Ratiometric PCR in a Portable Sample-to-Result Device for Broad-Based Pathogen Identification

ABSTRACT: Polymerase chain reaction (PCR)-based diagnostic testing is the gold standard method for pathogen identification (ID) with recent developments enabling automated PCR tests for several pathogens in PCR assays typically requires optics for an equivalent number of fluorescence channels, increasing instrumentation's complexity and cost. In this study, we first developed ratiometric PCR that surpassed one target per color barrier to allow multiplexed identification while minimizing optical components for affordable POC use. We realized it by amplifying pathogenic targets with fluorescently labeled hydrolysis probes with a specific ratio of red-to-green fluorophores for each bacterial species. We then coupled ratiometric PCR and automated magnetic beadsbased sample preparation within a thermoplastic cartridge and a portable droplet magnetofluidic platform. We named the integrated workflow POC-ratioPCR. We demonstrated that the POC-ratioPCR could detect one out of six bacterial targets related to urinary tract infections (UTIs) in a single reaction using only two-color channels. We further evaluated POC-ratioPCR using mock bacterial urine samples spiked with good agreement. The POC-ratioPCR presents a simple and effective method for enabling broad-based POC PCR identification of pathogens directly from crude biosamples with low optical instrumentation complexity.

Automated summary

In this study, a ratiometric PCR method called POC-ratioPCR was developed for fast and efficient identification of pathogens. The method amplifies targeted pathogens using fluorescently labeled hydrolysis probes with a specific ratio of red-to-green fluorophores, and combines ratiometric PCR with automated sample preparation. This method holds promise for practical applications due to its simplicity and effectiveness in identifying pathogens directly from crude biosamples with minimal optical instrumentation complexity.