Molekularna detekcija virusa šarenila agruma

master's thesis

• Title (english): Molecular detection of citrus variegation virus
• Author: Željka Sarađen
• Mentor: Dijana Škorić (mentor)
• Committee member: Dijana Škorić (član povjerenstva)
• Committee member: Renata Šoštarić (član povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2011, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Cilj ovog rada bio je utvrditi prisutnost virusa šarenila agruma (Citrus variegation virus, CVV) izravno iz uzoraka s terena i njegovu eventualnu rasprostranjenost u Hrvatskoj primjenom suvremenih laboratorijskih metoda utemeljenih na otkrivanju virusnog genoma. Pri tome su obuhvaćeni uzorci različitih tipova agruma, od komercijalnih nasada, obiteljskih vrtova do kolekcijskog nasada agruma Instituta za jadranske kulture i melioraciju krša u Splitu. Provedena molekularna detekcija CVV-a u uzorcima tkiva izravno s terena bila je prvo testiranje ove vrste kod nas te je bilo potrebno pronaći najprikladnije metode i optimizirati protokole. Za detekciju gena kapsidnog proteina CVVa primijenjena je molekularna metoda RT-PCR i IC/RT-PCR, dok su za izolaciju ukupnih nukleinskih kiselina primijenjene 2 metode, RNeasy Plant Mini Kit i izolacija prema protokolu Bennani i sur. (2002). Kao prihvatljiva i ekonomična metodologija za dokazivanje gena za kapsidni protein ovog virusa pokazao se postupak izolacije nukleinskih kiselina prema navedenom protokolu nakon kojeg slijedi RT-PCR. Elektroforetske analize amplikona provedene su uspješno u jeftinijem puferskom sustavu koji ima manji utjecaj na okoliš (Brody i Kern, 2004). Niti jedan testirani uzorak agruma nije pokazao prisutnost CVV-a primjenom navedene metodologije.

Abstract (english)

The purpose of this study was to determine the presence of Citrus variegation virus (CVV) from field samples and its possible distribution in Croatia using laboratory methods based on the detection of viral capsid protein gene. Citrus samples of various origines were included: from commercial orchards, gardens and citrus collection planting of the Institute for Adriatic Crops and Karst Reclamation in Split. Conducted molecular detection of CVV was the first study of that kind in our country and it was necessary to find the most appropriate detection methods and optimize the protocols. For the detection of CVV molecular methods RT-PCR and IC/RT-PCR were applied, while for the nucleic acids isolation RNeasy Plant Mini Kit and the procedure described in Bennani et al. (2002) were evaluated. The methodology using extraction procedure by Bennani et al. including the amplification of CVV coat protein gene by RT-PCR proved to be adequate and cost effective method for this purpose. Additional improvements were made by introducing. Tris-free buffer system (Brody and Kern, 2004) that is also safer for the environment. All tested samples of citrus collected here were negative for the presence of CVV.

• Keywords: CVV; agrumi; RT-PCR; IC/RT-PCR
• Keywords (english): CVV; citrus; RT-PCR; IC/RT-PCR
• Language: croatian
• URN:NBN: urn:nbn:hr:217:650552
• Study programme: Title: Biology; specializations in: Ecology; Course: Ecology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra biologije (magistar/magistra biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-04-10 12:57:49