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Titre : | Utilité de la quantification de lADN proviral chez les patients infectés par le HIV-1 |
Auteurs : | Soukaïna BAKKIOUI, Auteur ; Géraldine DESSILLY, Promoteur |
Type de document : | Travail de fin d'études |
Editeur : | Bruxelles : Institut Paul Lambin, 2020 |
Langues: | Français |
Index. décimale : | TFE - Biologie médicale |
Résumé : |
An effective antiretroviral treatment is based on an undetectable HIV-1 viral loads in the plasma. Nevertheless, the virus persists in the organism in integrated or non-integrated DNA forms. If the treatment is interrupted, a viral rebound can be observed because there is a latent reservoir. This reservoir represents the challenge for the cure of HIV-1 infection and its characteristics influence the course of the disease. Quantifying HIV-1 DNA in the blood is currently the most practical approach to measuring this residual infection. The objectives of this work consists to develop an ultrasensitive assay (ddPCR) to quantify proviral DNA and residual viremia, the comparison between ddPCR and qPCR and the implications of quantifying proviral DNA.
Firstly, an extraction of viral genomic DNA from HIV-1 positive patients is made. Secondly, the mix is prepared with the kit ddPCR Supermix for Probes (No dUTP) of Bio-Rad. After that, oils are adding to the mix to be transferred to the Droplet Generator QX200 Bio-Rad. Each sample is partitioned into 20.000 droplets with a volume of 1nL. The plate is transferred to perform PCR on the C1000 Touch Thermal Cycler of Bio-Rad. Thousand copies of the target sequence are obtained by PCR. Finally, the plate is transferred to the Droplet Reader QX200 of Bio-Rad where each droplet is arranged one after one. They pass through a bicolor optical detector. The droplets will be read either as positive or negative depending on their amplitude of fluorescence. Real-time PCR has also been used to quantify HIV-1 DNA. Its a method based on an enzymatic reaction that produces fluorescence, which continuously follows the production of amplicons. The kinetics allows to have an absolute quantification of the initial quantity of DNA. Different conditions were tested to quantify the proviral DNA on ddPCR. First of all, globin was chosen to be tested (modifications in primers concentrations and temperature). We found out that if we increase the globin primers concentration, the separation of globin is better. However, HIV-1 is inhibited and the globin isnt amplified in the positive control. When the HIV-1 primers concentration is to 0,6μM, the separation between negative and positive droplets of HIV-1 is optimal. However, it is still difficult to settle the threshold for the globin at this concentration. Then, the second reference gene selected was EIF2C1. We have increased the HIV-1 primers and probe concentrations. At high concentration, the HIV-1 is inhibited. At low concentration, the separation between negative and positive droplets of HIV-1 and EIF2C1 is optimal. Nevertheless, there is a droplet rain of HIV-1 and one in two wells has been amplified. In conclusion, the optimal conditions of separation between the negative and positive droplets of HIV-1 and globin are globin primers concentration to 0,15μM and HIV-1 primers concentration to 0,6μM. The negative and positive droplets of HIV-1 and EIF2C1 are separated when the EIF2C1 concentration is to 1x, HIV-1 primers concentration is to 1μM and HIV-1 probe concentration is to 0,2μM. The ddPCR offers a direct measurement of the DNA concentration while the qPCR uses a calibration curve. The simplest and cheapest technique for quantifying proviral HIV-1 DNA is real-time PCR. However, the ddPCR has better sensitivity, better precision for low viral loads, is lesser influenced by inhibitory substances and by minimal changes (temperatures, ...) than qPCR. |
Accès : | Identifiez-vous avant d'accéder au document électronique |
Disponible en ligne : | Oui |
Lieu du stage : | Université catholique de Louvain IREC/ Pôle MBLG |
Département : | Biologie médicale |
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