Abstract
INTRODUCTION: Current Toxoplasma gondii (TOX), Epstein-Barr virus (EBV), rubella virus, human cytomegalovirus (HCMV), and herpes simplex virus types I and II (HSV I and II) (TORCHes) diagnostic methods are limited by challenges, such as multi-step workflows and cold-chain dependency. This study aimed to develop an integrated "sample-in-result-out" system combining rapid nucleic acid release, room-temperature-stable multiplex polymerase chain reaction (PCR), and automated interpretation to improve diagnostic accuracy. METHODS: We utilized a self-prepared lysis buffer for fast nucleic acid extraction from diverse sample types. Primers, probes, and reaction components for six TORCHes pathogens were fabricated into freeze-dried microspheres via vacuum freeze-drying, enabling single-tube multicolor melting curve analysis (MMCA). The companion software, TORCHes-MCAv1.0, was used to automate pathogen typing and result reporting. Evaluation metrics encompassed sensitivity, specificity, precision, stability, and anti-interference ability. The system was validated using 210 clinical samples compared to commercial single-target qPCR, with Sanger sequencing resolving any discrepancies. RESULTS: Nucleic acid extraction and release were completed within 1 min. Freeze-dried microspheres demonstrated exceptional repeatability, with T (m) intra- and inter-batch CVs of 0.03-0.18%. Limits of detection (LoD) were 200 copies/mL for HSV-I/II, HCMV, and TOX, and 500 copies/mL for EBV and RV. Specificity analysis showed no cross-reactivity with non-target pathogens. Stability testing confirmed the microspheres could be stored stably at room temperature for 1 year. Clinical validation showed near-perfect agreement with qPCR (Kappa = 0.965-1.000), with all seven discrepant results confirmed correct via sequencing. The TORCHes-MCAv1.0 software achieved 100% concordance with manual interpretation. CONCLUSION: We developed a robust, streamlined integrated TORCHes detection system. Its simplicity, stability, and high-throughput nature make it a valuable complementary tool for confirming active infection and differentiating pathogen subtypes in complex clinical scenarios.