Improved PCR detection and subtyping of CTX-M-beta-lactamase-encoding genes
Abstract number: 1134_01_242
Edelstein M., Pimkin M.
Rapid detection and differentiation of CTX-M beta-lactamases is of need in many laboratories as this family of ESBLs is rapidly disseminating. PCR has been used widely to detect blaCTX-M genes but detection of all the known variants usually required multiple reactions with specific primers for different CTX-M subtypes. Consensus primers have also been described but the recent emergence of new CTX-M variants (e.g. CTX-M-25, -26) made them no longer versatile. In this study we describe a novel real-time PCR technique for detection of all CTX-Ms and a single-enzyme post-PCR restriction analysis for differentiation between the 5 CTX-M genetic clusters.
A pair of primers matching conserved sequences at positions 205 to 227 and 724 to 706 with respect to the CTX-M translational starting point was designed to amplify a 519-bp fragment of all the known blaCTX-M genes using a real-time approach with SYBR Green product detection. A computer analysis was used to identify BseDI as a restriction endonuclease capable of distinguishing the subtypes of blaCTX-M genes. PCR products were digested with BseDI and digests analysed by agarose gel electrophoresis. Bacterial strains producing known beta-lactamases (CTX-M-2, -3, -4, -5, -9, -15; TEM-1; SHV-1; KluA) were used for quality control and assessment of specificity of PCR and discriminative power of the restriction analysis.
A single DNA fragment of the expected size was amplified in all CTX-M-positive control strains. Melting curve peaks were detected at 85, 86.5 and 88°C for CTX-M-2, -3 and CTX-M-9 genetic subtypes, respectively. No increase in fluorescence was detected for CTX-M-negative isolates. Further restriction analysis produced the predicted restriction patterns and, therefore, allowed to distinguish different CTX-M subtypes.
The proposed real-time PCR is a versatile and specific tool for rapid detection of CTX-M beta-lactamases. When needed, the post-PCR restriction analysis may be used to confirm differentiation of CTX-M genetic subtypes.
|Session name:||XXIst ISTH Congress|
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