English

Abstract

This study aimed at optimizing the immunocapture (IC) and simple-direct-tube (SDT) -reverse transcriptase polymerase chain reaction (RT-PCR) techniques for detection of Rice yellow mottle virus (RYMV) in order to avoid the extraction of high quality RNA required for molecular methods and avoid costs involved. Rice yellow mottle virus strains and phylotypes were obtained from infected rice leaf samples collected from Morogoro, Arusha and Kilimanjaro regions. The efficacy and sensitivity of IC and SDT methods was demonstrated using the aliquots from infected plant sap obtained by grinding rice leaves and binding onto PCR tube using coating buffer and in phosphate buffer saline with 0.5% Tween-20 (PBST 1X), respectively, and assayed by RT-PCR with RYMVIIIF/RYMVIIR primers. Analysis of the PCR product was performed by electrophoresis on 1% agarose gel, pre-stained with 2.5 μl of ethidium bromide (10 µg of ethidium bromide per ml of 0.5x Tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer) at 100 V cm-1 for 30 min and visualized under UV light. The results indicate that SDT-RT-PCR and IC-RT-PCR detected RYMV in all tested infected leaf samples at the expected band size of 720 bp and had the same sensitivity as virus extraction RNA-RT PCR technique, implying that the methods can be useful for detection of wide range of RYMV strains. The negative control did not yield any amplicons. The results also show that these techniques are rapid methods for characterization of RYMV strains and may be recommended for use soon after periodical surveys to quickly identify new strains for breeding purposes at low cost. However, SDT protocol was easier and faster than IC and it was also cost-effective in terms of reagents for the detection of RYMV.   Key words: Rice yellow mottle virus, detection, immunocapture-RT-PCR, simple-direct-tube-RT-PCR.