TEM 、CTX-M-1、CTX-M-2、CTX-M-8、CTX-M-9、OXA-1、OXA-2급OXA-10 공9대특이성인물,자비법제취DNA모판,건립병우화SYBR GREEN I실시형광정량PCR반응체계,병대기정밀도、선성범위진행측정.이용건립방법대51주표형음성적다중내약적대장애희균、폐염극뢰백균진행ESBLs내약기인검측,병여개량삼유실험진행대비.결과:종39주ESBLs표형양성균주급51주ESBLs표형음성다중내약균주중확증출제OXA-2외공8충내약기인형병경측서증실.선성검측범위3×10~3～3×10~8고패/mL , r ＝ -0.994 7 ;비간중복성시험변이계수(CV)위9.6%.형광정량PCR방법여개량삼유실험방법비교차이무통계학의의( χ2 = 1.125,P > 0.05).결론:SYBR GREEN Ⅰ실시형광정량PCR검측ESBLs적내약기인구유특이성강、령민도고、쾌속、간편적특점,괄우림상감측혁란음성간균산ESBLs 기인형.
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 multi- drug 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 (χ2 = 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.