Multiplex Solid-Phase Real-Time Polymerase Chain Reaction without DNA Extraction: A Rapid Intraoperative Diagnosis Using Microvolumes

Abstract

Current polymerase chain reaction (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 minute; total time, 33 minutes) multiplex real-time PCR test (Direct Strip PCR) that did not require DNA extraction to detect 9 pathogens that could cause uveitis in 20-μl samples. Multicenter prospective evaluation of a diagnostic PCR test. A total of 511 participants (patients with infectious uveitis and controls) were examined at 18 institutes worldwide. After validation, intraocular fluid samples were subjected to etiologic or exclusive diagnosis, including intraoperative rapid diagnosis. The concordance and correlations between Direct Strip PCR and quantitative PCR (qPCR) results. Direct Strip PCR exhibited rapid detection, good repeatability and specificity, long storage stability, and detection ability equal to that of qPCR. It also showed low interinstitutional variability compared with qPCR, even when PCR beginners used various real-time PCR machines. The Direct Strip PCR for 9 pathogens exhibited high concordance against the qPCR (positive concordance rate, 98.8%-100%; negative concordance rate, 99.8%-100%; κ coefficient, 0.969-1.000; P < 0.001-0.031). 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. The Direct Strip PCR test may improve the prognosis of various infectious diseases because it facilitates rapid etiologic evaluation at the first hospital visit and can be used for intraoperative diagnosis.