Development of a novel, rapid integrated Cryptosporidium parvum detection assay

The aim of this study was to develop a reverse transcription-PCR assay and lateral flow detection protocol for specific identification of Cryptosporid ium parvum. The method which we developed is sensitive and specific and has a low limit of detection. In our protocol a solid phase material, the Xtra Bind Capture System, was used for extraction and purification of double-st randed RNA (dsRNA) specific for C. parvum. The Xtra Bind Capture System int erfaced with pellets concentrated from water samples collected with previou sly developed filtration devices. The pellets were resuspended in reagent w ater (final volume, 0.5 ml), and an equal amount of rupture buffer and the Xtra Bind Capture System was added to the resuspended pellet mixture. The d sRNA target sequences in a 0.5-ml portion were captured by the solid phase material via hybridization. The debris and potential inhibitors were remove d by washing the Xtra Bind material several times with buffer. The Xtra Bin d material with its bound dsRNA was added directly to an amplification reac tion mixture, and the target was amplified without elution from the Xtra Bi nd material. A PCR was performed in the presence of the Xtra Bind Capture S ystem, which resulted in robust amplification of the target. The detection system which we used was adapted from lateral flow chromatography methods t ypically used for antigen-antibody reactions. The result was a colored line that was visible if the organism was present. When this method was used, w e were able to reproducibly and correctly identify 10 oocysts added to 0.5 mi of reagent water. When the protocol was evaluated with a small set of en vironmental samples, the Level of detection was as low as 1 oocyst/liter. T he total time from resuspension of the pellet to detection was about 3 h, w hich is considerably less than the 5 h required for immunomagnetic separati on followed by an indirect immunofluorescence assay and microscopy.