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Detecting and quantifying Veillonella by real-time quantitative PCR and droplet digital PCR
Zanbo Ding1,2, Jinghua Cui2, Qun Zhang2
1College of Life Sciences, Northwest A&F University, Yangling, China.
Abstract:
Veillonella spp. are Gram-negative opportunistic pathogens present in the respiratory, digestive, and reproductive tracts of mammals. An abnormal increase in Veillonella relative abundance in the body is closely associated with periodontitis, inflammatory bowel disease, urinary tract infections, and many other diseases. We designed a pair of primers and a probe based on the 16S rRNA gene sequences of Veillonella and conducted real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) to quantify the abundance of Veillonella in fecal samples. These two methods were tested for specificity and sensitivity using simulated clinical samples. The sensitivity of qPCR was 100 copies/μL, allowing for the accurate detection of a wide range of Veillonella concentrations from 103 to 108 CFU/mL. The sensitivity of ddPCR was 11.3 copies/μL, only allowing for the accurate detection of Veillonella concentrations from 101 to 104 CFU/mL because of the limited number of droplets generated by ddPCR. ddPCR is therefore more suitable for the detection of low-abundance Veillonella samples. To characterize the validity of the assay system, clinical samples from children with inflammatory bowel disease were collected and analyzed, and the results were verified using isolation methods. We conclude that molecular assays targeting the 16S rRNA gene provides an important tool for the rapid diagnosis of chronic and infectious diseases caused by Veillonella and also supports the isolation and identification of Veillonella for research purposes. KEY POINTS: • With suitable primer sets, the qPCR has a wider detection range than ddPCR. • ddPCR is suitable for the detection of low-abundance samples. • Methods successfully guided the isolation of Veillonella in clinical sample.
Insights
Molecular assays targeting Veillonella 16S rRNA genes offer rapid diagnosis for diseases like inflammatory bowel disease. Quantitative PCR (qPCR) provides a wider detection range, while droplet digital PCR (ddPCR) excels at identifying low-abundance bacterial samples.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Genetics
Background:
- Veillonella spp. are Gram-negative opportunistic pathogens implicated in various diseases, including periodontitis and inflammatory bowel disease (IBD).
- Accurate quantification of Veillonella abundance is crucial for understanding its role in disease pathogenesis and for developing effective diagnostic tools.
Purpose of the Study:
- To develop and evaluate molecular assays, specifically real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), for quantifying Veillonella in fecal samples.
- To compare the sensitivity and specificity of qPCR and ddPCR for detecting Veillonella, particularly in clinical samples from children with IBD.
Main Methods:
- Design of primers and a probe targeting the 16S rRNA gene of Veillonella.
- Quantification of Veillonella in simulated and clinical fecal samples using qPCR and ddPCR.
- Validation of molecular assay results using bacterial isolation methods.
Main Results:
- qPCR demonstrated a sensitivity of 100 copies/μL with a wide detection range (10^3 to 10^8 CFU/mL).
- ddPCR exhibited higher sensitivity (11.3 copies/μL) but a narrower detection range (10^1 to 10^4 CFU/mL), making it suitable for low-abundance samples.
- Both methods were successfully applied to clinical samples from children with IBD, aiding in Veillonella isolation and identification.
Conclusions:
- Molecular assays targeting the Veillonella 16S rRNA gene are valuable tools for the rapid diagnosis of chronic and infectious diseases.
- qPCR and ddPCR offer complementary approaches for quantifying Veillonella, with qPCR suited for broader ranges and ddPCR for detecting low bacterial loads.
- These molecular methods support both disease diagnosis and the isolation/identification of Veillonella for further research.
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