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

Infectious diseases caused by pathogenic bacteria and fungi continuously pose a significant threat worldwide. The occurrence of polymicrobial infections, including polybacterial, polyfungal or bacteria-fungal co-infections further complicates diagnosis and treatment. Current diagnostic methods, heavily reliant on culture methods, are slow and often inefficient. This inefficiency underscores the urgent need for new diagnostic approaches that can swiftly identify a wide array of pathogens across both the bacterial and fungal kingdoms. In response to this need, our study introduces a duplex universal PCR and high-resolution melt (HRM) method that enables the detection of both bacterial and fungal pathogens within a single PCR-HRM procedure. This method uses two universal primer sets designed to target bacterial and fungal genomic DNA respectively, facilitating broad-range detection of 16 pathogens flagged by the World Health Organization (WHO). Moreover, this assay can be adapted in microfluidic-based digital reaction format and when analyzed via a one-versus-one support vector machine classifier achieved a detection accuracy exceeding 99.9%. This digital duplex PCR-HRM method has the capacity to quantitatively detect co-infections with varying pathogen ratios in simulated samples, demonstrating its versatility and multiplexed capacity. When applied to clinical bronchoalveolar lavage (BAL) samples, digital duplex PCR-HRM successfully identified both monomicrobial and polymicrobial infections. This development marks a significant advancement in the field of infectious disease diagnostics, offering a rapid, accurate, and comprehensive method for identifying a broad spectrum of bacterial and fungal pathogens, thus potentially improving patient management and outcomes.