Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage
Probiotics are typically enumerated by agar plate counting (PC) techniques. PC has several limitations including poor specificity, high variability, inability to enumerate dead cells, viable but non-culturable cells and cells in complex matrices. Viability droplet digital polymerase chain reaction (...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-10-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.966264/full |
| _version_ | 1797989881153060864 |
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| author | Anthony Kiefer Phillip M. Byrd Peipei Tang Gregory Jones Kevin Galles Vincenzo Fallico Connie Wong |
| author_facet | Anthony Kiefer Phillip M. Byrd Peipei Tang Gregory Jones Kevin Galles Vincenzo Fallico Connie Wong |
| author_sort | Anthony Kiefer |
| collection | DOAJ |
| description | Probiotics are typically enumerated by agar plate counting (PC) techniques. PC has several limitations including poor specificity, high variability, inability to enumerate dead cells, viable but non-culturable cells and cells in complex matrices. Viability droplet digital polymerase chain reaction (v-ddPCR) is an emerging enumeration technique with improved specificity, precision, and the ability to enumerate cells in varying states of culturability or in complex matrices. Good correlation and agreement between v-ddPCR and PC is well documented, but not much research has been published on the comparison when enumerating freeze-dried (FD) probiotics during storage. In this study, v-ddPCR utilizing PE51 (PE51-ddPCR), a combination of propidium monoazide (PMA) and ethidium monoazide (EMA), was evaluated as alternative enumeration technique to PC on blends of four FD probiotic strains over the course of a 3-month storage study with accelerated conditions. When PMA and EMA are combined (PE51), this study demonstrates agreement (bias = 7.63e+9, LOA = 4.38e+10 to 5.9e+10) and association (r = 0.762) between PC and v-ddPCR, at or above levels of an accepted alternative method. Additionally, v-ddPCR with individual dyes PMA and EMA provide insight into how they individually contribute to the viable counts obtained by PE51-ddPCR and provide a more specific physiological understanding of how probiotics cope with or experience damage during storage. |
| first_indexed | 2024-04-11T08:27:39Z |
| format | Article |
| id | doaj.art-268f95f48b4844189cab315f8f5c7321 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-11T08:27:39Z |
| publishDate | 2022-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-268f95f48b4844189cab315f8f5c73212022-12-22T04:34:40ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-10-011310.3389/fmicb.2022.966264966264Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storageAnthony KieferPhillip M. ByrdPeipei TangGregory JonesKevin GallesVincenzo FallicoConnie WongProbiotics are typically enumerated by agar plate counting (PC) techniques. PC has several limitations including poor specificity, high variability, inability to enumerate dead cells, viable but non-culturable cells and cells in complex matrices. Viability droplet digital polymerase chain reaction (v-ddPCR) is an emerging enumeration technique with improved specificity, precision, and the ability to enumerate cells in varying states of culturability or in complex matrices. Good correlation and agreement between v-ddPCR and PC is well documented, but not much research has been published on the comparison when enumerating freeze-dried (FD) probiotics during storage. In this study, v-ddPCR utilizing PE51 (PE51-ddPCR), a combination of propidium monoazide (PMA) and ethidium monoazide (EMA), was evaluated as alternative enumeration technique to PC on blends of four FD probiotic strains over the course of a 3-month storage study with accelerated conditions. When PMA and EMA are combined (PE51), this study demonstrates agreement (bias = 7.63e+9, LOA = 4.38e+10 to 5.9e+10) and association (r = 0.762) between PC and v-ddPCR, at or above levels of an accepted alternative method. Additionally, v-ddPCR with individual dyes PMA and EMA provide insight into how they individually contribute to the viable counts obtained by PE51-ddPCR and provide a more specific physiological understanding of how probiotics cope with or experience damage during storage.https://www.frontiersin.org/articles/10.3389/fmicb.2022.966264/fullprobioticstoragev-ddPCRviabilityenumerationPMA |
| spellingShingle | Anthony Kiefer Phillip M. Byrd Peipei Tang Gregory Jones Kevin Galles Vincenzo Fallico Connie Wong Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage Frontiers in Microbiology probiotic storage v-ddPCR viability enumeration PMA |
| title | Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage |
| title_full | Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage |
| title_fullStr | Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage |
| title_full_unstemmed | Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage |
| title_short | Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage |
| title_sort | viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage |
| topic | probiotic storage v-ddPCR viability enumeration PMA |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.966264/full |
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