Cross-kingdom pathogen detection via duplex universal PCR and high-resolution melt

Area of Science:

• Microbiology
• Molecular Diagnostics
• Infectious Diseases

Background:

• Polymicrobial infections, involving bacteria and fungi, present diagnostic and treatment challenges.
• Current culture-based methods for pathogen detection are slow and inefficient.
• There is a critical need for rapid, broad-spectrum diagnostic approaches for infectious agents.

Purpose of the Study:

• To develop a single, rapid diagnostic method for detecting both bacterial and fungal pathogens.
• To address the limitations of existing culture-based diagnostic techniques.
• To improve the identification of monomicrobial and polymicrobial infections.

Main Methods:

• A duplex universal PCR and high-resolution melt (HRM) assay was developed.
• The method utilizes two universal primer sets targeting bacterial and fungal genomic DNA.
• The assay was adapted to a microfluidic-based digital format with machine learning analysis.

Main Results:

• The duplex PCR-HRM method accurately detected 16 WHO-flagged pathogens.
• Digital duplex PCR-HRM achieved over 99.9% detection accuracy.
• The method quantitatively detected co-infections and identified pathogens in clinical bronchoalveolar lavage samples.

Conclusions:

• The digital duplex PCR-HRM assay is a rapid, accurate, and comprehensive diagnostic tool.
• This method significantly advances infectious disease diagnostics for bacterial and fungal pathogens.
• The technology holds potential for improved patient management and outcomes in polymicrobial infections.