[논문]Identification and Monitoring of Lactobacillus delbrueckii Subspecies Using Pangenomic-Based Novel Genetic Markers

Kim, Eiseul; Cho, Eun-Ji; Yang, Seung-Min; Kim, Hae-Yeong

doi:10.4014/jmb.2009.09034

Identification and Monitoring of Lactobacillus delbrueckii Subspecies Using Pangenomic-Based Novel Genetic Markers 원문보기

Journal of microbiology and biotechnology, v.31 no.2, 2021년, pp.280 - 289

Kim, Eiseul (Institute of Life Sciences and Resources and Department of Food Science and Biotechnology, Kyung Hee University) , Cho, Eun-Ji (Institute of Life Sciences and Resources and Department of Food Science and Biotechnology, Kyung Hee University) , Yang, Seung-Min (Institute of Life Sciences and Resources and Department of Food Science and Biotechnology, Kyung Hee University) , Kim, Hae-Yeong (Institute of Life Sciences and Resources and Department of Food Science and Biotechnology, Kyung Hee University)

Abstract ▼ AI-Helper

Genetic markers currently used for the discrimination of Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, our objective in this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. We evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genomes. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited at NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing the core and pangenomes. 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 detecting 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.

주제어

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제안 방법

Real-time PCR assay was conducted to determine the specificity and accuracy of primer pairs using a 7500 Real-time PCR system. Each reaction contained 20 ng of genomic DNA, 10 μl of 2X Thunderbird SYBR qPCR Mix (Toyobo, Japan), 500 nM of primer pairs, and distilled water up to 20 μl total volume.
The method to identify L. delbrueckii subspecies with genetic markers selected in this study requires WGS and bioinformatics analysis to confirm the presence of their markers. This method can accurately identify L.
These genetic markers were present in all genomes of the same subspecies but absent in genomes of other subspecies and bacterial strains. To rapidly and cost-effectively identify L. delbrueckii subspecies, subspecies specific primer pairs for three subspecies mainly isolated from food samples were designed. The real-time PCR method using these genes could accurately identify L.

대상 데이터

To test the developed real-time PCR method, 64 isolates, 15 probiotic products, and 11 dairy products were used. L.

이론/모형

According to the developer’s recommendation, a genome database for pangenome analysis was constructed using Anvi’o genome storage. The pangenome was then analyzed using the NCBI BLASTp and MCL algorithm. Subsequently, a phylogenetic tree was constructed based on pangenome cluster frequencies.

성능/효과

from probiotic and dairy products were used. A total of 54 strains of lactic acid bacteria including L.delbrueckii subspecies were obtained from the Korean Agricultural Culture Collection (KACC, Korea), the Korean Collection for Type Cultures (KCTC, Korea), the Korean Culture Center of Microorganisms (KCCM, Korea), the NITE Biological Resource Center (NBRC, Japan), and the Laboratory Isolates (LI, Korea) (Table 2). All reference strains were grown in MRS broth (Difco, Becton & Dickinson, USA) for extraction of genomic DNA.
By comparing genomes of the same subspecies, 995 to 1, 628 protein-coding genes were found in common in the genomes of each subspecies and considered as the core-genome for each subspecies. After comparing each core-genome with pangenome for protein-coding genes present in all genomes except for target genomes, 5 to 50 protein-coding genes were selected as candidate genetic markers for each subspecies. These candidate genetic markers were aligned with 59, 196, 562 genome sequences.
As a result of pangenome analysis, a total of 67, 178 genes from 41 L. delbrueckii subspecies yielded a pangenome size of 3, 456 genes. The core-genome, accessory-genome, and unique-genome had 749, 2, 071, and 636 genes, respectively.
In conclusion, pangenome analysis was performed to select genetic markers for six L. delbrueckii subspecies. These genetic markers were present in all genomes of the same subspecies but absent in genomes of other subspecies and bacterial strains.
lactis (n = 17) and bulgaricus (n = 47) (Table 4). These results confirmed that the real-time PCR developed in this study could accurately identify strains present in probiotic and dairy products and bacterial isolates to the subspecies level.

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