Comparative evaluation of ligation-mediated PCR and spoligotyping as screening methods for genotyping of...

Spoligotyping has been suggested as a screening test in multistep genotyping of Mycobacterium tuberculosis strains. Relying on restriction fragment length polymorphism (RFLP) analysis with IS6110 (IS6110 RFLP analysis) as a "gold standard," we performed a comparative evaluation of spoligotyping and ligation-mediated PCR (LMPCR), a recently described PCR-based typing method, as rapid screening tests for fingerprinting of 158 M. tuberculosis strains collected in Verona, Italy. LMPCR seemed to be comparable to spoligotyping in terms both of feasibility with rapidly extracted DNA and of generation of software-analyzable images. Moreover, LMPCR grouped considerably fewer strains than spoligotyping (38 versus 67%) and was found to reduce the cluster overestimation rate (26.3 versus 58%) and to give a better discriminatory index (0.992 versus 0.970) compared to spoligotyping. In our geographical region, where there was no evidence of clustered strains carrying fewer than six IS6110 copies, LMPCR was found to be more discriminatory than spoligotyping. We also evaluated two models of three-step typing strategies, involving the use of spoligotyping and LMPCR as screening methods and IS6110 RFLP analysis as a further supporting test. LMPCR proved to be a more effective first-step test than spoligotyping, significantly reducing the need for subtyping. LMPCR should be considered an alternative to spoligotyping as a rapid screening method for M. tuberculosis fingerprinting, particularly in areas with a low prevalence of M. tuberculosis strains carrying few copies of IS6110.