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Updated: Jul 12, 2026
A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors
Published on: December 7, 2014
Validación del kit de amplificación cuantitativa alélica específica Primerdesign para la detección de la mutación
Deborah Vaz1,2, Tshiphiri Netshidzivhani1,2, Johan Potgieter1,2
1National Health Laboratory Service - Tshwane Academic Division, Pretoria, South Africa.
Introduction:
Janus Kinase 2 (JAK2) is a tyrosine kinase involved in cellular signalling. A point mutation in codon 617 of JAK2 (JAK2V617F) causes a gain-of-function effect that stimulates proliferation of myeloid cells. The aim of this study was to evaluate the Primerdesign quantitative allele-specific amplification (Quasa) kit for the detection of JAK2V617F.
Methods:
Specimens previously evaluated using real-time PCR with melting curve analysis were analysed using the Quasa kit on the Bio-Rad CFX 96 platform. Results of the two PCR tests were compared to evaluate the accuracy of the kit. For intra-assay precision, samples were analysed in duplicate on the same run, and for inter-assay variation, the same known samples were tested on three non-consecutive days. The analytical sensitivity and quantitative analysis of the assay were determined using the World Health Organization (WHO) 1st International Reference Panel for Genomic JAK2V617F (United Kingdom Official Medicines Control Laboratory) and the United Kingdom National External Quality Assessment Service (UK NEQAS) results of their JAK2 p.val617phe (V617F) Mutation Status Programme.
Results:
Concordance of the qualitative test results between the test methods was 100%. Precision experiments confirmed repeatability and reproducibility. Analytical sensitivity was confirmed at 0.1%.
Conclusion:
The PrimerDesign Quasa kit for the detection of JAK2V617F is a sensitive and reliable assay that yields reproducible qualitative results. The kit is commercially available and has, in addition, the potential for quantitative analysis when samples are analysed in duplicate. This kit assay validation may be valuable to laboratories offering or interested in offering JAK2V617F mutational testing.
