Loop-mediated isothermal amplification (LAMP)-based method for detecting factor V Leiden and factor II G20210A common va
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Loop‑mediated isothermal amplification (LAMP)‑based method for detecting factor V Leiden and factor II G20210A common variants Giovanni Luca Tiscia1 · Donatella Colaizzo1 · Patrizia Vergura1 · Giovanni Favuzzi1 · Elena Chinni1 · Charlotte Vandermeulen2 · Liselot Detemmerman2 · Elvira Grandone1,3
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Automated methodologies allowing for rapid detection of Factor V Leiden and Factor II G20210A variants are desirable, due to a high number of tested patients. Here, we report a preliminary validation of a CE-marked in vitro diagnostic (IVD) certified method for simultaneous detection of Factor V Leiden and Factor II G20210A variants on whole blood samples. The novel method is based on Loop-mediated isothermal AMPlification (LAMP) applied for a duplex detection of Factor V Leiden and Factor II G20210A variants without requiring prior DNA extraction, whereas the routine one is a TaqMan SNP genotyping targeting genomic DNA. We tested routine patients for both variants using novel and current methods and estimated concordance rate. Patients were tested under similar laboratory procedures. One hundred and eight patients referred for the thrombophilia testing in the period between 9th December 2019 to 27th February 2020 represented the study population. We routinely identified for the Factor V Leiden variant 163 wild-type, 17 heterozygotes and no homozygote. Concerning the Factor II G20210A variant, we identified 170 wild-type, nine heterozygotes and one homozygous carrier. Two heterozygotes carried both variants (double heterozygotes). The LAMP method showed a 100% concordance rate, detecting rightly all genotypes. The LAMP for a duplex detection of common thrombophilia variants shows analytic performances as good as those of the standard method. Keywords Factor V Leiden · Factor II G20210A · Mutation · Thrombophilia · Detection · Molecular investigations
Highlights • Number of patients tested for Factor V Leiden and Factor
II G20210A variants is high
• The Loop-mediated isothermal AMPlification showed
good analytic performances for the detection of Factor V Leiden and Factor II G20210A
• Loop-mediated isothermal AMPlification applied for
Introduction
* Elvira Grandone [email protected]
Factor V Leiden (FVL) due to Arg506Gln exchange and causing resistance to activated protein C was described in 1994 [1]. Since then a great deal of studies documented an association between this variant and venous thromboembolism (VTE), defined as the occurrence of deep vein thrombosis (DVT) with/without pulmonary embolism (PE) [2]. In 1996, a molecular investigation, aiming at contributing to enhancing theory on VTE, allowed to identify a novel genetic determinant—G20210A variant—in gene (F2) coding for prothrombin (FII) [3]. The FII G20210A in F2 3′-untranslated region is associated with high protein levels and confers susceptibility to VTE [3]. The FVL and FII G20210A variants are included among the conditions which
a duplex detection can be a
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