Development of a sensitive polymerase chain reaction method for the detection of Toxoplasma gondii in water

Toxoplasma gondii is becoming a potential threat for public water supplies worldwide, as demonstrated by the occurrence of waterborne toxoplasmosis outbreaks in developing countries as well as industrialised countries. The aim of the present study was to develop a sensitive molecular approach (PCR) for the detection of Toxoplasma oocysts in water. Sporulated and unsporulated T. gondii oocysts (strains DX and AHC1) were isolated from faeces of laboratory-infected cats. After purification and enumeration, oocysts were spiked into 1-L water replicates and concentrated using centrifugation, Al₂(SO₄)₃ or Fe₂(SO₄)₃ flocculation. DNA was extracted from the concentrated pellets, and a universal primer and a T. gondii-specific primer were selected to amplify a region at the small subunit ribosomall RNA gene. A theoretical detection limit of 0.1 oocysts was achieved for samples that had been concentrated using centrifugation or Al₂(SO₄)₃ flocculation. No PCR products were generated for samples that had been pretreated using Fe₂(SO₄)₃ flocculation. The final target would be the development of a complete technique able to work as a diagnostic tool for the detection of Toxoplasma in environmental and drinking water.