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Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples

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Abstract

Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the β-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower Ct values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier Ct values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.

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Acknowledgments

This work was supported by the Spanish Ministerio de Ciencia e Innovación grants AGL2005-07776-C03-03 and AGL2008-05275-C03-03/ALI). Part of this work was also funded by the Fondo Europeo de Desarrollo Regional (Feder) grant POICV 2000–2006. We thank staff at the wastewater treatment plants and the Entidad de Saneamiento de Aguas for assistance in sample collection.

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Authors and Affiliations

  1. Instituto de Ingeniería del Agua y Medio Ambiente, Ciudad Politécnica de la Innovación, Ed. 8G, Acceso D, planta 2, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022, Valencia, Spain

    José L. Alonso & Inmaculada Amorós

  2. Centro Avanzado en Microbiología de Alimentos, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022, Valencia, Spain

    Irene Cañigral

Authors
  1. José L. Alonso
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  2. Inmaculada Amorós
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  3. Irene Cañigral
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Correspondence to José L. Alonso.

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Table 6

Mean C t values for four WWTP samples (sample 1, sample 2, sample 3, and sample 4) seeded with 60,000 cysts of G. lamblia for detection of Giardia with β-giardin P241 probesa (DOC 24 kb)

Table 7

Mean C t values for three WWTP samples (WWTP1, WWTP2, and WWTP3) seeded with 40,000 oocysts of C. parvum for detection of Cryptosporidium with COWP P702 probes* (DOC 27 kb)

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Alonso, J.L., Amorós, I. & Cañigral, I. Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples. Appl Microbiol Biotechnol 89, 1203–1211 (2011). https://doi.org/10.1007/s00253-010-2984-6

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  • DOI: https://doi.org/10.1007/s00253-010-2984-6

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