Dataset Information

Development of a real-time quantitative PCR method for detection and quantification of Prevotella copri.

ABSTRACT:

Background

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 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.

Results

In 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.

Conclusions

This 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.

SUBMITTER: Verbrugghe P

PROVIDER: S-EPMC7798335 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

Development of a real-time quantitative PCR method for detection and quantification of Prevotella copri.

Verbrugghe Phebe P Van Aken Olivier O Hållenius Frida F Nilsson Anne A

BMC microbiology 20210111 1

<h4>Background</h4>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 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.<h4>Results</h4>In this study, a Sybr Green qPCR protocol for P. copri detection and quantification was ...[more]

PMID: 33430782

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Project description:BackgroundQuantitative Polymerase Chain Reaction (qPCR) is a well-established method for quantifying levels of gene expression, but has not been routinely applied to the detection of constitutional copy number alterations of human genomic DNA. Microdeletions or microduplications of the human genome are associated with a variety of genetic disorders. Although, clinical laboratories routinely use fluorescence in situ hybridization (FISH) to identify such cryptic genomic alterations, there remains a significant number of individuals in which constitutional genomic imbalance is suspected, based on clinical parameters, but cannot be readily detected using current cytogenetic techniques.ResultsIn this study, a novel application for real-time qPCR is presented that can be used to reproducibly detect chromosomal microdeletions and microduplications. This approach was applied to DNA from a series of patient samples and controls to validate genomic copy number alteration at cytoband 22q11. The study group comprised 12 patients with clinical symptoms of chromosome 22q11 deletion syndrome (22q11DS), 1 patient trisomic for 22q11 and 4 normal controls. 6 of the patients (group 1) had known hemizygous deletions, as detected by standard diagnostic FISH, whilst the remaining 6 patients (group 2) were classified as 22q11DS negative using the clinical FISH assay. Screening of the patients and controls with a set of 10 real time qPCR primers, spanning the 22q11.2-deleted region and flanking sequence, confirmed the FISH assay results for all patients with 100% concordance. Moreover, this qPCR enabled a refinement of the region of deletion at 22q11. Analysis of DNA from chromosome 22 trisomic sample demonstrated genomic duplication within 22q11.ConclusionIn this paper we present a qPCR approach for the detection of chromosomal microdeletions and microduplications. The strategic use of in silico modelling for qPCR primer design to avoid regions of repetitive DNA, whilst providing a level of genomic resolution greater than standard cytogenetic assays. The implementation of qPCR detection in clinical laboratories will address the need to replace complex, expensive and time consuming FISH screening to detect genomic microdeletions or duplications of clinical importance.

Project description:Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R(2) > 0.98) over a 7-log-unit dynamic range down to 10(1) B. atrophaeus cells or spores. Quantification of S. marcescens (R(2) > 0.98) was linear over a 6-log-unit dynamic range down to 10(2) S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

Dataset Information

Development of a real-time quantitative PCR method for detection and quantification of Prevotella copri.

Background

Results

Conclusions

Publications

Development of a real-time quantitative PCR method for detection and quantification of Prevotella copri.

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