Novel quantitative TaqMan real-time PCR assays for detection of Cryptosporidium at the genus level and genotyping of major human and cattle-infecting species

Authors

  • J.B. Burnet,

    1. Department of Environment and Agro-biotechnologies (EVA), Centre de Recherche Public - Gabriel Lippmann, Belvaux, Luxembourg
    2. Department of Environmental Sciences and Management, Université de Liège (ULg), Arlon, Belgium
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  • L. Ogorzaly,

    1. Department of Environment and Agro-biotechnologies (EVA), Centre de Recherche Public - Gabriel Lippmann, Belvaux, Luxembourg
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  • A. Tissier,

    1. Department of Environment and Agro-biotechnologies (EVA), Centre de Recherche Public - Gabriel Lippmann, Belvaux, Luxembourg
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  • C. Penny,

    1. Department of Environment and Agro-biotechnologies (EVA), Centre de Recherche Public - Gabriel Lippmann, Belvaux, Luxembourg
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  • H.M. Cauchie

    Corresponding author
    • Department of Environment and Agro-biotechnologies (EVA), Centre de Recherche Public - Gabriel Lippmann, Belvaux, Luxembourg
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Correspondence

Henry-Michel Cauchie, Department of Environment and Agro-biotechnologies (EVA), Centre de Recherche Public - Gabriel Lippmann, 41 rue du Brill, L-4422 Belvaux, Luxembourg.

E-mail: cauchie@lippmann.lu

Abstract

Aims

Development of TaqMan MGB real-time PCR assays for quantitative typing of major cattle and human-pathogenic Cryptosporidium species.

Methods and Results

Three specific TaqMan MGB real-time PCRs, based on the SSU rRNA gene, were directed towards livestock-restricted Cryptosporidium andersoni and Cryptosporidium bovis as well as both human-pathogenic Cryptosporidium parvum and Cryptosporidium hominis. A generic TaqMan assay further identified all known Cryptosporidium species and simultaneously monitored PCR inhibition through an external amplification control. The generic and specific assays were highly reproducible, and all displayed a detection limit of one oocyst per reaction. The specific TaqMan protocols also proved valuable for specifically detecting and quantifying target DNA in the presence of non-target DNA in environmental samples.

Conclusions

All TaqMan MGB real-time PCR assays fulfilled the required specificity and sensitivity criteria, both on laboratory strains and on a surface water matrix.

Significance and Impact of the Study

No molecular-based method was yet available for the quantitative detection of C. andersoni and the cluster formed by C. bovis, Cryptosporidium ryanae and Cryptosporidium xiaoi. This work provides a novel tool to evaluate the parasite load from domestic ruminants and humans, and to improve assessment and management of microbial risk through better appraisal of the origin and fate of faecal pollutions.

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