Reverse Transcripion-Polymerase chain Reaction Mediated Detection and Identification of Tobacco mosaic virus- Egyption Strain
Reverse Transcripion-Polymerase chain Reaction Mediated Detection and Identification of Tobacco mosaic virus- Egyption Strain
Sahar. A. Shoman1 and B. A. Othman2
ABSTRACT
A procedure involving reverse transcription followed by polymerase chain reaction (RT-PCR) with availability of the database sequences (GenBank) provided a sensitive method for the detection or Tobacco mosaic virus- Egyptian strain (TMV-E). Total RNA extract of infected plant and RNA of purified TMV particles were subjected in this method with four degenerate and undegenerate primers. The primer pairs generated two specific PCR fragments of 3428 base pair (bp) and 3836 bp of the whole TMV genome. The intensity of these RT-PCR amplified products was estimated, and it indicated relatively to the amount of the virus in the infected plant, The specificity of amplification was verified using internal primers through nested—PCR (n-PCR) to demonstrate the specificity of this technique. the n-PCR-expected product (869bp) was sequenced, and this nucleotide sequence was analyzed using a software program. Sequence analysis of the amplified fragment revealed a high conserved sequence homology located at the 3'-terminal region of the 126KDa (ORF1) sequence of the published TMV strains. Detection of the TMV-E using RT-PCR raises the availability of species-specific primers for the TMV-E that will be helpful for diagnosis in mixed infection. In addition, it will be helpful in further molecular characterization of this strain.
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