Priprava, vrednotenje in uporaba kontrolnih materialov za določanje koncentracije virusa opičjih koz z verižno reakcijo s polimerazo

Šulin, Mija (author), Bogožalec Košir, Alexandra (mentor)

Za zanesljivo diagnostiko z molekularnimi metodami potrebujemo kontrolne materiale, ki med drugim omogočajo primerjavo rezultatov med laboratoriji. Uporaba kontrolnih materialov je zelo pomembna pri razvoju novih testov, natančni kvantifikaciji genetskega materiala, z njimi tudi upoštevamo in kompenziramo človeške napake med izvedbo analize. Za vrednotenje kontrolnih materialov, ki so nukleinske kisline, uporabljamo digitalno verižno reakcijo s polimerazo (dPCR), ki omogoča povezljivost na najvišjo meroslovno raven, do SI enot. Omogoča absolutno kvantifikacijo nukleinskih kislin, kar pomeni, da za kvantifikacijo ne potrebuje kalibracijske krivulje. Metoda je tudi ponovljiva, natančna in manj dovzetna za inhibicijo kot kvantitativni PCR (qPCR). Posledično ima dPCR velik pomen za raziskovalne in diagnostične aplikacije. V magistrskem delu smo pripravili, ovrednotili in uporabili kontrolni material za kvantifikacijo virusa opičjih koz s qPCR. Najprej smo pripravili kontrolni material iz sintetične DNA, in sicer: v vodi (H2O), v vodi z dodano nosilno DNA iz spermijev lososa (ssH2O) ter v vodi z dodano nosilno DNA iz priželjca teleta (ctH2O). Kontrolni material je bil shranjen pri dveh temperaturah (–20 °C in –80 °C). Z metodo dPCR smo materialu določili koncentracijo, ovrednotili tako homogenosti med kot znotraj vzorca ter testirali dolgotrajno in kratkotrajno stabilnost. Ugotovili smo, da je kontrolni material iz sintetične DNA najstabilnejši v ssH2O, shranjen pri temperaturi –80 °C, zato smo pri pripravi kontrolnega materiala iz genomske DNA (gDNA) uporabili te pogoje. Kontrolni material iz gDNA je pri teh pogojih stabilen, ohrani homogenost in koncentracijo. Pri ovrednotenju materiala iz gDNA smo primerjali tudi dve platformi za dPCR (QX200 Droplet Digital PCR (Bio-Rad) in Naica System 6-color Digital PCR (Stilla Technologies)), ki sta dali primerljive rezultate. Ko smo imeli pripravljen kontrolni material, smo ga uporabili za kvantifikacijo virusa opičjih koz in virusa vakcinije v vzorcih z metodo qPCR. Rezultate kvantifikacije virusov smo nato primerjali z metodo dPCR. Čeprav je med rezultati pristranskost višja od 25 %, so rezultati pri virusu opičjih koz primerljivi, ker je bila koncentracija virusa nizka in pomembno je bilo to, da smo virus zaznali. Za umeritveno krivuljo smo uporabili sintetično DNA, zato primerjava vzorcev ni bila optimalna, ker je pri vzorcih različnih kompleksnosti lahko učinkovitost pomnoževanja različna.

• Language: Slovenian
• Keywords: dPCR, kontrolni material, virus opičjih koz
• Work type: Master's thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2024
• PID: 20.500.12556/RUL-164033
• Publication date in RUL: 16.10.2024

Secondary language

• Language: English
• Title: Preparation, characterization and use of control materials for monkeypox virus quantification using polymerase chain reaction

Abstract

Reliable diagnostics using molecular methods requires control materials that, among other things, allow comparison of results between laboratories. The use of control materials is very important for the development of new tests, for the accurate quantification of genetic material, and to account for and compensate for human error during the analysis. Digital polymerase chain reaction (dPCR), which has connectivity to the highest metrological level, to SI units, is used to evaluate nucleic acid control materials. It allows absolute quantification of nucleic acids, which means that it does not require a calibration curve for quantification. The method is also reproducible, accurate and less susceptible to inhibition than real-time PCR (qPCR). Therefore, dPCR has a major impact on research and diagnostic applications. In this master’s thesis, we prepared, evaluated, and used control material for quantification of monkeypox virus by qPCR. First, we prepared control material from synthetic DNA in three different backgrounds: H2O, aqueous solution of salmon sperm DNA (ssH2O) and aqueous solution of calf thymus DNA (ctH2O), each stored at two temperatures (-20 °C and -80 °C). Using a dPCR method, we assigned a value to the material, evaluated its homogeneity between and within the sample and tested long-term and short-term stability. We conclude that the synthetic DNA control material is the most stable in the ssH2O background when stored at -80 °C, so we used these conditions when preparing the genomic DNA (gDNA) control material. The gDNA control material maintained homogeneity and concentration under these conditions. When evaluating gDNA material, we also compared two dPCR platforms (QX200 Droplet Digital PCR (Bio-Rad) and Naica System 6-color Digital PCR (Stilla Technologies)), which gave comparable results. Once we had prepared the control material, we used it to quantify monkeypox and vaccinia virus using the qPCR method. The results of virus quantification were then compared with the dPCR method. Although there is a high bias among the results, the results for monkeypox virus are comparable because the concentration of the virus itself was low and it was important that the virus was detected at all. Synthetic DNA was used for the calibration curve, so the comparison of samples was not optimal, because the amplification efficiency can be different for samples of different complexity.

• Keywords: dPCR, control material, monkeypox virus