Abstract

In mollusk aquaculture, a large number of Vibrio species are considered major pathogens. Conventional methods based on DNA amplification and sequencing used to accurately identify Vibrio species are unsuitable for monitoring programs because they are time-consuming and expensive. The aim of this study was, therefore, to develop the MALDI-TOF MS method in order to establish a rapid identification technique for a large panel of Vibrio species. We created the EnviBase containing 120 main spectra projections (MSP) of the Vibrio species that are potentially responsible for mollusk diseases, comprising 25 species: V. aestuarianus, V. cortegadensis, V. tapetis and species belonging to the Coralliilyticus, Harveyi, Mediterranei, and Orientalis clades. Each MSP was constructed by the merger of raw spectra obtained from three different media and generated by three collaborating laboratories to increase the diversity of the conditions and thus obtain a good technique robustness. Perfect discrimination was obtained with all of the MSP created for the Vibrio species and even for very closely related species as V. europaeus and V. bivalvicida. The new EnviBase library was validated through a blind test on 100 Vibrio strains performed by our three collaborators who used the direct transfer and protein extraction methods. The majority of the Vibrio strains were successfully identified with the newly created EnviBase by the three laboratories for both protocol methods. This study documents the first development of a freely accessible database exclusively devoted to Vibrio found in marine environments, taking into account the high diversity of this genus.

Key points

• Development of a MALDI-TOF MS database to quickly affiliate Vibrio species.

• Increase of the reactivity when faced with Vibrio associated with mollusk diseases.

• Validation of MALDI-TOF MS as routine diagnostic tool.

中文翻译：

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MALDI-TOF MS数据库，用于快速鉴定弧菌。对海洋软体动物潜在致病

摘要

在软体动物养殖中，许多弧菌被认为是主要的病原体。基于DNA扩增和测序的传统方法用于准确识别弧菌，不适合用于监视程序，因为它们既费时又昂贵。因此，本研究的目的是开发MALDI-TOF MS方法，以便为大型弧菌物种建立快速鉴定技术。我们创建了一个EnviBase，其中包含120种可能引起软体动物疾病的弧菌物种的主要光谱投影（MSP），其中包括25种：南美锥嘴兽（V. aestuarianus），V。cortegadensis，V。tapetis和属于Coralliilyticus，Harveyi，Mediterranei和Orientalis进化枝的物种。每个MSP都是通过合并从三种不同介质获得的原始光谱并由三个合作实验室生成的，从而增加条件的多样性而获得的，因此具有良好的技术鲁棒性。与所有的创建的MSP获得完美区分弧菌物种，甚至非常密切相关的物种如五europaeus和V. bivalvicida。新的EnviBase库通过100 Vibrio的盲测验证由我们的三位合作者使用直接转移和蛋白质提取方法进行的菌株。三个实验室针对这两种方案方法，使用新创建的EnviBase成功鉴定出大多数弧菌菌株。考虑到该属的高度多样性，本研究记录了专门针对海洋环境中弧菌的可免费访问的数据库的首次开发。

关键点

•开发MALDI-TOF MS数据库，以快速隶属弧菌。

•面对与软体动物疾病相关的弧菌时，反应性增加。

•验证MALDI-TOF MS作为常规诊断工具。