Improved Rapid Detection of Viable Waterborne Pathogens

Innovative Biotechnologies International, Inc., has reported previously a simple method to detect viable Cryptosporidium parvum oocysts in drinking water using the isothermic Nucleic Acid Sequence-Based Amplification (NASBA) method, coupled with a rapid liposome nanovesicle (Nanozome)-based biosensor technology. This assay system is based on the ability to immunocapture low numbers (i.e., five or more) oocysts and heat shock them to induce the production of a specific nRNA, which in turn can be amplified by NASBA. Then, the resulting single-stranded amplicons can be quantified directly by a rapid and visually detectable hybridization reaction employing a highly sensitive Nanozome-amplified lateral-flow assay. The entire assay, completed within 4-5 hours, can overcome many of the problems inherent in the current U.S. Environmental Protection Agency (EPA) Methods 1622 and 1623, particularly labor-intensity, inability to distinguish nonviable from viable oocysts, and decreasing the possibility of false-positive determinations caused by misreading of immunofluorescence slides. Although data already are available and thorough analyses have been carried out previously, a number of additional data points with sufficient statistical relevance are required to consider the NASBA/Nanozome method for possible routine use in drinking water monitoring. These analyses include confirmation that: The designed primer pairs and probes are specific for C. parvum and not prone to false-positive reactions against other Cryptosporidium species that do not infect humans or other microbial agents anticipated in water. The designed primer pairs and probes also can detect C. hominis. The test system is capable of detecting low numbers of C. parvum oocysts when they are present among a large background of other microbial agents. The entire test system can differentiate between viable and ultraviolet (UV)-irradiated oocysts. The entire test system will yield performance specifications (i.e., sensitivity and specificity) at least as good as EPA Methods 1622 and 1623. A team of scientists at Cornell University, Innovative Biotechnologies International, and Clancy Environmental Consultants, who have worked together previously on the development of the C. parvum test, will be assembled to perform the proposed work. Successful completion of the Phase I proposal will permit Innovative Biotechnologies International to propose a Phase II effort to perform more extensive performance-based evaluation of the test protocol at a large number of water treatment utilities with the goal of achieving EPA approval of a new method for routine monitoring of drinking water.