ViennaLab specializes in developing products for
in vitro diagnosis (IVD) of several genetic diseases and predispositions. I frequently use their FV-PTH-MTHFR Strip Assay; this assays for Factor V Leiden, Prothrombin, and methylene tetrahydrofolate reductase. This combination is mainly used for the diagnosis of venous thrombosis (a blood clot that forms within a vein).
The FV-PTH-MTHFR Strip Assay is based on the reverse hybridization principle, and includes three successive steps: DNA is isolated from anticoagulated blood. Then, factor V, prothrombin and MTHFR gene sequences are simultaneously amplified in vitro and biotin-labelled in a single (multiplex) amplification reaction. Finally, the amplification products are selectively hybridized to a test strip, which contains oligonucleotide probes (wild type- and mutant-specific) immobilized as parallel lines. Bound biotinylated sequences are detected using streptavidin-alkaline phosphatase and color substrates. The assay covers 3 mutations: FV (G1691A), PTH (G20210A) and MTHFR (C677T).
The kit contains the following major components which are characterized into 3 divisions:
1. Lysis Solution and GenXTRACT Resin for extraction of DNA from whole blood.
Extraction requires just 100 uL of whole blood (fresh/frozen); an important recommendation is that the DNA concentration should not exceed then 20 – 200 ng/reaction. Extraction starts with incubating the blood and Lysis Solution for 15 minutes. This is followed by a binding step using the GenXTRACT Resin and another incubation.
2. Amplification Mix for amplification (Taq Polymerase is not supplied in the kit).
Taq DNA Polymerase should be diluted to 0.2 U/uL using the supplied Dilution buffer. The total PCR reaction volume is 25 ul: 15 ul Amplification Mix + 5 ul (diluted Taq Pol) + 5 ul extracted DNA (less than 40 ng/µl). Mentioned reaction mix is amplified on thermal cycler using a standard 3 step, 30 cycle protocol. Amplicons can be checked via gel electrophoresis; if bands don’t appear, the entire protocol needs to be repeated.
3. For hybridization & washing: DNAT, Typing Trays, TestStrips, Hybridization Buffer, Wash Solution A, Conjugate solution, Wash Solution B, Color Developer.
This post-PCR step needs a couple of dedicated instruments: a water bath with a shaking platform and adjustable temperature (45°C ± 0.5°C), and an orbital shaker. This entire step has 2 incubations and takes 120 minutes. During this time, several samples can be processed using the typing trays. Each typing tray has 6 reservoirs and thus can process 6 samples simultaneously.
Results are easily interpreted visually; there is no need for any documentation system. A positive reaction of the control indicates the correct function of the Conjugate Solution and Color Developer. This line should always stain positive. Results are reported as normal genotype, heterozygous genotype or homozygous mutant genotype.
In our laboratory, we found this assay to be a robust, sensitive and single multiplex assay for venous thrombosis.
Research Associate
Molecular Genetics
DNA Lab