Real-time Fluorescence Quantitative PCR for Detecting Extended Spectrum-β-Lactamases Genotype
10.3969/j.issn.0253-9896.2009.10.008
- VernacularTitle:实时荧光定量PCR检测ESBLs基因型方法的研究
- Author:
Rui LIU
;
Xiaoyan MU
;
Huimin LIU
;
Lin FAN
;
Chen WANG
- Publication Type:Journal Article
- Keywords:
polymerase chain reaction;
beta - lactamases;
genotype;
drug resistance;
multiple;
gram - negative bacteria
- From:
Tianjin Medical Journal
2009;37(10):839-842,后插5
- CountryChina
- Language:Chinese
-
Abstract:
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.
