genesig Real-Time PCR Detection Kit for Simian Virus 40 (Large T-Antigen Gene), Standard Kit

Kits and Assays / Kits-PCR, genesig™

-20°C

The genesig® Kit for Simian Virus 40 (SV40) is designed for the in vitro quantification of SV40 genomes. The kit is designed to have the broadest detection profile possible while remaining specific to the SV40 genome.

The primers and probe sequences in this kit have 100% homology with a broad range of SV40 sequences based on a comprehensive bioinformatics analysis.

PRINCIPLE OF THE TEST:

Real-time PCR
A SV40 specific primer and probe mix is provided and this can be detected through the FAM channel.
The primer and probe mix provided exploits the so-called TaqMan® principle. During PCR amplification, forward and reverse primers hybridize to the SV40 DNA. A fluorogenic probe is included in the same reaction mixture which consists of a DNA probe labeled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved and the reporter dye and quencher are separated. The resulting increase in fluorescence can be detected on a range of real-time PCR platforms.

Positive Control
For copy number determination and as a positive control for the PCR set up, the kit contains a positive control template. This can be used to generate a standard curve of SV40 copy number / Cq value. Alternatively the positive control can be used at a single dilution where full quantitative analysis of the samples is not required. Each time the kit is used, at least one positive control reaction must be included in the run. A positive result indicates that the primers and probes for detecting the target SV40 gene worked properly in that particular experimental scenario. If a negative result is obtained the test results are invalid and must be repeated. Care should be taken to ensure that the positive control does not contaminate any other kit component which would lead to false-positive results. This can be achieved by handling this component in a Post PCR environment. Care should also be taken to avoid cross-contamination of other samples when adding the positive control to the run. This can be avoided by sealing all other samples and negative controls before pipetting the positive control into the positive control well.

Negative Control
To validate any positive findings a negative control reaction should be included every time the kit is used. For this reaction the RNAse/DNAse free water should be used instead of template. A negative result indicates that the reagents have not become contaminated while setting up the run.

Carry-over prevention using UNG (optional)
Carry over contamination between PCR reactions can be prevented by including uracil-N- glycosylase (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. We recommend the application of 0.2U UNG per assay with a 15 minute incubation step at 37°C prior to amplification. The heat-labile UNG is then inactivated during the Taq polymerase activation step.

STANDARD KIT COMPONENTS:

• SV40 specific primer/probe mix (150 reactions BROWN); FAM labeled

• SV40 positive control template (RED); for standard curve

• RNAse/DNAse free water (WHITE); for resuspension of primer/probe mixes

• Template preparation buffer (YELLOW); for resuspension of positive control template and standard

REAGENTS AND EQUIPMENT TO BE SUPPLIED BY THE USER:

• Real-Time PCR Instrument

• DNA extraction kit
This kit is recommended for use with genesig EASY DNA/RNA Extraction kit. However, it is designed to work well with all processes that yield high quality DNA with minimal PCR inhibitors.

• oasigTM Lyophilised or PrecisionPLUSTM 2 x qPCR Mastermix
This kit is designed to work well with all commercially available Mastermixes. However, we recommend the use of oasigTM or PrecisionPLUSTM 2x qPCR MasterMix.

• Pipettors and Tips

• Vortex and centrifuge

• Thin walled 1.5 ml PCR reaction tubescurve preparation

KIT STORAGE AND STABILITY:
This kit is stable at room temperature but should be stored at -20 °C on arrival. Once the lyophilised components have been re-suspended they should not be exposed to temperatures above -20°C for longer than 30 minutes at a time and unnecessary repeated freeze/thawing should be avoided. The kit is stable for six months from the date of re-suspension under these circumstances.
If a standard curve dilution series is prepared this can be stored frozen for an extended period. If you see any degradation in this serial dilution a fresh standard curve can be prepared from the positive control.
We do not recommend using the kit after the expiry date stated on the pack.

SUITABLE SAMPLE MATERIAL:
All kinds of sample material suited for PCR amplification can be used. Please ensure the samples are suitable in terms of purity, concentration, and DNA integrity. Always run at least one negative control with the samples. To prepare a negative control, replace the template DNA sample with RNAse/DNAse free water.

DYNAMIC RANGE OF TEST:
Under optimal PCR conditions genesig® SV40 detection kits have very high priming efficiencies of >95% and can detect less than 100 copies of target template.

PRODUCT MANUAL:
http://www.genesig.com/assets/files/sv40_std.pdf

INSTRUMENTATION REQUIREMENTS:
Designed for use with real-time PCR instruments. . For use in genesig® q16, look for the “Easy” nomenclature.

ABOUT THE genesig® KITS:
genesig® kits are sold for research use only and are not licensed for diagnostic procedures.

Nucleic acid testing (NAT) is the method of choice for detection and quantification of a wide range of micro organisms. Primerdesign manufactures and supplies high quality quantitative Real-Time PCR kits for the detection and simultaneous quantification of numerous significant pathogens. A copy number standard curve is provided for quantification and an the internal extraction template (DNA or RNA), controls for the quality of the nucleic acid extraction and eliminates false negative results.

The kit is designed with the broadest possible detection profile to ensure that all clinically relevant strains and subtypes are detected. Target sequences are selected by working with data from key opinion leaders in the field. Multiple sequence alignments and unprecedented Real-Time PCR expertise in design and validation ensure the best possible kit. 

Details of the target and priming specificity are included in the individual handbooks above. Packaged, optimized and ready to use.

genesig Standard Kit:
Product features:
• Exceptional value for money
• Rapid detection of all clinically relevant subtypes
• Positive copy no. standard curve for quantification
• Highly specific detection profile
• High priming efficiency
• Broad dynamic detection range (>6 logs)
• Sensitive to < 100 copies of target
• Accurate controls to confirm findings

DNA/RNA EXTRACTION:
genesig® Easy DNA/RNA extraction kits are a simple to use, low cost and powerful extraction technology suitable for virtually all sample types.