Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2
IntroductionReliable and accurate methods for probiotic identification and enumeration, at the strain level plays a major role in confirming product efficacy since probiotic health benefits are strain-specific and dose-dependent. In this study, real-time PCR methods were developed for strain specifi...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-01-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1076631/full |
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| author | Hanan R. Shehata Hanan R. Shehata Hanan R. Shehata Basma Hassane Steven G. Newmaster |
| author_facet | Hanan R. Shehata Hanan R. Shehata Hanan R. Shehata Basma Hassane Steven G. Newmaster |
| author_sort | Hanan R. Shehata |
| collection | DOAJ |
| description | IntroductionReliable and accurate methods for probiotic identification and enumeration, at the strain level plays a major role in confirming product efficacy since probiotic health benefits are strain-specific and dose-dependent. In this study, real-time PCR methods were developed for strain specific identification and enumeration of L. paracasei 8700:2, a probiotic strain that plays a role in fighting the common cold.MethodsThe assay was designed to target a unique region in L. paracasei 8700:2 genome sequence to achieve strain level specificity. The identification assay was evaluated for specificity and sensitivity. The enumeration viability real-time PCR (v-qPCR) method was first optimized for the viability treatment, then the method was evaluated for efficiency, limit of quantification, precision, and its performance was compared to plate count (PC) and viability droplet digital PCR (v-ddPCR) methods.ResultsThe identification method proved to be strain specific and highly sensitive with a limit of detection of 0.5 pg of DNA. The optimal viability dye (PMAxx) concentration was 50 μM. The method was efficient (> 90% with R2 values > 0.99), with a linear dynamic range between 6*102 and 6*105 copies. The method was highly precise with a relative standard deviation below 5%. The Pearson correlation coefficient (r) was 0.707 for PC and v-qPCR methods, and 0.922 for v-qPCR and v-ddPCR. Bland-Altman method comparison showed that v-qPCR always gave higher values compared to PC method (relative difference ranging from 119% to 184%) and showed no consistent trend (relative difference ranging from −20% to 22%) when comparing v-qPCR and v-ddPCR methods.DiscussionThe difference between PC and v-PCR methods can potentially be attributed to the proportion of cells that exist in a viable but non culturable (VBNC) state, which can be count by v-PCR but not with PC. The developed v-qPCR method was confirmed to be strain specific, sensitive, efficient, with low variance, able to count VBNC cells, and has shorter time to results compared to plate count methods. Thus, the identification and enumeration methods developed for L. paracasei 8700:2 will be of great importance to achieve high quality and efficacious probiotic products. |
| first_indexed | 2024-04-10T22:18:23Z |
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| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-10T22:18:23Z |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-345d7ebc34214dadbf4fdfd62fe71beb2023-01-18T05:26:10ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-01-011310.3389/fmicb.2022.10766311076631Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2Hanan R. Shehata0Hanan R. Shehata1Hanan R. Shehata2Basma Hassane3Steven G. Newmaster4Natural Health Product Research Alliance, Department of Integrative Biology, College of Biological Science, University of Guelph, Guelph, ON, CanadaDepartment of Microbiology, Faculty of Pharmacy, Mansoura University, Mansoura, EgyptPurity-IQ Inc., Guelph, ON, CanadaPurity-IQ Inc., Guelph, ON, CanadaNatural Health Product Research Alliance, Department of Integrative Biology, College of Biological Science, University of Guelph, Guelph, ON, CanadaIntroductionReliable and accurate methods for probiotic identification and enumeration, at the strain level plays a major role in confirming product efficacy since probiotic health benefits are strain-specific and dose-dependent. In this study, real-time PCR methods were developed for strain specific identification and enumeration of L. paracasei 8700:2, a probiotic strain that plays a role in fighting the common cold.MethodsThe assay was designed to target a unique region in L. paracasei 8700:2 genome sequence to achieve strain level specificity. The identification assay was evaluated for specificity and sensitivity. The enumeration viability real-time PCR (v-qPCR) method was first optimized for the viability treatment, then the method was evaluated for efficiency, limit of quantification, precision, and its performance was compared to plate count (PC) and viability droplet digital PCR (v-ddPCR) methods.ResultsThe identification method proved to be strain specific and highly sensitive with a limit of detection of 0.5 pg of DNA. The optimal viability dye (PMAxx) concentration was 50 μM. The method was efficient (> 90% with R2 values > 0.99), with a linear dynamic range between 6*102 and 6*105 copies. The method was highly precise with a relative standard deviation below 5%. The Pearson correlation coefficient (r) was 0.707 for PC and v-qPCR methods, and 0.922 for v-qPCR and v-ddPCR. Bland-Altman method comparison showed that v-qPCR always gave higher values compared to PC method (relative difference ranging from 119% to 184%) and showed no consistent trend (relative difference ranging from −20% to 22%) when comparing v-qPCR and v-ddPCR methods.DiscussionThe difference between PC and v-PCR methods can potentially be attributed to the proportion of cells that exist in a viable but non culturable (VBNC) state, which can be count by v-PCR but not with PC. The developed v-qPCR method was confirmed to be strain specific, sensitive, efficient, with low variance, able to count VBNC cells, and has shorter time to results compared to plate count methods. Thus, the identification and enumeration methods developed for L. paracasei 8700:2 will be of great importance to achieve high quality and efficacious probiotic products.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1076631/fullreal-time PCRprobe-based assaystrain specific PCR assayprobioticsviability PCRPMAxx |
| spellingShingle | Hanan R. Shehata Hanan R. Shehata Hanan R. Shehata Basma Hassane Steven G. Newmaster Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2 Frontiers in Microbiology real-time PCR probe-based assay strain specific PCR assay probiotics viability PCR PMAxx |
| title | Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2 |
| title_full | Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2 |
| title_fullStr | Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2 |
| title_full_unstemmed | Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2 |
| title_short | Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2 |
| title_sort | real time polymerase chain reaction methods for strain specific identification and enumeration of strain lacticaseibacillus paracasei 8700 2 |
| topic | real-time PCR probe-based assay strain specific PCR assay probiotics viability PCR PMAxx |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1076631/full |
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