A nested PCR-based point-of-care multiplex test for detection of bacterial pathogens in cerebrospinal fluids.

Objective: To integrate a multiplex one-tube nested real-time polymerase chain reaction (mOTN-PCR) with a point-of-care testing (POCT) instrument for rapid, sensitive detection of Method: The sensitivity of the mOTN-PCR was assessed using recombinant plasmids, and its specificity was evaluated using common pathogens associated with CSF infections. Simulated CSF samples were prepared using both manual nucleic acid extraction and fully automated nucleic acid extraction (POCT instrument), and then detected in parallel by mOTN-PCR and conventional real-time PCR. The clinical performance of the mOTN-PCR-POCT assay was further evaluated using 109 clinical samples (36 clinical CSF samples and 73 non-CSF clinical samples), and compared with the manual nucleic acid extraction followed by the conventional real-time PCR method. Results: The mOTN-PCR achieved detection limits (LOD) of 5, 10, and 10 copies/μL for SPN, GBS, and GAS plasmids, respectively, with no cross-reactivity. In simulated CSF samples, the LODs were 20, 10, and 20 CFU/mL for SPN, GBS, and GAS, respectively, using manual extraction, within 4 hours. The mOTN-PCR-POCT method consistently detected all three pathogens at 20 CFU/mL, reducing total testing time to approximately 2 hours. Clinical sample testing revealed that the mOTN-PCR-POCT identified three additional positives in samples previously deemed negative by conventional real-time PCR, demonstrating superior detection capability and a more streamlined operational workflow of the mOTN-PCR-POCT. Conclusion: The mOTN-PCR-POCT was demonstrated to be simple to operate, requires no culture, and enables rapid, highly sensitive detection of SPN, GBS, and GAS in CSF.