Rapid differentiation of bacterial species by high resolution melting curve analysis

Molecular based differentiation of various bacterial species is important in phylogenetic studies, diagnostics and epidemiological surveillance, particularly where unusual phenotype makes the classical phenotypic identification of bacteria difficult. Molecular approach based on the sequence of 16S ribosomal RNA gene analysis can achieve fast and reliable identification of bacteria. High resolution melting (HRM) curve analysis has been developed as an attractive novel technique for DNA sequence discrimination but it's application for bacteria differentiation has not been well studied yet. We have developed HRM assay for differentiation of sixteen pathogenic or opportunistic bacterial species. Amplified partial 16S ribosomal RNA gene region between 968 and 1401 positions (E. coli reference numbering) was subsequently used in high resolution melting curve analysis of PCR products for bacterial species differentiation. Sixteen bacterial species were simultaneously discerned by difference plot of normalized and temperatures shifted melting curves, without need for spiking of DNA, hetero-duplexing experiments or application of several primer pairs. High resolution melting curve analysis of duplex DNA is simple, fast and reliable tool for bacterial species differentiation and may efficiently complement phenotypic identification of bacteria.