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Quantification of Cryptosporidium
18S Ribosomal gene
For general laboratory and research use only
Introduction to Cryptosporidium
Cryptosporidium is a protozoan pathogen of the Apicomplexa phylum and causes a diarrheal illness called cryptosporidiosis. Cryptosporidium does not utilise an insect vector and is capable of completing its life cycle within a single host. Cryptosporidiosis is typically an acute short-term infection but can become severe and nonresolving in children and immunocompromised individuals such as AIDS patients. The parasite is transmitted by environmentally hardy cysts (oocysts) that, once ingested, excyst in the small intestine and result in an infection of intestinal epithelial tissue. Transmission can occur via cysts excreted in faeces entering water supplies. Treatment of water by filtration can remove these cysts. After infection, symptoms may manifest within 2 days and last up to 2 weeks. These include a low fever, stomach pains and watery diarrhea. Some individuals may be asymptomatic and are, along with those with symptoms, contagious for several weeks. A number of species of Cryptosporidium infect mammals. In humans, the main causes of disease are C. parvum and C. hominis (previously C. parvum genotype 1). C. canis, C. felis, C. meleagridis, and C. muris can also cause disease in humans. The genome of Cryptosporidium parvum was sequenced in 2004 and was found to be unusual amongst Eukaryotes in that the mitochondria seem not to contain DNA.
Specificity
The PrimerDesign genesig Kit for Cryptosporidium (Crypto) Genomes is designed for the in vitro quantification of Crypto genomes. The kit is designed to have the broadest detection profile possible whilst remaining specific to the Crypto genome. The primers have 100% homology with all reference sequences included in the polygenetic tree below and therefore have a very broad quantification profile.
Kit Contents
Crypto specific primer/probe mix (150 reactions BROWN)
FAM labeled, BHQ quenched
Crypto positive control template (for Standard curve RED) RNAse/DNAse free water
Trademarks
PrimerDesign is a trademark of PrimerDesign Ltd. The PCR process is covered by US Patents 4,683,195, and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche AG. ABI, ABI PRISM GeneAmp and MicroAmp are registered trademarks of the Applera Genomics (Applied Biosystems Corporation). BIOMEK is a registered trademark of Beckman Instruments, Inc.; iCycler is a registered trademark of Bio-Rad Laboratories, Rotor-Gene is a trademark of Corbett Research. LightCycler is a registered trademark of the Idaho Technology Inc. GeneAmp, TaqMan and AmpliTaqGold are registered trademarks of Roche Molecular Systems, Inc., The purchase of the PrimerDesign reagents cannot be construed as an authorization or implicit license to practice PCR under any patents held by Hoffmann-LaRoche Inc.
Carry-over prevention using UNG (optional) Carry over contamination between PCR reactions can be prevented by including uracil-Nglycosylase (UNG) in the reaction mix. Some commercial mastermix preparations contain UNG or alternatively it can be added as a separate component. UNG can only prevent carry over from PCR reactions that include deoxyuridine triphosphate (dUTP) in the original PCR reaction. PrimerDesign recommend the application of 0.2U UNG per assay with a 15 minute incubation step at 37C prior to amplification. The heat-labile UNG is then inactivated during the Taq polymerase activation step (95C for 10 minutes).
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* This component contains high copy number template and is a VERY significant contamination risk. It must be opened and handled in a separate laboratory environment, away from the other components.
Crypto detection mix Component 2 x PrecisionTM MasterMix Crypto Primer/Probe mix (BROWN) RNAse/DNAse free water (WHITE) Final Volume 2. Volume 10 l 1 l 4 l 15 l
Pipette 15l of this mix into each well according to your real-time PCR experimental plate set up. Prepare sample DNA templates for each of your samples (suggested concentration 5ng/l) in RNAse/DNAse free water. If the concentration of DNA is not known, then dilute your DNA sample reactions 1:20 (10l of sample DNA and 190l of water). Pipette 5l of diluted DNA template into each well, according to your experimental plate set up. For negative control wells use 5l of RNAse/DNAse free water. The final volume in each well is 20l.
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Preparation of standard curve dilution series. 1) Pipette 900l of RNAse/DNAse free water into 5 tubes and label 2-6 2) Pipette 100l of Positive Control Template (RED) into tube 2 3) Vortex thoroughly 4) Change pipette tip and pipette 100l from tube 2 into tube 3 5) Vortex thoroughly
Standard Curve Tube 1 Positive control (RED) Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 6.
Copy Number 2 x 105 per l 2 x 104 per l 2 x 103 per l 2 x 102 per l 20 per l 2 per l
Pipette 5l of standard template into each well, according to your experimental plate set up. The final volume in each well is 20l.
Amplification Protocol
Amplification conditions using PrimerDesign 2X PrecisionTM MasterMix. Standard Curve Step UNG treatment (if required) ** Enzyme activation (if required) *** Denaturation 50 Cycles DATA COLLECTION * Time 15 mins 10 mins 10s 60s Temp 37 oC 95 oC 95 oC 60 oC
* Fluorogenic data for the control DNA should be collected during this step through the FAM and VIC channels ** Required if your Mastermix includes UNG to prevent PCR carryover contamination *** Not all Mastermixes require this enzyme activation step. Follow the manufacturers instructions for your Mastermix.
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