Abstract
BackgroundSince its discovery in 2007, the importance of the human gut bacterium Prevotella copri (P. copri) has been widely recognized with its links to diet and health status and potential as next generation probiotic. Therefore, precise, convenient and cost-effective diagnostic tools for the detection and quantification of P. copri from clinical and environmental samples are needed.ResultsIn this study, a Sybr Green qPCR protocol for P. copri detection and quantification was developed and tested on P. copri-spiked murine faeces samples targeting both the 16S rRNA gene and P. copri genome specific genes. The use of one 16S rRNA primer pair and 2 genome specific primer pairs resulted in at least 10x higher specificity and sensitivity than the primer-only PCR currently cited in the literature, reaching a sensitivity of 103 CFU/mL. Furthermore, we showed that the new 16S rRNA primer set provided the best balance of detection of a wide range of P. copri strains, while avoiding off-target detection of other Prevotella genus species. The quantification of P. copri in human stool samples using the new 16S rRNA primers also correlated well with 16S rRNA high throughput MiSeq sequencing data (r2 = 0.6604, p = 0.0074). The two genome specific primer pairs on the other hand uniquely detect the DSM18205 reference strain, allowing differential detection of indigenous and experimentally administered P. copri populations. Finally, it was shown that SYBR green qPCR mixes have an influence on sensitivity and specificity, with Biorad SsoAdvanced Universal SYBR Green Supermix performing the best under our test conditions of six commercially available SYBR green master mixes.ConclusionsThis improved qPCR-based method will allow accurate P. copri identification and quantification. Moreover, this methodology can also be applied to identify other bacterial species in complex samples.
Highlights
Since its discovery in 2007, the importance of the human gut bacterium Prevotella copri (P. copri) has been widely recognized with its links to diet and health status and potential as generation probiotic
Verbrugghe et al BMC Microbiology (2021) 21:23 subjects, increased P. copri abundance was correlated with improved glucose tolerance after barley consumption; and in mice, oral administration of live P. copri resulted in improved glucose metabolism [10], suggesting a causal relationship
Though the primers showed good specificity and sensitivity for pure P. copri Genomic DNA (gDNA) samples, this was not the case in the presence of murine faeces, where non-specific amplification was observed in the melt curves at low P. copri concentrations even under stringent PCR conditions (Fig. 1)
Summary
Since its discovery in 2007, the importance of the human gut bacterium Prevotella copri (P. copri) has been widely recognized with its links to diet and health status and potential as generation probiotic. One. Verbrugghe et al BMC Microbiology (2021) 21:23 subjects, increased P. copri abundance was correlated with improved glucose tolerance after barley consumption; and in mice, oral administration of live P. copri resulted in improved glucose metabolism [10], suggesting a causal relationship. Verbrugghe et al BMC Microbiology (2021) 21:23 subjects, increased P. copri abundance was correlated with improved glucose tolerance after barley consumption; and in mice, oral administration of live P. copri resulted in improved glucose metabolism [10], suggesting a causal relationship In line with these findings, P. copri has been identified as a candidate for a generation probiotic to prevent and treat metabolic diseases [11]. Considering faecal P. copri has been associated with both health benefits and some inflammatory states, it is important to have accurate and rapid methods for testing its abundance
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
