The development of molecular-biological system for the identification of RNA-1 of ukrainian isolates of beet necrotic yellow vein virus

Abstract

By using published data and genetic database of NCBI we searched nucleotide sequence of the gene encoding the polyprotein P237 and nucleotide sequences 3' – noncoding RNA-1 region for the bio-informative analysis and determination conserved sequences of RNA-1 of BNYVV.

         On the basis of analysis the consensus nucleotide sequence of RNA-1 was made and conservative and polymorphic fragments of genomic RNA-1 of BNYVV was shown. For appropriate primers design strictly conserved fragments were selected. The polymorphic genome regions were excluded from the analysis. This ensures a universal identification system of RNA-1 all existing and potential pathotypes isolates of BNYVV.

         On the matrix consensus sequence of P237 was created design primers with optimal characteristics: Forward 5’-AGCGGAATCAGTGGCAAGAA-3’, Reverse 5’-ACCATCATCGCCCTTCATGG-3’. Appropriate primers with optimal characteristics were synthesized, with optimum parameters of GC composition and similar temperature annealing of two primers. The PCR was performed using cDNA of Ukrainian isolate of BNYVV under the following conditions: initial denaturation 5 min – 94 ⁰C; 30 cycles: denaturation 30 sec – 94 ⁰C, annealing of primers 30 sec – 60 ⁰C, elongation 30 sec – 72 ⁰C, 72 ⁰C final synthesis – 7 min. As a result the visualization of PCR amplification products revealed fragments of size – 803 nucleotides pairs. An availability on the electrophoregram expected reaction product indicates that the developed system for the identification of RNA-1 of BNYVV is effective.

         On the next stage of the study we had to pick up the operating temperature annealing of primers. It was made series of reactions and multi-temperature annealing of primers from 50 ⁰C to 64 ⁰C. Although primers annealing temperature conditions were not significantly affected the efficiency of amplification reactions, we have found that the most optimal temperature is in the range of 50 ⁰C – 60 ⁰C. Under these conditions nonspecific amplification products were not observed and the amount of amplicons was sufficient for clear visualization.