PARAMETERS AFFECTING POLYMERASE CHAIN-REACTION DETECTION OF WATERBORNE CRYPTOSPORIDIUM-PARVUM OOCYSTS

Cryptosporidium parvum is an enteric protozoan parasite of medical and veterinary importance. Dissemination of environmentally resistant ooc ysts in surface water plays an important role in the epidemiology of c ryptospridiosis. Although the polymerase chain reaction (PCR) is a wel l-established technique and is widely used for detecting microorganism s, it is not routinely applied for monitoring waterborne C. parvum. In order to facilitate the application of PCR to the detection of waterb orne C. parvum oocysts, a comparison of published PCR protocols was un dertaken and different sample-preparation methods tested. The sensitiv ity of a one-step PCR method, consisting of 40 temperature cycles, was 10 purified oocysts or fewer than 100 oocysts spiked in raw lake wate r. The detection limit of two primer pairs, one targeting the ribosoma l small subunit and another specific for a C. parvum sequence of unkno wn function, was approximately ten-fold lower than achieved with a pri mer pair targeting an oocyst shell protein gene. Three cycles of freez ing/thawing were sufficient to expose oocyst DNA and resulted in highe r sensitivity than proteinase K digestion, sonication or electroporati on. Inhibition of PCR by surface water from different local sources wa s entirely associated with the soluble fraction of lake water. Membran e filtration was evaluated in bench-scale experiments as a means of re moving lake water inhibitors and improving the detection limit of PCR. Using gel and membrane filtration, the molecular size of inhibitory s olutes from lake water was estimated to less than 27 kDa.