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Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM(®) and Bifidobacterium animalis subsp. lactis Bl-04(®) via Chip-Based Digital PCR

The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically...

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Autores principales: Hansen, Sarah J. Z., Morovic, Wesley, DeMeules, Martha, Stahl, Buffy, Sindelar, Connie W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904286/
https://www.ncbi.nlm.nih.gov/pubmed/29696008
http://dx.doi.org/10.3389/fmicb.2018.00704
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author Hansen, Sarah J. Z.
Morovic, Wesley
DeMeules, Martha
Stahl, Buffy
Sindelar, Connie W.
author_facet Hansen, Sarah J. Z.
Morovic, Wesley
DeMeules, Martha
Stahl, Buffy
Sindelar, Connie W.
author_sort Hansen, Sarah J. Z.
collection PubMed
description The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR) to enumerate Bifidobacterium animalis subsp. lactis Bl-04 and Lactobacillus acidophilus NCFM both as single strains and in combination. Primers and probes were designed to differentiate the target strains against other strains of the same species using known single copy, genetic differences. The assay was optimized to include propidium monoazide pre-treatment to prevent amplification of DNA associated with dead probiotic cells as well as liberation of DNA from cells with intact membranes using bead beating. The resulting assay was able to successfully enumerate each strain whether alone or in multiplex. The cdPCR method had a 4 and 5% relative standard deviation (RSD) for Bl-04 and NCFM, respectively, making it more precise than plate counts with an industry accepted RSD of 15%. cdPCR has the potential to replace traditional plate counts because of its precision, strain specificity and the ability to obtain results in a matter of hours.
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spelling pubmed-59042862018-04-25 Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM(®) and Bifidobacterium animalis subsp. lactis Bl-04(®) via Chip-Based Digital PCR Hansen, Sarah J. Z. Morovic, Wesley DeMeules, Martha Stahl, Buffy Sindelar, Connie W. Front Microbiol Microbiology The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR) to enumerate Bifidobacterium animalis subsp. lactis Bl-04 and Lactobacillus acidophilus NCFM both as single strains and in combination. Primers and probes were designed to differentiate the target strains against other strains of the same species using known single copy, genetic differences. The assay was optimized to include propidium monoazide pre-treatment to prevent amplification of DNA associated with dead probiotic cells as well as liberation of DNA from cells with intact membranes using bead beating. The resulting assay was able to successfully enumerate each strain whether alone or in multiplex. The cdPCR method had a 4 and 5% relative standard deviation (RSD) for Bl-04 and NCFM, respectively, making it more precise than plate counts with an industry accepted RSD of 15%. cdPCR has the potential to replace traditional plate counts because of its precision, strain specificity and the ability to obtain results in a matter of hours. Frontiers Media S.A. 2018-04-11 /pmc/articles/PMC5904286/ /pubmed/29696008 http://dx.doi.org/10.3389/fmicb.2018.00704 Text en Copyright © 2018 Hansen, Morovic, DeMeules, Stahl and Sindelar. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Hansen, Sarah J. Z.
Morovic, Wesley
DeMeules, Martha
Stahl, Buffy
Sindelar, Connie W.
Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM(®) and Bifidobacterium animalis subsp. lactis Bl-04(®) via Chip-Based Digital PCR
title Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM(®) and Bifidobacterium animalis subsp. lactis Bl-04(®) via Chip-Based Digital PCR
title_full Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM(®) and Bifidobacterium animalis subsp. lactis Bl-04(®) via Chip-Based Digital PCR
title_fullStr Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM(®) and Bifidobacterium animalis subsp. lactis Bl-04(®) via Chip-Based Digital PCR
title_full_unstemmed Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM(®) and Bifidobacterium animalis subsp. lactis Bl-04(®) via Chip-Based Digital PCR
title_short Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM(®) and Bifidobacterium animalis subsp. lactis Bl-04(®) via Chip-Based Digital PCR
title_sort absolute enumeration of probiotic strains lactobacillus acidophilus ncfm(®) and bifidobacterium animalis subsp. lactis bl-04(®) via chip-based digital pcr
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904286/
https://www.ncbi.nlm.nih.gov/pubmed/29696008
http://dx.doi.org/10.3389/fmicb.2018.00704
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