CP2 gene as a useful viability marker for Cryptosporidium parvum

Abstract

The validity of the CP2 gene of Cryptosporidium parvum as a viability marker was evaluated using absolute quantitative real-time polymerase chain reaction (qPCR) assays. Total ribonucleic acid (RNA) was isolated from live and heat-killed C. parvum oocysts, and complementary deoxyribonucleic acid was synthesized and used as a template. The most accurate number of viable C. parvum oocysts was predicted when the CP2 gene was used as a target gene. The lower detection limit of the CP2 gene was ten oocysts, which was the most sensitive among examined target genes. With heat shock induction, only hsp70 messenger RNA (mRNA) was induced, and the predicted viable oocyst number was increased by heat shock for this marker. The CP2, hsp70, Cryptosporidium oocyst wall protein, and beta-tubulin mRNAs were not detected in heat-killed oocysts, but the 18S ribosomal ribonucleic acid (rRNA) showed heat stability until 48 h after heat killing. Although the 18S rRNA demonstrated the fastest response in crossing point (CP) value among the examined primer sets in qPCR, overestimation of viable oocysts was noted in the analysis with this gene. In conclusion, the CP2 gene was identified as the most sensitive, reliable, and accurate candidate of a viability marker of C. parvum by qPCR evaluation.

Quantitative Evaluation of Infectivity Change of Cryptosporidium parvum after Gamma Irradiation

“…Total RNA was extracted in accordance with the manufacturer's instructions as described by Lee et al [13]. For reverse transcription (RT), the reaction mixtures included 0.5-1.0 μ g of total RNA, 10 pmol of oligo (dT)20 primer (Bioneer, Daejon, Korea), 1 × Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV) RT reaction buffer, 0.25 mM dNTP mixture (TaKaRa, Otsu, Shiga, Japan), 1 U of recombinant RNasin ribonuclease inhibitor (Promega, Madison, Wisconsin, USA), and 10 U of M-MLV RT enzyme (Enzynomics, Daejeon, Korea).…”

“…Plasmid pGEMT-CP2 was prepared as previously described [13]. Absolute qPCR reactions were performed in a 20-μ l volume using cDNA as a template.…”

“…In the present study, we investigated the reduction of the infectivity of C. parvum by gamma irradiation and the repair of the infectivity using absolute quantitative real-time PCR (qPCR). As it has been already known that in vitro infectivity of C. parvum using human ileocecal anenocarcinoma cell line (HCT-8) could be a useful substitute for animal infectivity study, and CP2 gene is a useful viability marker for quantitation of C. parvum [12,13], we evaluated infectivity change and repair of C. parvum after gamma irradiation quantitatively using an in vitro system. Several biological characteristics of non-irradiated C. parvum were also evaluated quantitatively, including changes in viability and in vitro infectivity during 12 months of storage, and the time point when the number of parasites was maximal in a cell culture system.…”

Overview of Cryptosporidium Presentations at the 10th International Workshops on Opportunistic Protists

“…Thus, heat-killed C. parvum oocysts remain detectable by FISH after 6 days (54), and FISH data have only modest agreement with results of cell culture and animal infectivity analyses (26). Therefore, more recent efforts have been concentrated on reverse transcriptase PCR (RT-PCR) detection of mRNA, which degenerates more quickly than rRNA (35). Several targeted genes have been used, including the ␤-tubulin, hsp70, amyloglucosidase, and CP2 genes (18,22,27,35).…”

“…Therefore, more recent efforts have been concentrated on reverse transcriptase PCR (RT-PCR) detection of mRNA, which degenerates more quickly than rRNA (35). Several targeted genes have been used, including the ␤-tubulin, hsp70, amyloglucosidase, and CP2 genes (18,22,27,35). A current challenge is the improvement of sensitivity of RT-PCR detection protocols.…”