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Cited by 98Probiotics and Fermented FoodsBacterial Identification and Susceptibility TestingGenomics and Phylogenetic Studies

Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate

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Kim, Eiseul, Yang, Seung-Min, Lim, Bora, Park, Si Hong, Rackerby, Bryna, Kim, Hae-Yeong·2020·BMC Microbiology
DOI 10.1186/s12866-020-01781-zPMC PMC7160897

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Abstract

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.

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  • A quantitative real-time PCR standard curve was constructed to quantify HC-2 using L. plantarum-specific primers with a 128 bp amplicon.

    “a quantitative real-time PCR (qPCR) standard curve was constructed to quantify the number of HC-2 using L. plantarum-specific primers (F: 5′-TTATTCAAGCCGTCGGAGTG-3′; R: 5′-TCGCTGGTGCTAATGCAATG-3′.) with 128 bp amplicon (Kim et al., 2020).”

    From Adhesion and Colonization of the Probiotic Lactobacillus plantarum HC-2 in the Intestine of Litopenaeus Vannamei Are Associated With Bacterial Surface Proteins (2022) · cites this paper as Kim et al., 2020

    Table 1 in Results identifies the quoted primers and 128-bp amplicon as specific to L. paraplantarum, not L. plantarum; it lists a different L. plantarum primer pair with a 147-bp product. The Methods describe standard curves made from Lactobacillus reference-strain DNA to assess assay accuracy, not a curve to quantify HC-2. (LLM, conf=0.90)

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Authors
Kim, E., Yang, S.-M., Lim, B., Park, S. H., Rackerby, B., & Kim, H.-Y.
Affiliations
  • Kyung Hee University (KR)
  • Oregon State University (US)
Corresponding author
Kim, Hae-Yeong
ORCID
Venue
BMC Microbiology
Publisher
Springer Science and Business Media LLC
Published
2020-04-15
Volume
20
Issue
1
Pages
96-96
ISSN
1471-2180 (ISSN-L)
DOI
10.1186/s12866-020-01781-z
PMC
PMC7160897
OpenAlex
W3016402523
Cited by
98
References
42
Citations by year
2026: 18 · 2025: 15 · 2024: 19 · 2023: 22 · 2022: 12 · 2021: 8 · 2020: 4
FWCI
7.45
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Microbiology (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Ministry of Agriculture, Food and Rural Affairs (918005-4)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Bacterial Identification and Susceptibility Testing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Keywords
species-specific primers, comparative genomics, 16S-23S rRNA gene, probiotics, fermented dairy products, label verification, species-specific genes, 96-well plate assay
MeSH terms
DNA, Bacterial, DNA, Ribosomal, Food Microbiology, Lactobacillus, Phylogeny, RNA, Ribosomal, 16S, RNA, Ribosomal, 23S, Sensitivity and Specificity, Species Specificity, Bacterial Typing Techniques, Polymerase Chain Reaction, Sequence Analysis, DNA, DNA Primers, Genomics

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