Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate
| Autor: | Seung-Min Yang, Bora Lim, Bryna Rackerby, Eiseul Kim, Si Hong Park, Hae-Yeong Kim |
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| Rok vydání: | 2020 |
| Předmět: |
DNA
Bacterial 0106 biological sciences Microbiology (medical) In silico lcsh:QR1-502 Computational biology Probiotic product DNA Ribosomal Polymerase Chain Reaction Sensitivity and Specificity 01 natural sciences Microbiology Genome lcsh:Microbiology law.invention 03 medical and health sciences Species Specificity law RNA Ribosomal 16S 010608 biotechnology Lactobacillus Gene Phylogeny Polymerase chain reaction DNA Primers Comparative genomics 0303 health sciences biology 030306 microbiology Genomics Sequence Analysis DNA Ribosomal RNA biology.organism_classification 16S ribosomal RNA Bacterial Typing Techniques RNA Ribosomal 23S PCR Food Microbiology Species-specific primer 16S rRNA gene Research Article |
| Zdroj: | BMC Microbiology, Vol 20, Iss 1, Pp 1-14 (2020) BMC Microbiology |
| DOI: | 10.21203/rs.2.20588/v4 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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