Vpliv reverzne transkripcije na določanje količine tarčne RNA pri SARS-CoV-2

Nechevska, Anastasija (avtor), Milavec, Mojca (mentor)

Izbira pravilne diagnostične metode je ključna za zagotavljanje natančnih in zanesljivih rezultatov pri odkrivanju virusov. To predstavlja poseben izziv pri RNA-virusih, kot je SARS-CoV-2, saj je v tem primeru najprej treba izvesti reverzno transkripcijo (RT), kar lahko pomembno vpliva na rezultate analize. Za diagnozo virusa SARS-CoV-2 kot zlati standard velja metoda RT in kvantitativne verižne reakcije s polimerazo (RT-qPCR) zaradi svoje izjemne natančnosti, enostavnosti izvedbe ter občutljivosti pri zaznavanju in kvantifikaciji virusne RNA. Kljub tem prednostim lahko pri zelo nizkih koncentracijah virusa pride do lažno negativnih rezultatov. Alternativna metoda je RT in digitalne verižne reakcije s polimerazo (RT-dPCR), ki omogoča absolutno kvantifikacijo in zaznavanje virusa pri zelo nizkih koncentracijah, kar je ključno v zgodnjih fazah okužbe. V okviru magistrske naloge smo z metodama RT-qPCR in RT-dPCR izvedli analize na dveh vrstah vzorcev: (1) sintetičnih segmentih virusa SARS-CoV-2, ki vsebujejo značilna zaporedja genoma virusa, in (2) genomski RNA (gRNA), izolirani iz virusov gojenih na celičnih kulturah. Uporabili smo različne komercialno dostopne enostopenjske in dvostopenjske RT-komplete in primerjali njihovo učinkovitost in natančnost pri zaznavanju za virus SARS-CoV-2 značilnih nukleinskih zaporedij. Kot pozitivno kontrolo smo uporabili zapis za encim luciferazo (LUC), ki se pogosto uporablja kot kontrola prisotnosti inhibitorjev RT v reakciji. Ugotovili smo, da so enostopenjski kompleti boljša izbira pri obeh metodah, tako za segmente RNA kot za gRNA, saj smo dobili primerljive vrednosti za izbrane tarče virusa. Pri sintetičnih segmentih RNA smo z nekaterimi dvostopenjskimi kompleti dobili višje vrednosti kot z enostopenjskimi kompleti, a hkrati smo opazili visoko variabilnost med tarčami. Kontrola LUC se je izkazala kot neustrezna izbira, saj smo z obema pristopoma (enostopenjski in dvostopenjski) v primeru obeh metod (RT-qPCR in RT-dPCR) dobili visoke in med seboj neprimerljive rezultate. Pokazali smo, da obe metodi sicer učinkovito delujeta pri zaznavanju in kvantifikaciji virusne RNA, vendar je bila med rezultati (vrednosti Cq) posameznih metod RT-qPCR velika variabilnost, kar pomeni, da vrednosti Cq ne moremo neposredno uporabljati za diagnostične namene. Pokazali smo tudi, da metoda analize in izbira kompletov reagentov pomembno vplivata na zanesljivost in ponovljivost rezultatov, kar je ključno za učinkovito diagnosticiranje virusa SARS-CoV-2.

• Jezik: Slovenski jezik
• Ključne besede: RT-qPCR, RT-dPCR, SARS-CoV-2
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-167164
• COBISS.SI-ID: 226174723
• Datum objave v RUL: 11.02.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Effect of reverse transcription on target RNA quantification in SARS-CoV-2

Izvleček

Choosing the correct diagnostic method is crucial for providing accurate and reliable results in virus detection. This presents a particular challenge for RNA-viruses, such as SARS-CoV-2, as it is necessary to first perform reverse transcription (RT), which can significantly impact the results of the analysis. The gold standard for diagnosing SARS-CoV-2 is the method of RT quantitative polymerase chain reaction (RT-qPCR) due to its exceptional accuracy, ease of implementation, and sensitivity in detecting and quantifying viral RNA. Despite these advantages, very low virus concentrations can lead to false-negative results. On the other hand, the method of RT digital polymerase chain reaction (RT-dPCR) allows for absolute quantification and detection of the virus at very low concentrations, which is crucial in the early stages of infection. In this master's thesis, we performed analyses using RT-qPCR and RT-dPCR methods on two types of samples: (1) synthetic segments of SARS-CoV-2 virus containing characteristic virus sequences, and (2) genomic RNA (gRNA) isolated from the SARS-CoV-2 grown on cell cultures. For this purpose, we used various commercially available single-step and two-step RT kits. The aim was to compare their effectiveness and accuracy in detecting SARS-CoV-2 specific nucleic acid sequences. As positive control for RT, we used gene for the luciferase enzyme (LUC), which is commonly used as a control for the presence of RT inhibitors in the reaction. We found that single-step kits are a better choice for both methods, both for segments and for gRNA, as we obtained comparable values for selected viral targets. With certain two-step kits, we obtained higher values for RNA segments compared to one-step kits, but at the same time, we observed high variability among the targets. The LUC control proved to be an inadequate choice, because of the resulting high and thus incomparable Cq values for both methods. We demonstrated that both methods are effective in detecting and quantifying viral RNA, however, there was significant variability in the results (Cq values) of the individual RT-qPCR methods, indicating that Cq values cannot be directly used for diagnostic purposes. We also demonstrated that the method of analysis and type of used kit, significantly influence the reliability and repeatability of the results, which is crucial for the effective diagnosis of the SARS-CoV-2 virus.

• Ključne besede: RT-qPCR, RT-dPCR, SARS-CoV-2