Estimating viability of Cryptosporidium parvum oocysts using reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding amyloglucosidase

Mark C. Jenkins, James Trout, Mitchell S. Abrahamsen, Cheryl A. Lancto, James Higgins, Ron Fayer

Research output: Contribution to journalArticlepeer-review

45 Scopus citations

Abstract

The purpose of the present study was to determine if reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding the enzyme amyloglucosidase (CPAG) could serve as a indicator for C. parvum oocyst viability. Oocysts were stored for 1-11 months in the refrigerator and at monthly intervals extracted for total RNA for RT-PCR analysis. An aliquot of these C. parvum oocysts was inoculated into neonatal mice which were necropsied 4 days later for ileal tissue that was analyzed by semi-quantitative PCR to determine the level of parasite replication. The CPAG RT-PCR assay detected RNA from as few as 103 C. parvum oocysts. An effect of storage time on both RT-PCR signal and mouse infectivity was observed. RNA from oocysts stored for 1-7 months, unlike oocysts stored for 9 or 11 months, contained CPAG mRNA that was detectable by RT-PCR. A gradual decrease in the RT-PCR signal intensity was observed between 5 and 7 months storage. The intensity of RT-PCR product from oocysts and the signal from semi-quantitative PCR of ileal tissue DNA from mice infected with these same aged oocysts were comparable. The RT-PCR assay of CPAG mRNA in cultured cells infected with viable C. parvum oocysts first detected expression at 12 h with highest expression levels observed at 48 h post-infection. These results indicate that CPAG RT-PCR may be useful for differentiating viable from non-viable C. parvum oocysts and for studying the expression of the gene for amyloglucosidase in vitro. Copyright (C) 2000 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)97-106
Number of pages10
JournalJournal of Microbiological Methods
Volume43
Issue number2
DOIs
StatePublished - Dec 15 2000

Bibliographical note

Funding Information:
This study was supported in part by a grant (RFP 2502) from the American Water Works Association Research Foundation.

Keywords

  • Cryptosporidium
  • Reverse transcriptase-polymerase chain reaction
  • Viability

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