Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of mycobacteria in routine clinical practice.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of mycobacteria in routine clinical practice.
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DOI：
10.1371/journal.pone.0024720
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Drancourt M
中科院分区：
文献类型：
--
作者：
El Khéchine A;Couderc C;Flaudrops C;Raoult D;Drancourt M
从呼吸道标本中回收的非结核分枝杆菌是全球结核病实验室诊断中新兴的混杂生物。迫切需要新技术来快速识别临床实践中分离的分枝杆菌。基质辅助激光解吸飞行时间质谱 (MALDI-TOF MS) 先前已被证明可以有效地识别藏品中高浓度接种物中生长的分枝杆菌。然而，尚未对其在常规实验室实践中的使用进行彻底评估。我们建立了一个原始方案，用于在 Tween-20 中解离、机械破坏细胞壁以及用甲酸和乙腈提取蛋白质后，对热灭活的分枝杆菌进行 MALDI-TOF MS 鉴定。通过将该协议应用于结核分枝杆菌、鸟分枝杆菌和 20 个其他分枝杆菌物种的参考分离株的少至 105 个菌落形成单位，我们获得了用于创建本地数据库的物种特异性质谱。使用该数据库，我们的方案能够通过 MALDI-TOF MS 在 2.5 小时内识别出 87 株结核分枝杆菌、25 株鸟结核分枝杆菌和 12 株非结核分枝杆菌临床分离株，鉴定分数≥2。我们的数据表明，MALDI-TOF MS 可用作热灭活分枝杆菌常规鉴定的一线方法。 MALDI-TOF MS 是一种在发达国家和发展中国家的临床微生物实验室中实施的有吸引力的方法。
Non-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed. We set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 105 colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25 M. avium and 12 non-tuberculosis clinical isolates with identification scores ≥2 within 2.5 hours. Our data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heat-inactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories in both developed and developing countries.
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