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