Kim Hyeon-Be, Kim Eiseul, Yang Seung-Min, Lee Shinyoung, Kim Mi-Ju, Kim Hae-Yeong
Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University, Yongin, South Korea.
Front Microbiol. 2020 Aug 28;11:2087. doi: 10.3389/fmicb.2020.02087. eCollection 2020.
species are used as probiotics to provide beneficial effects to humans. These effects are specific to some species or subspecies of . However, some species or subspecies are not distinguished because similarity of 16S rRNA and housekeeping gene sequences within species is very high. In this study, we developed a real-time polymerase chain reaction (PCR) assay to rapidly and accurately detect 22 species by selecting genetic markers using comparative genomic analysis. A total of 210 genome sequences were compared to select species- or subspecies-specific genetic markers. A phylogenetic tree based on pan-genomes generated clusters according to species or subspecies except that two strains were not grouped with their subspecies. Based on pan-genomes constructed, species- or subspecies-specific genetic markers were selected. The specificity of these markers was confirmed by aligning these genes against 210 genome sequences. Real-time PCR could detect 22 specifically. We constructed the criterion for quantification by standard curves. To further test the developed assay for commercial food products, we monitored 26 probiotic products and 7 dairy products. Real-time PCR results and labeling data were then compared. Most of these products (21/33, 63.6%) were consistent with their label claims. Some products labeled at species level only can be detected up to subspecies level through our developed assay.
一些物种被用作益生菌，为人类带来有益效果。这些效果特定于某些物种或亚种。然而，由于某些物种内16S rRNA和管家基因序列的相似性非常高，一些物种或亚种难以区分。在本研究中，我们通过比较基因组分析选择遗传标记，开发了一种实时聚合酶链反应（PCR）检测方法，以快速准确地检测22种特定物种。共比较了210个基因组序列，以选择物种或亚种特异性遗传标记。基于泛基因组构建的系统发育树根据物种或亚种生成聚类，除了两个菌株未与其亚种归为一组。基于构建的泛基因组，选择了物种或亚种特异性遗传标记。通过将这些基因与210个基因组序列比对，确认了这些标记的特异性。实时PCR能够特异性地检测22种特定物种。我们通过标准曲线构建了定量标准。为了进一步测试所开发的检测方法对商业食品的适用性，我们监测了26种益生菌产品和7种乳制品。然后比较了实时PCR结果和标签数据。这些产品中的大多数（21/33，63.6%）与其标签声明一致。一些仅在物种水平标注的产品，通过我们开发的检测方法可以检测到亚种水平。
