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- Title
PRIMERJAVA METOD ZA IZOLACIJO RNA Z NAMENOM DOLOČANJA VIROIDOV V HMELJU Z RT-PCR IN RT-qPCR.
- Authors
GUČEK, Tanja; JAKŠE, Jernej; RADIŠEK, Sebastjan
- Abstract
RT-PCR and RT-qPCR (real-time RT-PCR) methods are routinely used to determine viroids in hops, allowing rapid, reliable, and sensitive detection. The main limitation is RNA isolation, which can result in false negative results, due to low viroid concentration or the presence of PCR inhibitors. Six commercial RNA extraction reagents (Sigma-Aldrich TRIzol, SpectrumTM Plant Total kit, New England Biolabs (NEB) Monarch Total RNA Miniprep kit, Macherey-Nagel (MN) NucleoSpin® RNA Plus kit, Bioline ISOLATE II RNA Plant Kit, Qiagen RNeasy® Plant Mini Kit) and total nucleic acids isolation method using CTAB reagent were compared for their ability to extract hop viroids. Infected (CBCVd + HLVd) and healthy hop plants, different tissue (leaves and cones) and homogenization methods (use of press and mortar) were tested. The concentration of the samples was measured and analyzed by RT-PCR and RT-qPCR. With RTPCR, the strongest signals were detected after CTAB isolation. In the case of the use of commercial extraction kits, only RNA was isolated, and samples isolated with Qiagen, NEB and MN kits, were amplified by RT-qPCR without non-specific signals. For less sensitive determination of viroids by RT-PCR, the use of CTAB isolation is sufficient. While in the case of testing samples with lower viroid concentrations, the use of commercially available reagents is recommended.
- Publication
Hmeljarski Bilten, 2020, Issue 27, p5
- ISSN
0350-0756
- Publication type
Article