Real-time PCR Diagnostic Kit for Giardia and Cryptosporidium

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DESCRIPTION (provided by applicant): This two-year proposal seeks small business support for IDX Labs LLC to continue collaborating with Dr. Houpt at the University of Virginia to bring sensitive real-time PCR (qPCR) tests for Giardia and Cryptosporidium to U.S. labs. According to the CDC, these parasites infect up to 2 million Americans annually, and we have developed robust sensitive qPCR tests for both infections that have been used successfully on hundreds of patients in Bangladesh and Tanzania. During Phase I these assays were converted into a Cryptosporidium/Giardia multiplex assay that detected all known human subspecies and was workable on two major qPCR platforms (Roche LightCycler and BioRad iCycler). During Phase I we also determined the most sensitive DNA extraction procedure among the commercially available kits, and then improved the sensitivity of PCR detection further through magnetic target-specific DNA capture. This Phase II application will now finalize the target DNA extraction procedure from stool for highest throughput and collect validation data from the field at our site in Tanzania in order to prepare an FDA 501(k) application, which we predict will allow a strong market entry. We hypothesize that our technique will be more sensitive than the typical gold standard Merifluor Cryptosporidium/Giardia IFA, and we will utilize a third tiebreaker PCR test to resolve any discrepant results. Secondarily, the test will also be validated on water samples versus the EPA's Giardia/Cryptosporidium method #1623, given the market and need for improved tests for these protozoa on drinking and recreational water samples. The innovation of this application lies in the DNA capture extraction technology, a platform translatable to other clinical specimens besides stool, and the concept of bringing state-of-the-art diagnostics beyond antigen detection to under-funded parasitic infections of waterborne health importance. This Phase II application will advance our development of a Giardia/Cryptosporidium real time PCR kit that incorporates highly sensitive target-specific DNA extraction with a highly optimized multiplex qPCR assay. During Phase II the final sample-to-result kit will be completed and performance will be validated on clinical samples against the existing gold standard IFA to enable a 501(k) submission to FDA.