Applied Microbiology and Biotechnology

, Volume 89, Issue 4, pp 1203–1211

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

  • José L. Alonso
  • Inmaculada Amorós
  • Irene Cañigral
Methods and Protocols

DOI: 10.1007/s00253-010-2984-6

Cite this article as:
Alonso, J.L., Amorós, I. & Cañigral, I. Appl Microbiol Biotechnol (2011) 89: 1203. doi:10.1007/s00253-010-2984-6
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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.

Keywords

Cryptosporidium Giardia LightCycler LNA probe qPCR Sewage TaqMan 

Supplementary material

253_2010_2984_MOESM1_ESM.doc (24 kb)
Table 6Mean Ct 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)
253_2010_2984_MOESM2_ESM.doc (28 kb)
Table 7Mean Ct 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)

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • José L. Alonso
    • 1
  • Inmaculada Amorós
    • 1
  • Irene Cañigral
    • 2
  1. 1.Instituto de Ingeniería del Agua y Medio Ambiente, Ciudad Politécnica de la Innovación, Ed. 8G, Acceso D, planta 2Universidad Politécnica de ValenciaValenciaSpain
  2. 2.Centro Avanzado en Microbiología de AlimentosUniversidad Politécnica de ValenciaValenciaSpain

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