USE OF RT-PCR TECHNIQUES IN DETECTION OF VIRUSES OF PEPPER IN REPUBLIC OF MACEDONIA

Апстракт

Plant viruses can be a limiting factor and cause economically significant damage in the agricultural production. That is why, early and precise detection is very important in prevention of significant yield losses. In this study, early detection of plant viruses was performed on pepper, as one of the economically most important crops in Republic of Macedonia. Pepper plants were tested for Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV) and Potato virus Y (PVY), most common RNA viruses in pepper production. Virus antigen accumulation was measured with the use of DAS-ELISA method on 259 plant samples. CMV was detected as most prevalent virus on pepper (51% in 2012, 34% in 2013 and 61% in 2014). In 2012, AMV was detected as second, while in 2013 and 2014 was PVY. Detection was performed using Reverse Transcription – Polymerase Chain Reaction (RT-PCR).
The RNA was extracted using TRIzol method from the collected leaf samples. RT-PCR was performed for each of the viruses separately due to the differences in the annealing temperatures of the used primers. In order to prevent RNA degradation of the plant viruses, each step of manipulation with the plant material was carefully optimized. The gained amplicons after RT-PCR, associated with different viruses were: for CMV the amplicon was 773 bp, for AMV was 669 bp, while for PVY 902 bp, respectively. The RT-PCR method exhibited high sensitivity, detecting the viruses in the early stages of infection, before observation of any visible symptoms on the pepper plants and measuring negative OD absorbance by DAS-ELISA.