Detection and speciation of Cryptosporidium spp. in environmental water samples by immunomagnetic separation, PCR and endonuclease restriction

Current methods for the detection of Cryptosporidium oocysts in water sampl es are both time-consuming and subject to variation in sensitivity. A genus -specific PCR assay was designed for the specific amplification of a 552-bp region of the 18S rRNA gene. Post-amplification endonuclease restriction g enerated unique digest patterns that enabled differentiation between the th ree species, C, muris, C, baileyi and C. parvum, the major human pathogen. Theoretical restriction profiles for other Cryptosporidium species were als o predicted, The assay routinely detected 10 oocysts in 10-ml purified oocy st preparations, but sensitivity was found to be 10(3)-10(4)-fold lower in environmental water samples. The use of Chelex resin and an immunomagnetic separation procedure overcame this inhibition. This provided detection leve ls of 10(1)-10(3) oocysts, depending on water turbidity, Rapid and sensitiv e pathogen detection methods are essential for the water industry. The resu lts of this study demonstrate that PCR has the potential to improve current detection capabilities greatly by differentiating the major human pathogen s from non-pathogenic species. This will greatly facilitate a closer examin ation of the epidemiology of this important pathogen.