Cryptosporidium Genotyping for Source Tracking Improving our ability to manage the safety of our water supplies.

Invitrogen Proprietary & Confidential 2 Source Tracking Made Easier! An all in one kit containing everything you need to genotype your Cryptosporidium samples! Kit contains 1.Slide extraction reagents 2.Simple safe DNA isolation and Purification reagents 3.Primary, Secondary, and Platinum PCR SuperMix. 4.Restriction Enzymes (SspI, VspI, DdeI, plus buffers) 5.C. parvum PCR-RFLP Positive Control Template. 6.Simple to use standardised Electrophoresis reagents with 2% and 4% buffer free E-gel casettes. 7.I-Gel PowerBase (Part of kits and ). 8.Dynabeads anti-Cryptosporidium kit (Part of kit ). 9.Spot on Slides (Part of kit ). 10.L10 tubes (Part of kit ).

Invitrogen Proprietary & Confidential 3. BASIC PROTOCOL  50 L of water was passed through a Filta-max™ filter.  Processed parasite enumeration using Method 1623  Immunomagnetic separation (IMS) carried out using Dynabeads  -GC Combo Kit  Oocysts enumerated using immunofluorescent antibodies (IFA),  PCR-RFLP analysis. Xiao et al.  DNA extracted Ruecker et al  All positive reactions digested with Ssp I, Vsp I and Dde I  Fractionated by 2% agarose gel electrophoresis and visualized  PCR products excised from the electrophoresis gel,  Purified and sequenced (if necessary)

Invitrogen Proprietary & Confidential 4 FIG. 2. Molecular forensic profiling of Cryptosporidium parasites by repetitive nested PCR-RFLP analysis using microscope slides containing three water samples processed by USEPA method 1623.

Invitrogen Proprietary & Confidential 5 FIG. 2. Molecular forensic profiling of Cryptosporidium parasites by repetitive nested PCR-RFLP analysis using microscope slides containing three water samples processed by USEPA method 1623.

Invitrogen Proprietary & Confidential 6 Simple DNA Extraction protocol, no solvents or alcohols All reagents included Pre-poured Agarose gel cassettes and standardised buffer-less electrophoresis running system Call off charts for fragment sizes/patterns