Multiplex solid-phase real-time polymerase chain reaction without DNA extraction: A rapid intraoperative diagnosis using microvolumes

PURPOSE Current PCR methods for the diagnosis of infections are time-consuming and require large sample volume and skilled technicians. We developed a novel, easy-to-use, and rapid (processing time: 1 min; total time: 33 min) multiplex real-time PCR test (Direct Strip PCR), which did not require DNA extraction, to detect nine pathogens that could cause uveitis in 20 μL samples. DESIGN Multi-center prospective evaluation of a diagnostic PCR test. SUBJECTS AND CONTROLS A total of 511 subjects (patients with infectious uveitis, and controls) were examined at 18 institutes in the world. METHODS After validation, intraocular fluid samples were subjected to etiological or exclusive diagnosis, including intraoperative rapid diagnosis. MAIN OUTCOME MEASURES The concordance and correlations between Direct Strip PCR and quantitative (q)PCR. RESULTS Direct Strip PCR exhibited rapid detection, good repeatability and specificity, long storage stability, and detection ability equal to qPCR. It also showed low inter-institutional variability compared to qPCR even when PCR beginners using various types of real-time PCR machine. The Direct Strip PCR for 9 pathogens exhibited high concordance against the qPCR (positive concordance rate, 98.8 to 100%; negative concordance rate, 99.8 to 100%; κ coefficient, 0.969 to 1.000, P value: <0.001 to 0.031). Additionally, results obtained using Direct Strip PCR and qPCR were highly correlated (ρ=0.748, P<0.001). This assay was used for rapid intraoperative diagnosis. CONCLUSIONS The Direct Strip PCR test may improve the prognosis of various infectious diseases as it facilitates rapid etiological evaluation at the first hospital visit and can be used for intraoperative diagnosis.