RT-PCR specific for Cryspovirus is a highly sensitive method for detecting Cryptosporidium parvum oocysts

Abstract

Cryptosporidium parvum represents a considerable health risk to humans and animals because the parasite has a low infectious dose and is usually present at low numbers in environmental samples, which makes detection problematic. The purpose of this study was to evaluate Cryspovirus as a target for sensitive detection of C. parvum in clinical samples. Semi-quantitative RT-PCR (sqRT-PCR) and quantitative RT-PCR (qRT-PCR) directed to Cryspovirus sequences could detect less than 5 Cryptosporidium oocysts in RNA extracted from C. parvum-containing calf feces. Of interest was that a similar level of sensitivity was observed using RNA present in DNA extracts of the same C. parvum fecal samples. There was a strong correlation between both the sqRT-PCR and qRT-PCR product and number of C. parvum oocysts. Analysis of DNA extracted from a similar number of oocysts using PCR targeting the Cryptosporidium SSU rDNA gene sequence found that nested PCR was necessary to obtain a detectable PCR signal. The availability of DNA allowed for Cryptosporidium genotyping based on SSU rDNA sequencing as well as C. parvum subtyping through GP60 sequencing. By using DNA that contains viral RNA, the assay avoids two separate extractions — one for RNA and one for DNA. This two-step assay, first to detect Cryptosporidium by Cryspovirus-specific RT-PCR followed by nested SSU rDNA PCR for Cryptosporidium genotyping may represent an important tool for identifying the parasite in clinical samples.