Quantification and genotyping of cryptosporidium spp. in river water by quenching probe PCR and denaturing gradient gel electrophoresis

Y. Masago, K. Oguma, H. Katayama, S. Ohgaki

Research output: Chapter in Book/Report/Conference proceedingChapter

18 Citations (Scopus)

Abstract

A new detection method was developed for the simultaneous quantification and genotyping of Cryptosporidium spp. in river water. Several modifications made to the US EPA Method 1623 enabled high and stable recovery of Cryptosporidium from 40L of river water (geometric mean = 35%, standard deviation = 8.7%). Ouenching probe PCR (QProbe PCR) was used to quantify the 18S rRNA gene of Cryptosporidium spp. This method could successfully detect single oocysts in a sample, and the lower quantitation limit was as low as 2.5 oocysts/sample. In addition, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing was used to identify the genotypes. These methods were applied to detect Cryptosporidium spp. in the Koyama River, Japan. The positive ratio was 69% (11/16) with the maximum concentration of 59 oocysts/1 00 L. Seven genotypes including two novel ones were identified. These results showed that this detection method could provide valuable information on Cryptosporidium in river water, both in the concentration and in the genotypes, which is essential for the precise assessment of waterborne risk to human health.

Original languageEnglish
Title of host publicationWater Science and Technology
EditorsJoan Rose, Gertjan G.
Pages119-126
Number of pages8
Edition3
DOIs
Publication statusPublished - 2006

Publication series

NameWater Science and Technology
Number3
Volume54
ISSN (Print)0273-1223

Keywords

  • Cryptosporidium
  • Denaturing gradient gel electrophoresis (DGGE)
  • Genotyping
  • Quenching probe PCR (QProbe PCR)
  • Real time PCR

ASJC Scopus subject areas

  • Environmental Engineering
  • Water Science and Technology

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