Genetic markers currently used for the discrimination of Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, the objective of this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. This study evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genome. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited in NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing pan and core-genome. Genetic markers were confirmed to be specific for 59,196,562 genome sequences via in silico analysis. They were found in all strains of the same subspecies, but not in other subspecies or bacterial strains. These genetic markers also could be used to accurately identify genomes at the subspecies level for genomes known at the species level. A real-time PCR method for the detection of three main subspecies (L. delbrueckii subsp. delbrueckii, lactis, and bulgaricus) was developed to cost-effectively identify them using genetic markers. Results showed 100% specificity for each subspecies. These genetic markers could differentiate each subspecies from 44 other lactic acid bacteria. This real-time PCR method was then applied to monitor 26 probiotics and dairy products. It was also used to identify 64 unknown strains isolated from raw milk samples and dairy products. Results confirmed that unknown isolates and subspecies contained in the product could be accurately identified using this real-time PCR method.

中文翻译：

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使用基于泛基因组的新型遗传标记鉴定和监测德氏乳杆菌亚种。

目前用于区分德氏乳杆菌亚种的遗传标记在亚种水平上的鉴定效率较低。因此，本研究的目的是基于泛基因组分析选择新的遗传标记，用于准确鉴定和区分 6 个L. delbrueckii亚种。本研究评估了L. delbrueckii基因组，以避免在选择遗传标记的过程中由于错误标记的基因组而得出错误的结论。基因组分析显示L. delbrueckii的两个基因组保藏在 NCBI 中的亚种被错误识别。基于这些结果，通过比较泛基因组和核心基因组来选择亚种特异性遗传标记。通过计算机分析证实遗传标记对 59,196,562 个基因组序列具有特异性。它们存在于同一亚种的所有菌株中，但不存在于其他亚种或细菌菌株中。这些遗传标记还可用于准确识别物种水平已知基因组的亚种水平基因组。用于检测三个主要亚种（ L. delbrueckii subsp. delbrueckii、lactis和bulgaricus）的实时 PCR 方法) 的开发是为了使用遗传标记经济有效地识别它们。结果显示每个亚种的特异性为 100%。这些遗传标记可以将每个亚种与其他 44 种乳酸菌区分开来。然后应用这种实时 PCR 方法监测 26 种益生菌和乳制品。它还用于鉴定从原料奶样品和乳制品中分离出的 64 种未知菌株。结果证实，使用这种实时 PCR 方法可以准确鉴定产品中包含的未知分离株和亚种。