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

Oncomelania hupensis quadrasi is the intermediate host of S. japonicum, the causative species for schistosomiasis in the Philippines. The World Health Organization promotes snail control with molluscicides, physical removal, and environmental modification to eliminate this neglected tropical disease as a public health problem. Conventionally, risk areas are identified by a series of malacological surveys, microscopic testing, and stool collection to confirm transmission of the disease in humans. However, these procedures require highly skilled personnel and regularly conducting them for higher surveillance efficiency. Recent developments in disease diagnostics explore the utilization of environmental DNA as targets for polymerase chain reactions for 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 from a microsatellite region of O. h. quadrasi. Amplification is optimum at 5µM primer concentration with 63°C annealing temperature. The optimized PCR reaction condition is 95°C for 3 minutes of enzyme activation, and 40 cycles of template denaturation, annealing, and extension at 95°C for 30 seconds and 63°C for 25 seconds, respectively, followed by a melt curve analysis. The assay exhibited a detection limit of 1 copy number per microliter. The assay furthermore exhibited 99.4% efficiency, R2= 0.999, which specifically amplifies O .h. quadrasi DNA only. Validation of this assay in environmental water samples demonstrated 100% PPV and 100% NPV values, suggesting a potential tool for identifying risk areas, pathogen-directed surveillance, policy making, and disease control.