Détection moléculaire du parasite Cryptosporidium dans des échantillons d eau ; Texte imprimé / Mélanie Fontaine ; sous la dir de Michel Miegeville Jean Paul Moisan
Par : Fontaine , Mélanie -- 1975
Document archivé le : 10/03/2010
The protozoan parasite Cryptosporidium is known to occur widely in both raw and drinking water and is the cause of water-borne outbreaks of gastroenteritis throughout the world. The routinely used method for the detection of Cryptosporidium oocysts in water is based on an immunofluorescence assay which is non-specific for the human pathogenic species, C. parvum. We have developed a TaqMan PCR test that accurately quantifies C. parvum oocysts in water samples. The protocol consisted of the following successive steps: Envirochek» capsule filtration, immunomagnetic-separation (IMS), thermal lysis followed by DNA purification and finally real-time PCR using TaqMan technology. Quantification was accomplished by comparing the fluorescence signals obtained from test samples with those from standard dilutions of C. parvum oocysts. This IMS-TaqMan PCR assay permits rapid and reliable quantification over six orders of magnitude, with a quantification limit of 5 oocysts. Replicate samples of spiked tap water and Seine river water samples were tested and oocyst recoveries were range respectively from 69,7 % (+- 22,3) to 84,5 % (+- 9,7) and from 46,4 % (+- 7,3) to 57,6 % (+- 10,7). We also report on a TaqMan reverse transcription-PCR method that targets and quantifies C. parvum 18S rRNA for detecting viable oocysts in water. This test performed in water samples obtained similar recoveries. To study the suitability of 18S rRNA as an indicator of Cryptosporidium oocysts viability, the stability of 18S rRNA and rDNA was monitored by real-time RT-PCR following various heat treatments. Our results indicate that 18S rRNA detection may not be directly associated with viability following heat inactivation of oocysts even if in all the experiments 18S rRNA was less stable than rDNA. These new molecular methods offer a rapid, sensitive and specific alternative for C. parvum oocyst quantification in water and can be useful for better health risk assessment during routine controls of drinking water quality, evaluation of treatment efficiency as well as identification of risk resources.
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