High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD–HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, interserovar convergence, and evidence of attenuation in Leptospira reference collections

Abstract

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD–HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD–HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD–HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD–HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD–HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD–HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.