Background: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is widely used world wide (557 references in Pubmed on September 21st, 2010). 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 for. Nine hundred and twenty-seven isolates were analyzed totaling 39,904 data points. Samples were received from 11 international laboratories with a worldwide distribution and represented either pure CTAB extracted or thermolyzates DNA. Results: Seven out of the 11 laboratories (63 %), perfectly typed over 90% of isolates, 3 scored between 80-90% and a single one center was under 80% reaching 51% concordance only, however, this was mainly due to discordance in a single spacer, likely having an 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. 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 thermolyzates) did not affect the results in this study. Performance was, however, 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 occur randomly either in the process of re-using membranes, reusing procedure or during the reading of the results and transferring of data from the film to a digital file. Last, the validity of the microbead-based was high, as conformed by the proper detection of spacer 15 that is known to give weak signals in the classical spoligotyping.
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
GARZELLI, CARLO;
2011-01-01
Abstract
Background: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is widely used world wide (557 references in Pubmed on September 21st, 2010). 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 for. Nine hundred and twenty-seven isolates were analyzed totaling 39,904 data points. Samples were received from 11 international laboratories with a worldwide distribution and represented either pure CTAB extracted or thermolyzates DNA. Results: Seven out of the 11 laboratories (63 %), perfectly typed over 90% of isolates, 3 scored between 80-90% and a single one center was under 80% reaching 51% concordance only, however, this was mainly due to discordance in a single spacer, likely having an 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. 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 thermolyzates) did not affect the results in this study. Performance was, however, 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 occur randomly either in the process of re-using membranes, reusing procedure or during the reading of the results and transferring of data from the film to a digital file. Last, the validity of the microbead-based was high, as conformed by the proper detection of spacer 15 that is known to give weak signals in the classical spoligotyping.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
