Verifikacija komercialnega sestava za hitro detekcijo variant omikron SARS-Cov-2

Halilović, Edin (author), Jeras, Matjaž (mentor), Ćelap, Ivana (comentor)

Od odkritja leta 2019 pa do konca leta 2021 so pri povzročitelju bolezni COVID-19, virusu SARS-CoV-2, potrdili na tisoče različnih mutacij, zaradi katerih so nastale njegove različice, ki vzbujajo skrb zaradi pospešenega širjenja in potencialno hujše klinične slike same bolezni. Ena zadnjih tovrstnih, nevarnih različic je različica omikron. Vsake različice SARS-CoV-2 seveda ne moremo zaznati s standardno metodo RT-PCR, sekvenciranje virusne RNA pa je drago in časovno precej zahtevno. Cilj magistrskega dela je bila verifikacija komercialnega sestava NovaplexTM SARS-CoV-2 Variants VII Assay in njegova primerjava z referenčno metodo sekvenciranja, za zaznavanje različic SARS-CoV-2, z namenom hitrejšega ugotavljanja širjenja posameznih različic virusa v populaciji. V raziskavi smo uporabili vzorce RNA 43 bolnikov z, v obdobju od 1. 12. 2021 do 30. 1. 2022 ugotovljeno prisotnostjo virusa SARS-CoV-2, in sicer z metodo RT-PCR v obliki komercialnega sestava TaqPath␢ COVID-19 CE-IVD RT-PCR. Izbrane vzorce smo analizirali s testom NovaplexTM SARS-CoV-2 Variants VII, obenem pa smo jih poslali še na sekvenciranje na Hrvaški zavod za javno zdravstvo. Pričakovali smo visoko stopnjo primerljivosti rezultatov (> 95 %), pridobljenih s testiranim reagenčnim kompletom in z referenčno metodo, glede na to, da je prvi namenjen prav zaznavanju različic omikrona. Poleg tega smo preverili tudi zanesljivost kompleta TaqPath␢ COVID-19 CE-IVD RT-PCR kot presejalne metode za odkrivanje različic omikron BA.1 in BA.2, saj je ta namenjen zaznavi gena S virusa SARS-CoV-2, ki pa pri varianti BA.1, zaradi mutacije, ni več sposoben pomnoževanja. S testiranjem smo dejansko ugotovili 95-odstotno primerljivost rezultatov, pridobljenih s testiranim reagenčnim kompletom in z referenčno metodo sekvenciranja. Prav tako smo potrdili, da lahko komercialni komplet TaqPath␢ služi kot presejalni test za različico omikron BA.1, za katero je značilen izpad pomnoževanja gena S, obenem pa ugotovili, da ni uporaben za presejalno testiranje na prisotnost različice omikron BA.2 in njene podvariante BA.2.3, saj se pri njiju obeh v tem reagenčnem kompletu poteče podvajanje gena S.

• Language: Slovenian
• Keywords: virus SARS-CoV-2, varianta omikron, pomnoževanje z verižno reakcijo s polimerazo v realnem času (RT-PCR), sekvenciranje
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-150944
• Publication date in RUL: 26.09.2023

Secondary language

• Language: English
• Title: Verification of a commercial kit for rapid omicron SARS-Cov-2 variants detection

Abstract

From the discovery in 2019 until the end of 2021, thousands of different mutations have been confirmed in the causative agent of the disease COVID-19, the SARS-CoV-2 virus, from which variants of the virus have arisen, which cause concern due to their rapid spread and potentially more severe clinical presentation. One of the last variants causing concern is the omicron variant. RT-PCR cannot differentiate SARS-CoV-2 variants, and the method of choice for variant detection, sequencing, is expensive and time-consuming. The goal of the master's thesis was the verification of the commercial NovaplexTM SARS-Cov-2 Variants VII Assay kit by comparison with the reference method for detecting SARS-CoV-2 variants, i.e. sequencing, with the aim of faster detection of variants of interest and its spread in the population. In the research, we used the remaining RNA samples (43 samples) from patients in whom the presence of the SARS-CoV-2 virus was detected in the period from 1.12.2021 until 30.1.2022 by RT-PCR method using the commercial TaqPath␢ COVID-19 RT-PCR kit. The selected samples were then analyzed using the NovaplexTM SARS-CoV-2 Variants VII Assay and were sent for sequencing to the Croatian Institute of Public Health. We expected a high degree of comparability of the tested kit with the reference method (95%), given that the kit was designed to detect mutations specific to the omicron variant. In addition, we wanted to verify the reliability of the TaqPath␢ kit as a screening method for the detection of BA.1 and BA.2 omicron variants, given that the kit detects the S gene of the SARS-CoV-2 virus, which in the BA.1 omicron variant results in the drop-out of S gene (inability to reproduce). Through testing, we found a high degree of comparability of the tested kit with the reference method, i.e. sequencing of 95%. Through testing, we also determined that the commercial TaqPath␢ kit can serve as a screening test for the Omicron BA.1 variant because there was drop-out of the S gene, but it cannot serve as a screening test for the Omicron variant BA.2 and subvariant BA.2.3, because they have duplication of the S gene on the same kit.

• Keywords: SARS-CoV-2, omicron SARS-CoV-2, RT-PCR, Novaplex, TaqPath␢, sequencing