Specific differentiation of recombinant PVYN:O and PVYNTN isolates by multiplex RT-PCR
作者： Xianzhou Nie , Rudra P. Singh
DOI: 10.1016/S0166-0934(03)00221-0
关键词:
摘要: The recombinant isolates of tobacco veinal necrotic strain Potato virus Y (PVYN) and potato tuber group (PVY(NTN)) contain segments the PVYO PVY(N) genome. Three major junctions (RJ) are present in genome PVY(NTN) at sites HC/Pro-P3, 6K2-NIa, C-terminal region CP gene one RJ HC/Pro-P3 site some PVYN (termed PVY(N:O)). Protocols for specific differentiation PVY(N:O) from non-recombinant described. Specific primer pairs were designed to target three RJs so that sense antisense primers completely matched nucleotide sequences either side RJ. In a uniplex reverse transcription-polymerase chain reaction (RT-PCR), first pair amplified fragment 641bp PVY(N:O). second third exclusively fragments 448 290bp, respectively PVY(NTN). multiplex (triplex) RT-PCR, when all used simultaneously, (641, 290bp) PVY(NTN), while only (641bp) was isolates, clearly differentiating two isolates. No amplification observed PVY, including North American (NA)-PVY(N/NTN). For further improvement effects cDNA preparation using primers, random or oligo(dT) plus investigated. prepared by increased overall band intensity.
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