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

Abstract

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 Pc and P2 variants were amplified from plasmids pACW (PcW) and pACWP2 (PcW-P2) respectively, then these purified PCR products and P2 promoters were analyed full-length amplicon by HRM. Eight DNA fragments containing different Pc promoters were amplified and site-specific mutated from plasmids pACS (PcS), pACH2 (PcH2), pACH1 (PcH1), pACW (PcW), genomic DNA of Klebsiellar pneumonia HS07-68(PcWTGN-10)and HS05-1792(PcH2TGN-10)respectively. The purified PCR products and eight Pc variants were characterized by HRM analyses of an unlabeled probe and full-length amplicon. This assay was applied to the differentiate Pc and P2 variants in 109 class 1 integrons from 95 urine clinical Escherichia coli isolates in Huashan Hospital during 2004-2007. The differentiation results were compared with that determined by direct sequencing. Results P2 promoter with a significant higher melting temperature (Tm) can be identified by HRM analysis clearly. P2 promoters were identified in 2 class 1 integrons and consistent with direct sequencing results. Eight Pc variants were classified into three groups: PcS, PcSTGN-10, PcW, PcWTGN-10, PcH1, PcH1TGN-10. Using direct HRM analysis. PcH2, PcH2TGN-10 were classified into four groups: PcS, PcH1, PcH2, PcW, PcSTGN-10, PcH1TGN-10, PcH2TGN-10, PcWTGN-10 according to the melting curves of the unlabeled probe. Combined the HRM analyses of the whole amplicon and unlabeled probe, the eight Pc variants can be differentiated from each other. Five different Pc variants, PcS, PcW, PcH1, PcH2TGN-10 and PcWTGN-10, were identified and consistent with direct sequencing results. Conclusions This developed a simple Pc and P2 variants differentiation method via simultaneous HRM analyses of an unlabeled probe and full-length amplicon. This method is cost-effective and accurate, could be used in differentiation of Pc and P2 variants of class 1 integrons in clinical isolates.(Chin J Lab Med, 2017, 40: 95-100) Key words: Integrons; Promoter regions, genetic; Drug rsistance, bacterial; Polymerase chain reaction; High resolution melting