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A highly sensitive method for detecting Cryptosporidium parvum oocysts recovered from source and finished water using RT-PCR directed to Cryspovirus RNA

Author:
de Souza, Milena Sato, O'Brien, Celia, Santin, Monica, Jenkins, Mark
Source:
Journal of microbiological methods 2019 v.156 pp. 77-80
ISSN:
0167-7012
Subject:
Cryptosporidium parvum, Cryspovirus, Protozoa, RNA, capsid, fluorescent antibody technique, genes, human diseases, oocysts, reverse transcriptase polymerase chain reaction
Abstract:
Sensitive detection of Cryptosporidium oocysts is important because the protozoan can cause clinical infection in humans at extremely low numbers. In the present study, 1.5 × 102, 1.0 × 103, or 1.0 × 104C. parvum oocysts were spiked into 10 l of source or finished water in triplicate followed by recovery using Envirochek HV sampling capsules. One subsample of the recovered oocysts was analyzed by commercial immunofluorescence assay (IFA), while a second subsample was subjected to DNA-RNA extraction, followed by RT-PCR using primers directed to the gene encoding Cryspovirus capsid. IFA analysis of Envirochek filter eluates of finished water detected oocysts at all 3 C. parvum oocyst doses, but only at the 1.0 × 103 and 1.0 × 104 doses in source water. Cryspovirus RT-PCR appeared to offer greater sensitivity than IFA because C. parvum oocysts were detected using this molecular technique in both source and finished water concentrates at all 3 spiking levels. A linear relationship was observed between log oocysts spiking dose and the relative intensity of the Cryspovirus RT-PCR signal for finished water, but not for source water. These data indicate that Cryspovirus RT-PCR is a sensitive method for detecting C. parvum oocysts in source and finished water.
Agid:
6254297