High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira

A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis ( which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans ( serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii ( serovar Arborea), L. kirschneri ( serovar Cynopteri) and L. weilii ( serovar Celledoni), were typed against 13 previously published RAPD primers, using a real-time cycler ( the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit ( Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars. These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' ( plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potential.