A novel method based on high resolution melting (HRM) analysis for MIRU-VNTR genotyping of Mycobacterium tuberculosis.

The mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method is one of the most important methods that have been used in recent years for genotyping Mycobacterium tuberculosis. Agarose gel electrophoresis and capillary electrophoresis have been used to determine the size of amplicons, however, both of these methods have shortcomings. Here, we develop and evaluate a novel method for MIRU-VNTR typing based on high resolution melting (HRM) analysis. The MIRU40 locus was selected to evaluate different real-time PCR machines and the accuracy of our method; the Roche LightCycler 480 provided greatest consistency between the T(m) value and repeat number and was used in subsequent evaluations. Our method gives greater accuracy in comparison with conventional agarose gel electrophoresis (98.9% vs. 90.9%, p=0.017), and, with the help of fitting formulae, can be used to obtain the number of MIRU tandem repeats from the T(m) value. To validate our method we analyzed 12 classical MIRU loci to genotype 88 clinical isolates. The number of MIRU tandem repeats was determined accurately, quickly and conveniently. Crown