Development and Application of a Novel Conserved Signature Protein/Gene-Based qPCR Strategy for Improved Cryptosporidium Surveillance in Recreational Waters

Cryptosporidium is a major waterborne parasite that causes gastrointestinal illness. Conventional assays, including microscopy and immunological identification, often suffer from false positives or negatives due to non-specific binding or morphological differences between Cryptosporidium species. We developed a novel qPCR assay based on a Cryptosporidium-specific Conserved Signature Protein (CSP) to address the limitations of testing complex samples, including those from recreational waters. The CSP (hypothetical protein (cgd2_3830)) was identified as taxonomically unique to Cryptosporidium species. The CSP sequence and designed qPCR assay primers/probe demonstrated high specificity for the targeted Cryptosporidium species when tested against NCBI RefSeq databases. qPCR assay efficiency was determined as 95% and an R2 value of 0.99, with a slope and intercept of −3.4 and 40.1, respectively. Additionally, the Lower Limit of Detection (ALLOD) was determined as three gene copies, suggesting the potential to detect even a single oocyst. No non-specific amplification products or primer dimers were observed when the qPCR assay was evaluated using recreational water, fecal solution, and wastewater, while spike-in-control tests indicated minimal interference with the sensitivity of the assay, highlighting application for testing complex environmental DNA extracts. These findings highlight the application of the novel CSP-based qPCR assay for the rapid and sensitive detection of Cryptosporidium sp., thereby circumventing the sequence variability and multi-copy limitations associated with existing molecular markers. This proof-of-concept study presents a diagnostic framework utilizing CSP-based markers for developing water quality monitoring strategies, with scope for expansion to other microbial pathogens and potential applications in clinical and food safety settings.