The use of microbead-based spoligotyping for <it>Mycobacterium tuberculosis </it>complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes

<p>Abstract</p> <p>Background</p> <p>The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the <it>Mycobacterium tuberculosis </it>CRISPR locus, is used world-wide (598 references in Pubmed on April 8th,...

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Main Authors: Garzelli Carlo, Rastogi Nalin, Al-Hajoj Sahal, Herranz Marta, de Viedma Darío, Rasolofo-Razanamparany Voahangy, Stoffels Karolien, Fauville-Dufaux Maryse, Elias Atiná, Gomes Harrison, Rigouts Leen, Ruimy Raymond, Kremer Kristin, Ritacco Viviana, Zhang Jian, Abadia Edgar, Tortoli Enrico, Suffys Philip N, van Soolingen Dick, Refrégier Guislaine, Sola Christophe
Format: Article
Language:English
Published: BMC 2011-04-01
Series:BMC Infectious Diseases
Online Access:http://www.biomedcentral.com/1471-2334/11/110
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spelling doaj-2259e0564f4741df8127e98c901531222020-11-25T01:21:41ZengBMCBMC Infectious Diseases1471-23342011-04-0111111010.1186/1471-2334-11-110The use of microbead-based spoligotyping for <it>Mycobacterium tuberculosis </it>complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranesGarzelli CarloRastogi NalinAl-Hajoj SahalHerranz Martade Viedma DaríoRasolofo-Razanamparany VoahangyStoffels KarolienFauville-Dufaux MaryseElias AtináGomes HarrisonRigouts LeenRuimy RaymondKremer KristinRitacco VivianaZhang JianAbadia EdgarTortoli EnricoSuffys Philip Nvan Soolingen DickRefrégier GuislaineSola Christophe<p>Abstract</p> <p>Background</p> <p>The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the <it>Mycobacterium tuberculosis </it>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.</p> <p>Methods</p> <p>The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated <it>Mycobacterium tuberculosis </it>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.</p> <p>Results</p> <p>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).</p> <p>Conclusions</p> <p>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 <it>et al</it>. (J. Clin. Microbiol. 2004) and Zhang <it>et al</it>. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping.</p> http://www.biomedcentral.com/1471-2334/11/110
collection DOAJ
language English
format Article
sources DOAJ
author Garzelli Carlo
Rastogi Nalin
Al-Hajoj Sahal
Herranz Marta
de Viedma Darío
Rasolofo-Razanamparany Voahangy
Stoffels Karolien
Fauville-Dufaux Maryse
Elias Atiná
Gomes Harrison
Rigouts Leen
Ruimy Raymond
Kremer Kristin
Ritacco Viviana
Zhang Jian
Abadia Edgar
Tortoli Enrico
Suffys Philip N
van Soolingen Dick
Refrégier Guislaine
Sola Christophe
spellingShingle Garzelli Carlo
Rastogi Nalin
Al-Hajoj Sahal
Herranz Marta
de Viedma Darío
Rasolofo-Razanamparany Voahangy
Stoffels Karolien
Fauville-Dufaux Maryse
Elias Atiná
Gomes Harrison
Rigouts Leen
Ruimy Raymond
Kremer Kristin
Ritacco Viviana
Zhang Jian
Abadia Edgar
Tortoli Enrico
Suffys Philip N
van Soolingen Dick
Refrégier Guislaine
Sola Christophe
The use of microbead-based spoligotyping for <it>Mycobacterium tuberculosis </it>complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes
BMC Infectious Diseases
author_facet Garzelli Carlo
Rastogi Nalin
Al-Hajoj Sahal
Herranz Marta
de Viedma Darío
Rasolofo-Razanamparany Voahangy
Stoffels Karolien
Fauville-Dufaux Maryse
Elias Atiná
Gomes Harrison
Rigouts Leen
Ruimy Raymond
Kremer Kristin
Ritacco Viviana
Zhang Jian
Abadia Edgar
Tortoli Enrico
Suffys Philip N
van Soolingen Dick
Refrégier Guislaine
Sola Christophe
author_sort Garzelli Carlo
title The use of microbead-based spoligotyping for <it>Mycobacterium tuberculosis </it>complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes
title_short The use of microbead-based spoligotyping for <it>Mycobacterium tuberculosis </it>complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes
title_full The use of microbead-based spoligotyping for <it>Mycobacterium tuberculosis </it>complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes
title_fullStr The use of microbead-based spoligotyping for <it>Mycobacterium tuberculosis </it>complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes
title_full_unstemmed The use of microbead-based spoligotyping for <it>Mycobacterium tuberculosis </it>complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes
title_sort use of microbead-based spoligotyping for <it>mycobacterium tuberculosis </it>complex to evaluate the quality of the conventional method: providing guidelines for quality assurance when working on membranes
publisher BMC
series BMC Infectious Diseases
issn 1471-2334
publishDate 2011-04-01
description <p>Abstract</p> <p>Background</p> <p>The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the <it>Mycobacterium tuberculosis </it>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.</p> <p>Methods</p> <p>The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated <it>Mycobacterium tuberculosis </it>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.</p> <p>Results</p> <p>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).</p> <p>Conclusions</p> <p>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 <it>et al</it>. (J. Clin. Microbiol. 2004) and Zhang <it>et al</it>. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping.</p>
url http://www.biomedcentral.com/1471-2334/11/110
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