Usporedba kvantifikacije DNA virusa Epstein-Barr primjenom dvaju testova lančane reakcije polimerazom u stvarnom vremenu

master's thesis

• Title (english): Comparison of Epstein-Barr virus DNA quantification using two real-time polymerase chain reaction assays
• Author: Ivana Listeš
• Mentor: Snježana Židovec Lepej (mentor)
• Committee member: Snježana Židovec Lepej (predsjednik povjerenstva)
• Committee member: Petra Korać (član povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Committee member: Sandra Hudina (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2024-02-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Virus Epstein-Barr (EBV) onkogeni je virus koji pripada porodici Orthoherpesviridae i rodu Lymphocryptovirus čiji se genom sastoji od dvolančane molekule DNA koja sadrži oko 172 kb. Virus EBV umnožava se u epitelnim stanicama, B-limfocitima, T-limfocitima i NK-stanicama, a opisana su dva glavna tipa replikacijskog ciklusa EBV-a; latentni i litički ciklus. Cilj ovog istraživanja bio je usporediti broj kopija DNA EBV-a u uzorcima periferne krvi određenih primjenom dvaju standardiziranih kvantitativnih PCR testova u stvarnom vremenu te utvrditi jesu li rezultati procjene kinetike virusne replikacije usporedivi i primjenjivi u biološkim i biomedicinskim istraživanjima. Rezultati detekcije DNA EBV-a u 100 uzoraka periferne krvi qPCR testovima u stvarnom vremenu LightMix LightCycler 2.0 i LightMix LightCycler 480 II s adaptiranim protokolom bili su podudarni u 99 od 100 uzoraka (Kohenov kappa koeficijent 0,98 uz 95% interval pouzdanosti 0,94-1,00). Rezultati usporedbe kvantifikacije DNA EBV-a pokazali su statistički značajnu razliku u viremijama (medijan 49 400 vs. 128 000 kopija DNA EBV-a, p<0.001). Rezultati ovog istraživanja pokazuju da se praćenje kinetike replikacije virusa EBV u biomedicinskim istraživanjima obvezno mora temeljiti na primjeni identičnog standardiziranog molekularnog testa u kompletnom istraživanju te da rezultati između dostupnih standardiziranih testova nisu u potpunosti usporedivi.

Abstract (english)

Epstein-Barr virus (EBV) is an oncogenic virus that belongs to the family Orthoherpesviridae, genus Lymphocryptovirus, and has a double-stranded DNA genome that consists of 172 kb. The main cellular targets for viral replication via latent and lytic cycles include epithelial cells, B-lymphocytes, T-lymphocytes and NK-cells. The aim of this study was to compare the number of EBV DNA copies in peripheral blood samples determined by using two standardized real-time quantitative PCR test and to determine whether the results of the viral replication kinetics assessment are comparable and applicable in biological and biomedical research. EBV DNA detection in 100 peripheral blood samples by using 2 real-time PCR assays (LightMix LightCycler 2.0 and LightMix LightCycler 480 II with adapted protocol) showed concordant results in 99 of 100 samples (Kohen's kappa coefficient 0,98, 95% confidence interval 0,94-1,00). Comparison of EBV DNA quantification results obtained by the two assays has shown a statistically significant difference in measured viremias (median 49 400 vs. 128 000 EBV DNA copies/ml, p< 0.001). The results of this study have shown that monitoring of EBV quantification in biomedical research needs to be based on consistent use of identical molecular assays and that the results of standardised assays available today are not completely comparable.

• Keywords: EBV; viremija; real-time PCR
• Keywords (english): EBV; viremia; real-time PCR
• Language: croatian
• URN:NBN: urn:nbn:hr:217:180479
• Project: Number: IP-2020-02-8961; Title: Molekularna raznolikost virusa Epstein-Barr; Acronym: MRVEB; Leader: Snježana Židovec Lepej; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: Istraživački projekti 2020.g
• Study programme: Title: Molecular Biology-University graduate programme; Study programme type: university; Study level: graduate; Academic / professional title: sveučilišni/a magistar/magistra molekularne biologije (sveučilišni/a magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-03-08 12:10:51