Specific PCR primers for GLRaVs 1 through 5 were designed, tested and optimized. One step RT-PCR methodology was also developed and the reagents and testing conditions were optimized.
A simple blotting methodology for sample collection in the field was developed. Leaf petioles or young shoots are cut and blotted on specific nylon or nitrocellulose membrane and then these membranes are brought to the laboratory for processing and virus detection. The advantages of this method are: no technical training is required, and if necessary, the samples can be stored for a long time before they are prepared and tested.
In order to develop a reliable RT-PCR detection method, it is very important to identify variations among different isolates (or strains) of each one of these GLRaVs and accordingly design PCR primers that could detect all diverse isolates. In the past year, sequences from the coat protein gene from 10 and 5 different isolates of grapevine leafroll associated virus (GLRaV) -1 and -5, respectively, were compared. The number of nucleotides which were compared from the coat protein gene were 680 and 786 nt for GLRaV-1 and -5 respectively. All GLRaVs-1 isolates used in this study showed 95-99%homology in their compared sequences indicative of a homogeneous population. We had the same observation with GLRaV-5 with again 95-99%homology.
Reliability and sensitivity of RT-PCR for the detection of GLRaVs-1 to -5 was compared with ELISA and with symptom expression of leafroll disease on its biological indicator. From 137 vines tested in this experiment, 26, 16, and 29, respectively tested positive on biological indicator, ELISA, and RT-PCR. The results indicate that the RT-PCR is more sensitive than either indexing on biological indicator host or ELISA. Counting RT-PCR test for GLRaV-5 in this experiment (did not have access to a reliable ELISA reagent for this virus), we found that total of 41 vines were tested positive by PCR which again outnumbers biological index on indicator (27 plants).