In this study, we developed a multiplex one-tube nested real-time fluorescent quantitative PCR (mONRT-PCR) assay integrated with a portable, fully automated nucleic acid point-of-care testing (POCT) platform for the detection of Haemophilus influenzae (H. influenzae), Listeria monocytogenes (L. monocytogenes), and Cryptococcus neoformans (C. neoformans) in cerebrospinal fluid (CSF). The assay enables nested amplification within a closed system using conventional primers and probes, thereby reducing operational complexity and minimizing contamination risk. Analytical evaluation demonstrated limits of detection of 10⁰ copies/μL for H. influenzae and L. monocytogenes, and 10¹ copies/μL for C. neoformans using recombinant plasmids, as well as 10⁻⁷ to 10⁻⁶ ng/μL using genomic DNA. No cross-reactivity was observed when tested against a panel of 17 common non-target microorganisms encountered in clinical microbiology laboratories. In simulated CSF samples, the assay maintained detectable amplification at low pathogen concentrations. When implemented on the POCT platform, detection limits reached 5, 10, and 50 CFU/mL for the three pathogens, respectively. Clinical evaluation using 43 CSF samples showed almost perfect agreement with conventional qPCR (κ = 0.861, p < 0.001). Notably, additional C. neoformans detections were observed by mONRT-PCR-POCT compared with qPCR, suggesting improved sensitivity under clinical conditions. The assay yielded results within approximately 1 h and 47 min. These findings indicate that the proposed assay provides a rapid, sensitive, and integrated approach for meningitis pathogen detection, while maintaining a practical balance between analytical performance and operational simplicity. Further validation in larger cohorts is warranted.

Bloodstream infections (BSI) carry high mortality, but traditional blood culture is too slow for urgent clinical needs. This study aims to develop a rapid point-of-care testing assay based on one-tube multiplex nested polymerase chain reaction (PCR) (OM-NPCR-POCT) for early diagnosis of three pathogens in bloodstream infection patients: Escherichia coli (ECO), Klebsiella pneumoniae (KPN), and Streptococcus pneumoniae (SPN). The analytical sensitivity of the one-tube multiplex nested PCR (OM-NPCR) was evaluated using recombinant bacterial plasmids. The analytical sensitivity of the OM-NPCR-POCT assay was assessed using simulated samples. The clinical performance was evaluated in 90 clinical blood samples, with results compared to conventional quantitative PCR (qPCR). Finally, the agreement between the two detection methods was assessed via Kappa analysis. The limits of detection (LODs), calculated from serial dilution experiments, were 4, 2, and 1 copies/μL for plasmids ECO, KPN, and SPN, respectively. The OM-NPCR-POCT assay achieved LODs of 20, 10, and 10 CFU/mL for ECO, KPN, and SPN in simulated samples, with a total testing time of approximately 2 h. The clinical evaluation of OM-NPCR-POCT demonstrates consistency with conventional qPCR while exhibiting higher sensitivity. This method has potential as a rapid diagnostic tool for early bloodstream infection detection.