Articles
DETECTION OF APPLE STEM PITTING VIRUS AND PEAR VEIN YELLOWS VIRUS USING REVERSE TRANSCRIPTION – POLYMERASE CHAIN REACTION
Article number
472_7
Pages
87 – 96
Language
Abstract
Five sets of RT-PCR primers were designed based on the published nucleotide sequence of ASPV RNA. Two methods of preparation of nucleic acids from apple and pear tissues were applied: immunocapture of virus particles with specific antibodies and silicacapture of total nucleic acids.
Four primer pairs in four combinations: ASPF1-R2, ASPF6-R5, ASPF6-R2 and ASPF1-R5 initiated amplification of specific fragments (203, 585, 497 and 291 bp, respectively) from infected apple and pear tissues.
The best amplification results were usually obtained with primer pair ASPF1-R5. Nested PCR amplification, with ASPF6-R5 primers in the first round of PCR and ASPF1-R2 primers in the second one, was succesful although more laborious than standard PCR. It was possible to detect ASPV in the shoots of apple plants maintained in vitro. The amplified cDNA fragments of ASPV and PVYV were cloned into pCR-Script SK + vector and sequenced.
The sequences showed high homology with a published RNA sequence of ASPV. Few mismatches were observed in fragments corresponding to primers ASPF1 and ASPR2. It was also possible to obtain specific PCR products with ASPF8-R2 and ASPF8-R5 primers using cloned cDNA as template.
We expect that the developed assay can be useful for fast and sensitive detection of ASPV (PVYV), especially after testing the primers against a wider range of virus isolates.
Four primer pairs in four combinations: ASPF1-R2, ASPF6-R5, ASPF6-R2 and ASPF1-R5 initiated amplification of specific fragments (203, 585, 497 and 291 bp, respectively) from infected apple and pear tissues.
The best amplification results were usually obtained with primer pair ASPF1-R5. Nested PCR amplification, with ASPF6-R5 primers in the first round of PCR and ASPF1-R2 primers in the second one, was succesful although more laborious than standard PCR. It was possible to detect ASPV in the shoots of apple plants maintained in vitro. The amplified cDNA fragments of ASPV and PVYV were cloned into pCR-Script SK + vector and sequenced.
The sequences showed high homology with a published RNA sequence of ASPV. Few mismatches were observed in fragments corresponding to primers ASPF1 and ASPR2. It was also possible to obtain specific PCR products with ASPF8-R2 and ASPF8-R5 primers using cloned cDNA as template.
We expect that the developed assay can be useful for fast and sensitive detection of ASPV (PVYV), especially after testing the primers against a wider range of virus isolates.
Authors
T. Malinowski, B. Komorowska, T. Golis, B. Zawadzka
Keywords
ASPV, PVYV, RT-PCR
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