FV-PTH PCR Assay
The Viennalab FV-PTH mpx RealFast Assay detects the two most important thrombophilic variants in one multiplex real-time PCR run: Factor V Leiden (F5 1691G>A, p.R506Q) and the prothrombin variant F2 20210G>A. Both variants raise the risk of venous thrombosis. Factor V Leiden makes activated factor V less sensitive to breakdown by activated protein C, and the prothrombin variant raises the prothrombin concentration in plasma. Determining both in a single run gives the combined genotype, which matters because carriers of both variants have a higher risk than carriers of either one.
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This kit in detail
- Simultaneous detection of F5 1691G>A (Leiden, p.R506Q) and F2 20210G>A
- Multiplex real-time PCR, amplicons of 142 bp and 110 bp
- Four allele-specific probes: HEX and FAM for F5, Cy5 and ROX for F2
- WT-Control and MUT-Control supplied
- Whole peripheral blood, dried blood spots, buccal swabs or saliva
- 32 or 100 reactions per kit, REF 7-118 and REF 7-115
The Viennalab FV-PTH mpx RealFast Assay determines the genotype of the two most frequent hereditary risk factors for venous thrombosis in a single multiplex real-time PCR run.
Clinical background
Factor V Leiden is a point variant in the F5 gene, 1691G>A, which replaces arginine by glutamine at position 506 of the protein. Activated factor V then becomes less sensitive to breakdown by activated protein C, so thrombin formation continues for longer. The prothrombin variant 20210G>A in the F2 gene raises the prothrombin concentration in plasma. Both raise the risk of venous thromboembolism, and that risk is higher again in carriers of both variants and in combination with acquired factors such as immobilisation, surgery, pregnancy or oral contraceptive use.
Method
The assay amplifies a 142 bp fragment of F5 and a 110 bp fragment of F2 in one reaction, and reads four allele-specific hydrolysis probes: HEX and FAM for the wild-type and variant F5 allele, Cy5 and ROX for the wild-type and variant F2 allele. The kit supplies the RealFast 2x mpx Probe Mix, the FV-PTH mpx Assay Mix, a WT-Control and a MUT-Control. The recommended DNA input is 10 to 100 ng per reaction, at a concentration of 2 to 20 ng/µl. DNA isolated from whole peripheral blood, dried blood spots, buccal swabs or saliva can be used, and DNA extraction reagents are not supplied with the kit.
Instrument compatibility
Because the assay uses the ROX channel for one of its probes, it cannot be run on instruments that need ROX for normalisation: the Applied Biosystems StepOne, 7300 and 7900/7900HT are not suitable. The IFU lists the AB 7500 Fast, Bio-Rad CFX96, Roche LightCycler 480, bms MIC qPCR Cycler and Qiagen Rotor-Gene 6000 as suitable.
- RealFast™ 2x mpx Probe Mix: This mix contains HotStart Taq DNA polymerase and dNTPs in an optimized buffer system to enable precise amplification.
- FV-PTH mpx Assay Mix: This mix includes gene-specific primers for the F5 and F2 genes and four allele-specific, dual-labeled hydrolysis probes essential for detecting the mutations.
- FV-PTH mpx WT-Control: A control representing the wild type for both mutations is provided to ensure the accuracy of the assay.
- FV-PTH mpx MUT-Control: This control represents the homozygous mutant genotypes for the FV and PTH mutations, serving as a reference point for the assay.
Datasheets and regulatory documentation
Everything your quality team needs for evaluation and procurement.
Instructions for Use
Frequently paired with this kit
Other assays and instruments lab teams add alongside the FV-PTH PCR Assay.
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