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

摘要

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. Highlights ? RT-PCR assays that target Cryspovirus were developed to detect Cryptosporidium . ? The sqRT-PCR assay uses an internal standard to control for false negative reactions. ? A strong correlation between RT-PCR signal and numbers of C. parvum oocysts was seen.