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Master Thesis

Mise en place de la détection de la mutation JAK2 V617 en PCR digitale

Abstract : Vaquez's polycythemia, essential thrombocythemia and primary myelofibrosis form the group of Philadelphia-negative myeloproliferative neoplasias. These chronic haematological malignancies are associated with acquired genetic abnormalities that impart an advantage in proliferation and survival to haematopoietic stem cells. According to the new WHO 2016 classification, their diagnosis is based on several criterias, including the presence of the JAK2 gene V617F mutation. Currently, at the Saint Etienne University Hospital, the screening and quantification of the JAK2 V617F mutation are performed by quantitative PCR.The main aim of this study is to set up a new digital PCR technique for the detection of the JAK2 V617F mutation in order to apply for its accreditation. Indeed, since 2008, accreditation according to the NF EN ISO 15189 standard is mandatory. For this, laboratories must prepare method verification/validation files based on various documents proposed by COFRAC, such as SH GTA 04, which was used as a framework for this work. Digital PCR consists of distributing the sample in a large number of compartments either as micro-drops on a chip or as aqueous micro-drops suspended in a non-aqueous fluid. We have implemented the latter technology using Biorad® QXDx 200 droplet generator. Firstly, we optimised the operating protocol which allowed us to work in optimal conditions using 1.1µL of primer and 50ng of DNA per reaction well with an annealing temperature of 54°C. The results for repeatability, intermediate precision, accuracy and uncertainty of measurement complied with the laboratory requirements and literature data. The dPCR showed very good sensitivity and specificity values with 0.985 and 0.976 respectively. The tests to evaluate the limit of detection allowed us to obtain a value of 0.01% which is 10 times lower than the qPCR technique. Concomitantly with this work for the method validation file, we screened 30 patients with a history of thrombosis who had negative qPCR. None of these patients showed a positive signal in dPCR. Finally, we screened 26 patients for whom qPCR gave a very low allelic ratio (0.02% on average) below the detection limit. Of these, 17% were dPCR positive. We also performed a medico-economic study which showed that dPCR is 4 times less expensive than qPCR. For the detection of the JAK2 V617F mutation, we therefore replaced qPCR by dPCR, which not only is less expensive and less time-consuming but also has a better analytical performance.rformance.
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Master Thesis
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Submitted on : Tuesday, May 4, 2021 - 2:33:43 PM
Last modification on : Friday, May 7, 2021 - 3:19:58 AM
Long-term archiving on: : Thursday, August 5, 2021 - 7:39:46 PM


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  • HAL Id : dumas-03216930, version 1
  • PPN : 255147104


Manon Duprat. Mise en place de la détection de la mutation JAK2 V617 en PCR digitale. Sciences pharmaceutiques. 2021. ⟨dumas-03216930⟩



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