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Updated: Aug 24, 2026
Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Four-color multiplex 5' nuclease assay for the simultaneous detection of the factor V Leiden and the prothrombin
1Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547, USA. luis_ugozzoli@bio-rad.com
Abstract:
We developed a real-time multiplex four-color assay for the simultaneous detection of the factor V Leiden (FVL) and prothrombin (PT) G20210A mutations in one closed tube using a single thermocycling protocol. The assay combines the power of multiplex PCR with the specificity provided by allele-specific oligonucleotide (ASO) hybridization using the 5' nuclease assay format. Human genomic DNA is prepared from whole blood with standard procedures. A 97-bp DNA sequence of the coagulation factor V gene is co-amplified with a 111-bp DNA sequence of the coagulation factor II (PT) gene using four PCR primers. In addition, the reactions included four differentially labeled ASO probes for the specific detection of the different FVL/PT G20210A genotypes. To evaluate the assay's performance characteristics, we performed a comparison of two methods. We analyzed DNA samples from 52 individuals with known FVL/PT G20210A genotypes that were previously genotyped with an assay that combined PCR with the use of restriction fragment length polymorphisms. We found a 100% concordance between the results generated by both methodologies. We conclude that the four-color multiplex assay is specific and reproducible for the detection of the FVL/PT G20210A mutations, and it can be easily adapted for the detection of other SNPs.
Insights
A new multiplex assay enables simultaneous detection of factor V Leiden (FVL) and prothrombin (PT) G20210A mutations. This validated method offers a specific, reproducible, and efficient approach for genetic testing.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Factor V Leiden (FVL) and prothrombin (PT) G20210A mutations are common genetic risk factors for venous thromboembolism.
- Accurate and efficient detection of these mutations is crucial for clinical diagnosis and risk assessment.
Purpose of the Study:
- To develop and validate a real-time, multiplex, four-color assay for the simultaneous detection of FVL and PT G20210A mutations.
- To establish a single-tube, single-thermocycling protocol for enhanced diagnostic efficiency.
Main Methods:
- Developed a multiplex PCR assay combined with allele-specific oligonucleotide (ASO) hybridization using the 5' nuclease assay format.
- Co-amplified specific DNA sequences of the coagulation factor V and factor II genes.
- Utilized four differentially labeled ASO probes for genotype-specific detection.
Main Results:
- Achieved simultaneous detection of FVL and PT G20210A mutations in a single closed tube.
- Demonstrated 100% concordance with a previously established PCR-RFLP genotyping method.
- Validated the assay's specificity and reproducibility using 52 known DNA samples.
Conclusions:
- The developed four-color multiplex assay is a specific, reproducible, and efficient tool for detecting FVL and PT G20210A mutations.
- The assay format can be readily adapted for the detection of other single nucleotide polymorphisms (SNPs).
- This method offers potential for streamlined genetic testing in thrombophilia diagnostics.
