Developing a real-time PCR technique for Mycobacteria Tuberculosis strain typing using 15-Loci MIRU-VNTR based on high resolution melting - Step one

Abstract

Introduction: Tuberculosis (TB) is a major public health concern worldwide, with more than 2 billion people with latent infection. The precise identification of bacterial pathogens at the strain level is essential for epidemiological purposes. Consequently, constant efforts are undertaken to develop easy to use, low cost, and standardized methods, which can be applied routinely in a clinical laboratory. Mycobacterial interspersed repetitive unit – variable number of tandem repeat (MIRU-VNTR) typing is a rapid, highly reliable, and discriminative epidemiological typing methods has been adopted as a method of choice. Currently for MIRU-VNTR typing, size amplicon determination is based on agarose gel electrophoresis and capillary electrophoresis. The former is cheap and simple but size calling is very subjective and results are affected by distortion of lanes during electrophoresis. Capillary electrophoresis is subjective but very expensive. This study, developed and evaluated a 15-loci MIRU-VNTR real time PCR technique based on high resolution melting for strain typing of Mycobacterium tuberculosis complex (MTC) that was cheap, fast and of similar discriminatory power to the IS6110-RFLP.

Methods: Standard references containing five MIRU-VNTR tandem repeats and their flanking regions were designed by cloning. The standard references for each of the 15 loci MIRU-VNTR were used to determine a graphical and equation relationship between melting temperatures and number of tandem repeats. This was done using real time amplification and high resolution melting on the Rotor Gene Q machine and the data analyzed using the MatLab software. Seventy-one clinical isolates from HIV infected bacteremic individuals were used for the genotyping by agarose gel electrophoresis. The strain types were determined using the MIRU-VNTplus online database, to determine the diversity and clustering rates among the isolates.

Results: Melting temperatures (Tm) of various MIRU-VNTR loci were determined using the high resolution melting on the Rotor Gene Q machine using standards references constructed by cloning. Fitting curves and fitting equations with acceptable goodness of fit statistics were obtained relating the Tm to the number of MIRU-VNTR tandem repeats of standards. The fitted models were smoothly applicable to higher order number of MIRU-VNTR tandem repeats by extrapolation of the data due to the standards. By convention PCR and agarose gel electrophoresis, of the 71 clinical isolates from HIV infected bacteremic patients, 95.8% (68) were M. tuberculosis, 2.8% (2) M. africanum and 1.4% (1) M. caprae. Uganda genotype was the most predominant with 50% among the strains. There is very high strain diversity and low clustering rate among these clinical isolates from HIV infected bacteremic individuals. Among the 15 MIRU-VNTR loci five loci did not provide analyzable data with this technique.

Conclusion: Real time PCR and high resolution melting (HRM) is a cheap, faster, and precise technique that can be used for the 15 loci MIRU-VNTR genotyping of Mycobacteria tuberculosis complex.