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Method for Detection and Genotyping

Towards the Development of a National Reference Facility for Cryptosporidium Carolyn Read, Jenny Pender, Tom Murphy, John Egan, Grace Mulcahy and Theo de Waal. A Collaboration between the EPA, UCD and DAFF. Project Objectives. Method for Detection and Genotyping.

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Method for Detection and Genotyping

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  1. Towards the Development of a National Reference Facility for CryptosporidiumCarolyn Read, Jenny Pender, Tom Murphy, John Egan, Grace Mulcahy and Theo de Waal. A Collaboration between the EPA, UCD and DAFF Project Objectives Method for Detection and Genotyping • Establishment of a Cryptosporidium reference facility located at Central Veterinary Research Laboratory, DAFF, Celbridge, Co Kildare • Survey of Cryptosporidium monitoring in Irish drinking water supplies • Detection of Cryptosporidium In Water- US EPA Method 1622 • Genotyping of Cryptosporidium oocysts from slides using emerging methods Filtration • Pilot scheme focusing on detection and genotyping of Cryptosporidium oocysts in supplies on the the EPAs Remedial Action List IMS Progress to date • Survey of Cryptosporidium monitoring in Irish drinking water supplies complete and available on request. • USEPA method 1622 for the detection of Cryptosporidium oocysts in water has been established in the laboratory and validation is near completion. • Routine genotyping of Cryptosporidium oocysts off slides from method 1622 by PCR-Sequencing has been used successfully for the pilot scheme samples (Xiao et al., Ryan et al.) • Emerging methods using real time PCR followed by high resolution melt curve (HRM) analysis (DiGiovanni et al.) are being established. • Pilot scheme sampling and analysis commenced in February 2011- 9 months completed. DIC DAPI FITC Cryptosporidium oocysts Slide scraping Preliminary Pilot Scheme Results DNA extraction • *DNA not extracted from slides containing only 1 oocyst • **Genotype was not determined from some slides containing less than 5 oocysts Future of Laboratory: Real-time PCR Traditional PCR • Accreditation assessment by INAB due in March 2012 • Commence genotyping on oocysts from slides referred from water testing laboratories • Consultation with stakeholders to a establish model for future sustainability of a national reference facility for Cryptosporidium High Resolution melt curve analysis Sequencing References DiGiovanni et al, Water Research Foundation, Web Report #4099. Xiao et al, 2001, Applied and Environmental Microbiology, Mar 2001, p. 1097-1101 Ryan et al, 2003. Applied and Environmental Microbiolgoy, July 2003, P. 4302-4307

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