Differentiation of Pc and P2 variants in class 1 integron by high-resolution melting analysis

• DOI: 10.3760/cma.j.issn.1009-9158.2017.02.005
• Publication Date: 2017-02-11
• Authors: Quhao Wei; Gang Li; Xiaofei Jiang; Qingfeng Hu; Huoyang Lyu; Yonglie Zhou; Ming Guan
• Keywords: High Resolution Melt; Amplicon; genomic DNA

Objective To develop a simple high-resolution melting (HRM) analysis method for differentiation of Pc and P2 variants in class 1 integron.

Methods DNA fragments containing were amplified from plasmids pACW (PcW) pACWP2 (PcW-P2) respectively, then these purified PCR products promoters analyed full-length amplicon by HRM. Eight DNA different site-specific mutated pACS (PcS), pACH2 (PcH2), pACH1 (PcH1), (PcW), genomic Klebsiellar pneumonia HS07-68(PcWTGN-10)and HS05-1792(PcH2TGN-10)respectively. The eight characterized HRM analyses an unlabeled probe amplicon. This assay was applied to the differentiate 109 integrons 95 urine clinical Escherichia coli isolates Huashan Hospital during 2004-2007. results compared with that determined direct sequencing.

Results P2 promoter significant higher temperature (Tm) can be identified clearly. 2 consistent sequencing results. classified into three groups: PcS, PcSTGN-10, PcW, PcWTGN-10, PcH1, PcH1TGN-10. Using analysis. PcH2, PcH2TGN-10 four PcH1TGN-10, PcH2TGN-10, PcWTGN-10 according curves probe. Combined whole probe, differentiated each other. Five variants, results.

Conclusions This developed via simultaneous is cost-effective accurate, could used isolates.(Chin J Lab Med, 2017, 40: 95-100)

Key words: Integrons; Promoter regions, genetic; Drug rsistance, bacterial; Polymerase chain reaction; High resolution