Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™

Shehata, Hanan R.; Kiefer, Anthony; Morovic, Wesley; Newmaster, Steven G.

doi:10.3389/fmicb.2021.801795

Cited by 6 publications

(4 citation statements)

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“…The most common methods for probiotic strain identification are DNA based methods, such as conventional PCR and real-time PCR methods that can be designed to achieve species level or strain level identification ( Solano-Aguilar et al, 2008 ; Ahlroos and Tynkkynen, 2009 ; Achilleos and Berthier, 2013 ; Herbel et al, 2013 ; Morovic et al, 2016 ; Shehata et al, 2020 , 2021a ; Shehata and Newmaster, 2020 ; Zhang et al, 2021 ). Real-time PCR based methods have several advantages such as the ability to monitor reactions in real time, high sensitivity, high accuracy, and short time to results.…”

Section: Introductionmentioning

confidence: 99%

Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2

Shehata

Hassane²,

Newmaster

2023

Front. Microbiol.

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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.

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Section: Introductionmentioning

confidence: 99%

Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2

Shehata

Hassane²,

Newmaster

2023

Front. Microbiol.

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“…This method allows for highly accurate strain management. Until now, RT-PCR-based method targeting single nucleotide polymorphism or small deletion in the specific genes, CRISPER spacers (Shehata et al, 2021(Shehata et al, ), were 10.3389/fmicb.2022 established. Compared to these methods, our method is easy to identify the EF-2001 strain-specific region.…”

Section: Discussionmentioning

confidence: 99%

Establishment of a polymerase chain reaction-based method for strain-level management of Enterococcus faecalis EF-2001 using species-specific sequences identified by whole genome sequences

et al. 2022

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In the development and manufacture of fermented foods, it is crucial to control and manage the bacterial species used in the products. We previously reported a complete genome sequence analysis of the Enterococcus faecalis EF-2001 strain that was used for supplements. By comparing this sequence to the publicly available complete genome sequence of E. faecalis strains, we were able to identify specific sequences of the EF-2001 strain. We designed primer sets to amplify these specific regions and performed a polymerase chain reaction (PCR). We confirmed that the DNA fragments were specifically amplified in the genome of the EF-2001 strain, but not those of other lactic acid bacteria (LABs) or strains of the same genus. Furthermore, these primers amplified DNA fragments even in genomic DNA extracted from heat-treated bacteria at 121°C and foods containing the EF-2001 strain. These results suggest that this method allows for simple and highly accurate identification of specific fermentation strains, such as LABs at the strain level, which will be useful for controlling the quality of fermented foods.

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“…lactis Bb12 ( Solano-Aguilar et al, 2008 ), B. animalis subsp. lactis DSM 15954 and Bi-07™ ( Shehata et al, 2021a ), L. gasseri BNR17, and L. reuteri LRC (NCIMB 30242) ( Shehata et al, 2021b ).…”

Section: Real Time Pcr Based Methodsmentioning

confidence: 99%

Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques

Boyte,

Benkowski,

Pane

et al. 2023

Front. Microbiol.

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Probiotics are the largest non-herbal/traditional dietary supplements category worldwide. To be effective, a probiotic strain must be delivered viable at an adequate dose proven to deliver a health benefit. The objective of this article is to provide an overview of the various technologies available for probiotic enumeration, including a general description of each technology, their advantages and limitations, and their potential for the future of the probiotics industry. The current “gold standard” for analytical quantification of probiotics in the probiotic industry is the Plate Count method (PC). PC measures the bacterial cell’s ability to proliferate into detectable colonies, thus PC relies on cultivability as a measure of viability. Although viability has widely been measured by cultivability, there has been agreement that the definition of viability is not limited to cultivability. For example, bacterial cells may exist in a state known as viable but not culturable (VBNC) where the cells lose cultivability but can maintain some of the characteristics of viable cells as well as probiotic properties. This led to questioning the association between viability and cultivability and the accuracy of PC in enumerating all the viable cells in probiotic products. PC has always been an estimate of the number of viable cells and not a true cell count. Additionally, newer probiotic categories such as Next Generation Probiotics (NGPs) are difficult to culture in routine laboratories as NGPs are often strict anaerobes with extreme sensitivity to atmospheric oxygen. Thus, accurate quantification using culture-based techniques will be complicated. Another emerging category of biotics is postbiotics, which are inanimate microorganisms, also often referred to as tyndallized or heat-killed bacteria. Obviously, culture dependent methods are not suitable for these products, and alternative methods are needed for their quantification. Different methodologies provide a more complete picture of a heterogeneous bacterial population versus PC focusing exclusively on the eventual multiplication of the cells. Alternative culture-independent techniques including real-time PCR, digital PCR and flow cytometry are discussed. These methods can measure viability beyond cultivability (i.e., by measuring cellular enzymatic activity, membrane integrity or membrane potential), and depending on how they are designed they can achieve strain-specific enumeration.

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Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™

Cited by 6 publications

References 43 publications

Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2

Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2

Establishment of a polymerase chain reaction-based method for strain-level management of Enterococcus faecalis EF-2001 using species-specific sequences identified by whole genome sequences

Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques

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