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.
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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DOI：
10.1186/1471-2334-11-110
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发表时间：
2011-04-28
影响因子：
3.7
通讯作者：
Sola C
中科院分区：
文献类型：
--
作者：
Abadia E;Zhang J;Ritacco V;Kremer K;Ruimy R;Rigouts L;Gomes HM;Elias AR;Fauville-Dufaux M;Stoffels K;Rasolofo-Razanamparany V;Garcia de Viedma D;Herranz M;Al-Hajoj S;Rastogi N;Garzelli C;Tortoli E;Suffys PN;van Soolingen D;Refrégier G;Sola C
依赖于结核分枝杆菌CRISPR基因座的间隔区的膜反向线性印迹杂交的经典spoligotyping技术在世界范围内使用（Pubmed中的598篇参考文献，2011年4月8日）。然而，到目前为止，还没有进行实验室间质量控制研究来验证这种技术。我们通过将其与最近引入的高度稳健的基于微珠的spoligotyping进行比较来分析基于膜的spoligotyping的质量。分析了927株分离株，共计39，861个数据点。从分布在世界各地的11个国际实验室收到了样本。对来自基因分型参与中心的结核分枝杆菌复合群（MTC）分离菌株提取的CTAB和纯化物DNA进行基于微珠的高通量Spoligotyping。可获得关于各中心如何进行经典Spoligotyping方法的信息。通过比较两种方法获得的spoligotypes进行了基因型判别分析。采用非参数U-Mann Whitney同质性检验和斯皮尔曼秩相关检验验证观察结果。11个实验室中有7个（63%）对90%以上的分离株进行了完美分型，3个评分在80-90%之间，单个中心的评分低于80%，仅达到51%的一致性。然而，这主要是由于单个间隔区中的不一致性，可能在所使用的膜上具有非功能性探针。使用therapezate DNA的中心的表现以及中心使用更广泛的CTAB提取程序。很少有中心共享相同的有问题的间隔区，并且这些有问题的间隔区分散在整个CRISPR基因座上（主要是间隔区15、14、18、37、39、40）。我们证实，经典的spoligotyping是一个强大的方法，一般在大多数中心的可靠性高。所用的DNA提取程序（CTAB或therebezate）不影响本研究的结果。然而，性能是中心依赖的，这表明培训是Spoligotyping质量保证的关键组成部分。总体而言，没有特定的间隔物产生更高程度的偏离结果，这表明在重复使用膜的过程中或在结果的阅读和将数据从膜转移到数字文件期间随机发生错误。最后，如之前由科万等人（J.Clin.Microbiol. 2004）和Zhang等人（J. Med. Microbiol. 2009），并证明了间隔区15的正确检测，已知间隔区15在经典的spoligotyping中偶尔给出弱信号。
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.
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影响因子：
3
作者：
Jeffries DJ;Abernethy N;de Jong BC
通讯作者：
de Jong BC
影响因子：
3
作者：
Zhang, Jian;Abadia, Edgar;Sola, Christophe
通讯作者：
Sola, Christophe
影响因子：
9.4
作者：
VANSOOLINGEN, D;QIAN, LS;VANEMBDEN, JDA
通讯作者：
VANEMBDEN, JDA
影响因子：
9.4
作者：
Kamerbeek, J;Schouls, L;vanEmbden, J
通讯作者：
vanEmbden, J
影响因子：
11.8
作者：
Filliol I;Driscoll JR;Van Soolingen D;Kreiswirth BN;Kremer K;Valétudie G;Anh DD;Barlow R;Banerjee D;Bifani PJ;Brudey K;Cataldi A;Cooksey RC;Cousins DV;Dale JW;Dellagostin OA;Drobniewski F;Engelmann G;Ferdinand S;Gascoyne-Binzi D;Gordon M;Gutierrez MC;Haas WH;Heersma H;Källenius G;Kassa-Kelembho E;Koivula T;Ly HM;Makristathis A;Mammina C;Martin G;Moström P;Mokrousov I;Narbonne V;Narvskaya O;Nastasi A;Niobe-Eyangoh SN;Pape JW;Rasolofo-Razanamparany V;Ridell M;Rossetti ML;Stauffer F;Suffys PN;Takiff H;Texier-Maugein J;Vincent V;De Waard JH;Sola C;Rastogi N
通讯作者：
Rastogi N