New real-time PCR-based method for the joint genotyping of JAK2 (V617F) with inherited thrombophilic F5 and F2 mutations.
Clin Chim Acta
; 410(1-2): 59-63, 2009 Dec.
Article
em En
| MEDLINE
| ID: mdl-19778528
ABSTRACT
BACKGROUND:
During the last years the appearance of the acquired V617F mutation of the Janus Kinase 2 gene (JAK2) in patients suffering different thrombotic events has been described. We decided to develop a new and rapid multiplex real-time Polymerase Chain Reaction (PCR) in order to detect the V617F mutation together with the inherited prothrombotic mutations of factors F5 and F2. DESIGN ANDMETHODS:
The method was carried out on the LightCycler 2.0 (Roche Diagnostics, Mannheim, Germany) and consisted in a first step of simultaneous amplification by real-time PCR of the three genes to be genotyped, in a 20microl closed tube, using a primer pair together with the correspondent FRET-hybridization probes for each gene.RESULTS:
We assayed 41 samples in the multiplex PCR reaction, 19 were positive (46.34%) for V617F mutation. From the V617F positive samples we found 1 sample heterozygous for F2 (5.26%) and 1 sample heterozygous for F5 (5.26%), so a 10.52% of the samples tested combine V617F mutation with inherited thrombophilic mutations. Results were clear, rapid and reliable allowing a significant time saving.CONCLUSIONS:
The technique presented in this manuscript is a new achievement in the field of the molecular diagnosis that combines the genotyping of F5 and F2 with the assessment of the JAK2 (V617F) mutation load.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fator V
/
Protrombina
/
Reação em Cadeia da Polimerase
/
Trombofilia
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Janus Quinase 2
Tipo de estudo:
Evaluation_studies
/
Observational_studies
Limite:
Humans
Idioma:
En
Revista:
Clin Chim Acta
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Espanha