设计 PCR 检测方法，以在单个 96 孔板中特异性检测和鉴定 37 种乳杆菌。
Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate.
机构信息
Institute of Life Sciences & Resources and Department of Food Science and Biotechnology, Kyung Hee University, Yongin, 17104, South Korea.
Department of Food Science and Technology, Oregon State University, Corvallis, Oregon, 97331, USA.
出版信息
BMC Microbiol. 2020 Apr 15;20(1):96. doi: 10.1186/s12866-020-01781-z.
BACKGROUND
Lactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods.
RESULTS
To select species-specific sequences or genes, a total of 180 Lactobacillus genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S-23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S-23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the polymerase chain reaction (PCR) with the standard curve were confirmed. The PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This PCR assays were designed to detect and identify 37 Lactobacillus species. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label.
CONCLUSIONS
The method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.
背景
乳杆菌属被用作益生菌，在发酵食品生产中发挥着重要作用。然而，将 16S rRNA 基因序列用作乳杆菌属种的区分标准标记物的范围非常有限，因为一些乳杆菌属种的 16S rRNA 基因序列相似。在本研究中，我们开发了一种基于比较基因组分析的快速准确方法，用于鉴定常用于益生菌和发酵食品的 37 种乳杆菌属种。
结果
为了选择种特异性序列或基因，使用 Python 脚本比较了总共 180 个乳杆菌属基因组序列。在 37 个物种中的 14 个物种中，由于 16S-23S rRNA 基因的相似性，无法找到种特异性序列。通过比较基因组分析获得了选择的独特基因，并且通过计算机分析证实所有基因均特异性针对 52478804 个基因组；它们不是菌株特异性的，而是存在于同一物种的所有菌株中。从所选的 16S-23S rRNA 基因序列或物种的独特基因中设计了种特异性引物对。使用参考菌株确认了种特异性引物对的特异性，并通过标准曲线确认了聚合酶链反应（PCR）的准确性和效率。本研究中开发的 PCR 方法能够准确地区分仅使用 16S rRNA 基因无法区分的物种。该 PCR 方法设计用于检测和鉴定 37 种乳杆菌属。然后将开发的方法应用于 19 种益生菌和 12 种乳制品的监测。应用测试证实，在 17 种产品中检测到的物种与标签上指示的物种相匹配，而其余产品中含有标签上未显示的物种。
结论
本研究中开发的方法能够快速准确地区分不同的乳杆菌属种，可用于监测益生菌和乳制品等食品中的特定乳杆菌属种。
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