Development of a SYBR Green-Based Real-Time PCR Assay to Detect Oncomelania hupensis quadrasi DNA in Environmental Water Samples

Abstract

Oncomelania hupensis quadrasi is the intermediate host of S. japonicum, the causative species of schistosomiasis in the Philippines. Conventionally, risk areas are identified by procedures requiring highly skilled personnel and constant surveillance efforts. Recent developments in disease diagnostics explore the utilization of environmental DNA as targets for polymerase chain reactions in disease surveillance. In this study, a low-cost, specific, and efficient SYBR Green-based real-time PCR assay to detect O. h. quadrasi DNA from water samples was developed, optimized, and validated. Primers were designed based on the A18 microsatellite region of O. h. quadrasi. The assay exhibited a detection limit of one copy number per microliter at 99.4% efficiency and R² = 0.999, which specifically amplified O. h. quadrasi DNA only. Validation of this assay in environmental water samples demonstrated 100% PPV and NPV values, suggesting its potential as a tool for identifying risk areas, pathogen-directed surveillance, policy making, and disease control.