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THE USE OF MICROBEAD-BASED SPOLIGOTYPING FOR MYCOBACTERIUM TUBERCULOSIS COMPLEX TO EVALUATE THE QUALITY OF THE CONVENTIONAL METHOD: PROVIDING GUIDELINES FOR QUALITY ASSURANCE WHEN WORKING ON MEMBRANES
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Abstract
Background: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the
spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th,
2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique.
We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly
robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data
points. Samples were received from 11 international laboratories with a worldwide distribution.
Methods: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA
extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping
participating centers. Information regarding how the classical Spoligotyping method was performed by center was
available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both
methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were
performed to validate the observed results.
Results: Seven out of the 11 laboratories (63 %), perfectly typed more than 90% of isolates, 3 scored between 80-
90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to
discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using
thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers
shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus
(Mostly spacers 15, 14, 18, 37, 39, 40).
Conclusions: We confirm that classical spoligotyping is a robust method with generally a high reliability in most
centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study.
However performance was center-dependent, suggesting that training is a key component in quality assurance of
spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors.
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