Comparative evaluation of ligation-mediated PCR and spoligotyping as screening methods for genotyping of Mycobacterium tuberculosis strains

Abstract

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.

FIG. 1

Spoligotype dendrogram generated for the 158 M. tuberculosis strains and the corresponding patterns after computer analysis with GelCompar software. The scale on the left indicates the band-based similarity coefficients.

FIG. 2

Dendrogram and associated schematic representation of LMPCR patterns of the 158 M. tuberculosis strains after computer analysis with the Taxotron package. The scale on the left indicates the Dice index, which was used to compare the patterns with a linear tolerance error between 3.5 and 5%.

FIG. 3

Agarose gel (2.5%) electrophoresis of amplified M. tuberculosis DNA obtained by the LMPCR method. Lanes 1 and 16, molecular size marker (1-kb ladder; Gibco); lanes 2 to 15, M. tuberculosis study strains (strains 4 and 6 were grouped in the same cluster).

FIG. 4

Two models of sequential use of the three typing methods used to analyze the 158 M. tuberculosis strains. (A) Spoligotyping is the first-step test; (B) LMPCR is the screening method. In both cases IS6110 RFLP analysis is considered the most discriminatory test, following double-step typing by the PCR-based methods. For each step, the black and white bars on the left represent the fraction of clustered isolates and the fraction of isolates with unique patterns, respectively, while the percentages of clustered strains are shown on the right.