title: 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.
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 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.
authors.affiliation: Institute of Genetics and Microbiology UMR8621, CNRS Université Paris-Sud 11 Universud, Campus d'Orsay, F-91405 Orsay-Cedex, France.
authors.forename: Edgar
authors.initials: E
authors.lastname: Abadia
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Viviana
authors.initials: V
authors.lastname: Ritacco
authors.forename: Kristin
authors.initials: K
authors.lastname: Kremer
authors.forename: Raymond
authors.initials: R
authors.lastname: Ruimy
authors.forename: Leen
authors.initials: L
authors.lastname: Rigouts
authors.forename: Harrison Magdinier
authors.initials: HM
authors.lastname: Gomes
authors.forename: Atiná Ribeiro
authors.initials: AR
authors.lastname: Elias
authors.forename: Maryse
authors.initials: M
authors.lastname: Fauville-Dufaux
authors.forename: Karolien
authors.initials: K
authors.lastname: Stoffels
authors.forename: Voahangy
authors.initials: V
authors.lastname: Rasolofo-Razanamparany
authors.forename: Darío
authors.initials: D
authors.lastname: Garcia de Viedma
authors.forename: Marta
authors.initials: M
authors.lastname: Herranz
authors.forename: Sahal
authors.initials: S
authors.lastname: Al-Hajoj
authors.forename: Nalin
authors.initials: N
authors.lastname: Rastogi
authors.forename: Carlo
authors.initials: C
authors.lastname: Garzelli
authors.forename: Enrico
authors.initials: E
authors.lastname: Tortoli
authors.forename: Philip N
authors.initials: PN
authors.lastname: Suffys
authors.forename: Dick
authors.initials: D
authors.lastname: van Soolingen
authors.forename: Guislaine
authors.initials: G
authors.lastname: Refrégier
authors.forename: Christophe
authors.initials: C
authors.lastname: Sola
mesh_terms: D015373:Bacterial Typing Techniques / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D004269:DNA, Bacterial / Q000235:genetics; D005838:Genotype; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D011786:Quality Control; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial