Development and validation of qPCR assay targeting COWP conserved region for sensitive detection and quantification of Cryptosporidium infections.

Abstract

BACKGROUND: Cryptosporidium is a significant food and waterborne protozoan parasite. Molecular diagnosis and quantification play a crucial role in diagnosing infections, as well as in understanding the transmission dynamics. The Cryptosporidium oocyst wall protein (COWP) gene serves as an appropriate target for such assays since it remains conserved uniquely among significant species. OBJECTIVE: The current research focused on developing and validating a sensitive and specific quantitative PCR (qPCR) test targeting a conserved domain of the COWP gene to detect and quantify various Cryptosporidium species. METHODS: Design of selective degenerate primers on a conserved domain in the major Cryptosporidium spp. COWP gene. A COWP DNA library was constructed using molecular cloning into pET-15b vector to be used as a precursor for a standard curve absolute quantification strategy. RESULTS: The designed primers successfully amplified a 311-317&#x202f;bp product, with specificity tested using melt curve analysis. The slope of the standard curve was -3.279, efficiency of 100.8&#x202f;%, and R&#xb2; =&#x202f;0.95 (p&#x202f;<&#x202f;0.0001) with LOD equals 9.55&#x202f;&#xd7;&#x202f;10&#x2074; copies /&#xb5;L. CONCLUSION: A qPCR assay that is both sensitive and efficient was developed and validated. The method produces a reliable technique of absolute quantitation of Cryptosporidium DNA in samples of unknown quantity, from which infection rates may be estimated accurately.