Vibrio-Sequins - dPCR-traceable DNA standards for quantitative genomics of Vibrio spp

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Vibrio spp. are significant marine bacteria causing global foodborne gastroenteritis outbreaks.
• Traditional culture-based detection methods are being replaced by Next-Generation Sequencing (NGS).
• NGS methods face technical biases affecting genomic quantification accuracy.

Purpose of the Study:

• To develop a quantitative NGS-based method for precise Vibrio spp. enumeration.
• To establish absolute quantification using artificial DNA standards and digital PCR (dPCR).
• To improve the accuracy and reliability of genomic-based microbial detection.

Main Methods:

• Development of six artificial DNA standards (Vibrio-Sequins).
• Optimization of TaqMan assays and validation of three duplex dPCR methods.
• Application of a quantitative genomics approach combining NGS and dPCR for DNA quantification.

Main Results:

• Established limits of quantification (LOQ) from 20 to 120 cp/µl for Vibrio-Sequins.
• Achieved a consistent limit of detection (LOD) of approximately 10 cp/µl across all assays.
• Demonstrated enhanced quantitative power through the integration of NGS and dPCR in a proof-of-concept study.

Conclusions:

• Advanced quantitative (meta)genomic methods by ensuring metrological traceability in NGS DNA quantification.
• Provided a robust tool for future metagenomic studies requiring absolute microbial DNA quantification.
• Facilitated the development of statistical approaches for estimating measurement uncertainties in NGS data.