Quantitative assay for the detection of the V617F variant in the Janus kinase 2 (JAK2) gene using the Luminex xMAP technology

BMC Medical Genetics

Technical Advance

Daniel Gaudet¹  Raynald Simard¹  François W Paradis¹ 

[1] ECOGENE-21, department of médecine, Université de Montréal, Chicoutimi Hospital, 225 rue St-Vallier, G7H 7P2, Saguenay, Province of Québec, Canada;

关键词: Polymerase Chain Reaction Reaction;    Polycythemia Vera;    Essential Thrombocythemia;    Polymerase Chain Reaction Fragment;    JAK2V617F Mutation;   

DOI  :  10.1186/1471-2350-11-54

 received in 2009-10-09, accepted in 2010-04-01,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundThe availability of clinically valid biomarkers contribute to improve the diagnosis and clinical management of diseases. A valine-to-phenylalanine substitution at position 617 (V617F) in the Janus kinase 2 (JAK2) gene has been recently associated with key signaling abnormalities in the transduction of haemopoietic growth-factor receptors and is now considered as a useful clinical marker of myeloproliferative neoplasms. Several methods have recently been reported to detect the JAK2 V617F point mutation and show variable sensitivity.MethodsUsing the Luminex xMAP technology, we developed a quantitative assay to detect the JAK2V617F variant. The method was based on polymerase chain reaction (PCR) followed by hybridization to specific probes coupled with internally dyed microspheres. The assay comprises 3 steps: genomic DNA extraction, end point PCR reaction, direct hybridization of PCR fragments and quantification. It has been tested with different sources of nucleic acid.ResultsApplied to whole blood samples, this quantitative assay showed a limit of detection of 2%. A highly sensitive allele-specific primer extension reaction performed in parallel allowed to validate the results and to identify the specimens with values below 2%.ConclusionDirect hybridization assay using the Luminex xMAP technology allows sensitive quantification of JAK2V617F from blood spots. It is simple and can be easily performed in a clinical setting.

【 授权许可】

Unknown   
© Paradis et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.