Analytical and clinical validation of SYBR Green- and TaqMan-based multiplex real-time PCR assays for quantitative detection of intestinal protozoa in India

Intestinal protozoa such as Entamoeba histolytica , Giardia lamblia , and Cryptosporidium spp . are major causes of diarrhoeal disease worldwide, yet their accurate detection remains challenging because of the limitations of microscopy and antigen-based assays. This study developed and validated multiplex real-time PCR assays using SYBR Green and TaqMan chemistries for simultaneous detection and quantification of these pathogens in stool samples. A total of 540 patients with gastrointestinal disorders were examined using multiplex conventional PCR, SYBR-based qPCR, and probe-based qPCR. The TaqMan multiplex assay demonstrated the highest analytical sensitivity, with a limit of detection corresponding to the 10⁻⁸ serial dilution of the recombinant DNA standards with excellent linearity (R² ≈ 0.99) and diagnostic specificity (100%). Overall detection was 24.4% by probe-based and 23.1% by SYBR-based assays, with near-perfect inter-assay concordance (κ = 0.93–0.97). The probe-based assay additionally identified low-intensity and mixed infections confirmed by Sanger sequencing. These findings establish a sensitive, quantitative, and clinically applicable multiplex qPCR platform that improves the detection of intestinal protozoa, particularly in immunocompromised patients. By enabling simultaneous detection of three pathogens in a single reaction, the assay reduces reagent and consumable use, hands-on processing, instrument occupancy, and turnaround time compared with performing three separate single-plex qPCR assays. Under our laboratory conditions, the multiplex assay reduced reagent costs from approximately ₹1,800 per sample for three individual single-plex reactions to approximately ₹900 per sample, while decreasing the total assay time from approximately 4.5 h to 1.5 h, making it a practical and economical option for routine molecular diagnosis and surveillance in high-burden settings.