Development and evaluation of a multi-panel real-time PCR assay for rapid detection of six bacterial species causing...

Endophthalmitis is a severe intraocular infection with the potential for rapid progression and irreversible blindness. Early and accurate pathogen identification is critical to guide therapy and improve prognosis. Conventional microbiological methods, including culture, are limited by low sensitivity and slow turnaround times, particularly in patients who have received prior antibiotic treatment. This study reports the development and preliminary evaluation of a multi-panel real-time PCR assay designed to detect six clinically relevant bacterial species implicated in endophthalmitis (Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, and Streptococcus sanguinis). Unlike conventional multiplex PCR, the assay uses a panel of singleplex and duplex reactions that share a unified thermal cycling profile and rely on SYBR Green chemistry with melt curve analysis for species discrimination. This configuration eliminates the need for multichannel real-time PCR instruments and avoids the complexity of probe-based multiplexing, while maintaining acceptable efficiency and specificity. Analytical validation using reference strains demonstrated limits of detection between 10³-10⁴ CFU/mL, amplification efficiencies of 0.93-1.0, and no detectable cross-reactivity among the six targets. Preliminary testing on vitreous samples from suspected endophthalmitis patients demonstrated the feasibility of applying the assay to clinical specimens and showed detection of bacterial DNA in some culture-negative cases. The total turnaround time from DNA extraction to species identification was less than 4 h. Although the clinical sample size was small and the pathogen panel limited, these proof-of-concept data suggest that the proposed qPCR panel may complement conventional culture for more rapid pathogen detection in endophthalmitis.