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

1
Cryptosporidium Genotyping for Source Tracking
Improving our ability to manage the safety of
our water supplies.
2
Source Tracking Made Easier!

• An all in one kit containing everything you need
  to genotype your Cryptosporidium samples!
• Kit contains
• Slide extraction reagents
• Simple safe DNA isolation and Purification
  reagents
• Primary, Secondary, and Platinum PCR SuperMix.
• Restriction Enzymes (SspI, VspI, DdeI, plus
  buffers)
• C. parvum PCR-RFLP Positive Control Template.
• Simple to use standardised Electrophoresis
  reagents with 2 and 4 buffer free E-gel
  casettes.
• I-Gel PowerBase (Part of kits 740-05 and 740-06).
• Dynabeads anti-Cryptosporidium kit (Part of kit
  740-05).
• Spot on Slides (Part of kit 740-05).
• L10 tubes (Part of kit 740-05).

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)

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.
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.
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