Developing Two Rapid Protein Extraction Methods Using Focused-Ultrasonication and Zirconia-Silica Beads for Filamentous Fungi Identification by MALDI-TOF MS.
Developing Two Rapid Protein Extraction Methods Using Focused-Ultrasonication and Zirconia-Silica Beads for Filamentous Fungi Identification by MALDI-TOF MS.
复制标题
使用聚焦超声和氧化锆-二氧化硅珠开发两种快速蛋白质提取方法，通过 MALDI-TOF MS 鉴定丝状真菌
DOI：
10.3389/fcimb.2021.687240
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发表时间：
2021
影响因子：
5.7
通讯作者：
Xu YC
中科院分区：
文献类型：
--
作者：
Ning YT;Yang WH;Zhang W;Xiao M;Wang Y;Zhang JJ;Zhang G;Duan SM;Dong AY;Guo DW;Zou GL;Wen HN;Guo YY;Chen LP;Chai M;He JD;Duan Q;Zhang LX;Zhang L;Xu YC
由于缺乏简单快速的蛋白质提取方法以及数据库中物种覆盖率不足，利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）鉴定丝状真菌一直具有挑战性。在这项研究中，我们创建了两种丝状真菌的快速蛋白质提取方法：一步氧化锆-二氧化硅珠法 (ZSB) 和聚焦超声法 (FUS)。使用 VITEK MS 评估两种方法的鉴定准确性，并使用 M-Discover 100 MALDI-TOF MS 与常规方法相比，评估谱峰数量和信噪比 (S/N)。采用较好的方法为M-Discover 100 MS建立丝状真菌内部谱库，然后并行执行另一种方法和常规方法来验证内部谱库的准确性和通用性。使用这两种优化的方法，MALDI-TOF MS 分析之前的专用操作时间从每个样品的 30 分钟减少到 7 (ZSB) 或 5 (FUS) 分钟，每个额外的菌株仅增加几秒钟。两种方法对大多数霉菌类型的分离鉴定结果与常规方法相当或更好，VITEK MS 的总正确鉴定率分别为 79.67、76.42 和 76.42%。另一方面，与常规方法相比，两种快速方法通常对这些测试的分离株实现了更高的最大和最小信噪比。此外，ZSB 方法产生的最大和最小信噪比的总体平均值高于 FUS 的结果。使用 ZSB 方法成功地从 18 属 42 个物种的 135 个分离株中建立了 M-Discover MS 内部文库。通过对 467 个分离株的分析，ZSB 方法对分离株的物种水平鉴定正确率为 97.22%，而常规方法为 95.50%。这两种新颖的方法具有时间和成本效益，可以有效识别丝状真菌，同时提供建立内部图书馆的简化程序。因此，未来更多的临床实验室可能会考虑采用MALDI-TOF MS进行丝状真菌鉴定。
Filamentous fungi identification by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenging due to the lack of simple and rapid protein extraction methods and insufficient species coverage in the database. In this study, we created two rapid protein extraction methods for filamentous fungi: a one-step zirconia-silica beads method (ZSB) and a focused-ultrasonication method (FUS). The identification accuracy of two methods were evaluated with the VITEK MS, as well as number of spectra peaks and signal-to-noise ratio (S/N) with M-Discover 100 MALDI-TOF MS compared to the routine method. The better method was applied to build a filamentous fungi in-house spectra library for the M-Discover 100 MS, and then another one and routine method were performed in parallel to verify the accuracy and commonality of the in-house library. Using the two optimized methods, the dedicated operating time before MALDI-TOF MS analysis was reduced from 30 min to 7 (ZSB) or 5 (FUS) min per sample, with only a few seconds added for each additional strain. And both two methods identified isolates from most mold types equal to or better than the routine method, and the total correct identification rate using VITEK MS was 79.67, 76.42, and 76.42%, respectively. On the other hand, the two rapid methods generally achieved higher maximum and minimum S/N ratios with these isolates tested as compared to the routine method. Besides, the ZSB method produced overall mean of maximum and minimum S/N ratio higher than that by FUS. An in-house library of M-Discover MS was successfully built from 135 isolates from 42 species belonging to 18 genera using the ZSB method. Analysis of 467 isolates resulted in 97.22% correctly identified isolates to the species level by the ZSB method versus 95.50% by the routine method. The two novel methods are time- and cost-effective and allow efficient identification of filamentous fungi while providing a simplified procedure to build an in-house library. Thus, more clinical laboratories may consider adopting MALDI-TOF MS for filamentous fungi identification in the future.