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
1 Institute of Genetics and Microbiology UMR8621, CNRS Université Paris-Sud 11 Universud, Campus d'Orsay, F-91405 Orsay-Cedex, France
2 Instituto Nacional de Enfermedades Infecciosas ANLIS Carlos Malbrán, Vélez Sarsfield 563, 1281 Buenos Aires, Argentina
3 National Institute for Public Health and the Environment, Bilthoven, The Netherlands
4 EA 3964 Université Paris-Diderot & Microbiology Laboratory, Bichat-Claude Bernard Hospital AP-HP, Paris, France
5 Mycobacteriology Unit, Prince Leopold Institute of Tropical Medicine, 155 National Straat, 200 Antwerp, Belgium
6 Laboratory of Molecular Biology applied to Mycobacteria, Oswaldo Cruz Institute, Rio de Janeiro, Brazil
7 National Reference Centre of Tuberculosis and Mycobacteria, Scientific Institute of Public Health, Brussels, Belgium
8 Unité des Mycobactéries, Institut Pasteur de Madagascar, Antananarivo, Madagascar
9 Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital Gregorio Marañón, Madrid, Spain
10 CIBER Enfermedades Respiratorias (CIBERES), Spain
11 Department of Comparative Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
12 Unité de la Tuberculose et des Mycobactéries - WHO Supranational TB Reference Laboratory, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe
13 Dipartimento di Patologia Sperimentale Biotecnologie Mediche Infettivologia ed Epidemiologia, Università di Pisa, I-56127 Pisa, Italy
14 Regional Reference Center for Mycobacteria, Careggi Hospital, viale Morgagni 85, 50134 Firenze, Italy
15 Department of Pulmonary Diseases and Department of Microbiology, Radboud University Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
16 Unité de Génétique Mycobactérienne, Institut Pasteur, 25-28 rue du Dr. Roux, F-75724 Paris-Cedex 15, France
BMC Infectious Diseases 2011, 11:110 doi:10.1186/1471-2334-11-110Published: 28 April 2011
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.
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.
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).
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 occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping.