AWWA PATH50807

A simple and rapid molecular strategy for screening environmental samples for the presence of C. parvum oocysts with sensitivity and specificity is described. A buffer, capable of rupturing oocysts and releasing nucleic acid from sporozoites with >98% efficiency, was developed. This buffer interfaced with a solid phase binding material for capture of specific sequences from C. parvum. The solid phase binding of the target allowed for purification of the nucleic acid from the water matrix. The solid phase was added directly to the isothermal amplification reaction, eliminating losses from dilution and elution. After amplification, the results were visualized, a blue line if the target was present, the absence of the line indicated that results were negative. The specificity for C. parvum was ensured by design of the capture sequences for the dsRNA, amplification primers and detection probes. Includes 8 references, tables, figures.