Specific differentiation of recombinant PVYN:O and PVYNTN isolates by multiplex RT-PCR
Specific differentiation of recombinant PVYN:O and PVYNTN isolates by multiplex RT-PCR
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DOI：
10.1016/s0166-0934(03)00221-0
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发表时间：
2003-11-01
影响因子：
3.1
通讯作者：
Singh, RP
中科院分区：
文献类型：
--
作者：
Nie, XZ;Singh, RP
马铃薯Y病毒的烟草脉坏死株系（PVYN）和马铃薯块茎坏死群（PVYNTN）的重组分离物含有PVYO和PVYN基因组的片段。在重组PVYN分离物的基因组中，在HC/Pro-P3位点、6 K2-NIa位点和CP基因的C-末端区域存在三个主要的重组接头（RJ），并且在一些重组PVYN分离物中在HC/Pro-P3位点存在一个RJ（称为PVYN：O）。描述了用于将重组PVYNTN和PVYN：0与非重组PVYN特异性区分的方案。设计特异性引物对以靶向三个RJ，使得正义和反义引物完全匹配RJ任一侧的核苷酸序列。在单重逆转录-聚合酶链反应（RT-PCR）中，第一对引物从重组PVYNTN和PVYN：O中扩增出641 bp的片段。第二对和第三对引物分别从重组PVYNTN中专门扩增出448和290 bp的片段。在多重（三重）RT-PCR中，当同时使用所有三个引物对时，三个片段（641、448和290 bp）仅从重组PVYNTN扩增，而仅一个片段（641 bp）从PVYN：O分离物扩增，清楚地区分两个重组分离物。未观察到来自非重组PVY（包括PVYO和北美（NA）-PVYN/NTN）的扩增。为进一步完善多重RT-PCR方法，研究了特异性反义引物、随机引物和寡聚脱氧核糖核酸（dT）加随机引物制备cDNA的效果。随机引物加oligo（dT）制备的cDNA增加了总的条带强度。(C)2003 Elsevier B. V.保留所有权利。
The recombinant isolates of tobacco veinal necrotic strain of Potato virus Y (PVYN) and potato tuber necrotic group (PVYNTN) contain segments of the PVYO and the PVYN genome. Three major recombinant junctions (RJ) are present in the genome of the recombinant PVTNTN at sites HC/Pro-P3, 6K2-NIa, and the C-terminal region of CP gene and one RJ at HC/Pro-P3 site in some recombinant PVYN isolates (termed PVYN:O). Protocols for specific differentiation of the recombinant PVYNTN and PVYN:O from the non-recombinant PVYN are described. Specific primer pairs were designed to target the three RJs so that sense and antisense primers completely matched the nucleotide sequences at either side of the RJ. In a uniplex reverse transcription-polymerase chain reaction (RT-PCR), the first primer pair amplified a fragment of 641 bp from the recombinant PVYNTN and PVYN:O. The second and third primer pairs exclusively amplified fragments of 448 and 290 bp, respectively from the recombinant PVYNTN. In a multiplex (triplex) RT-PCR, when all three primer pairs were used simultaneously, the three fragments (641, 448 and 290 bp) were amplified exclusively from the recombinant PVYNTN, while only one fragment (641 bp) was amplified from the PVYN:O isolates, clearly differentiating the two recombinant isolates. No amplification was observed from the non-recombinant PVY, including PVYO and North American (NA)-PVYN/NTN. For further improvement of the multiplex RT-PCR, effects of cDNA preparation using specific antisense primers, random primers or oligo(dT) plus random primers were investigated. The cDNA prepared by random primer plus oligo(dT) increased the overall band intensity. (C) 2003 Elsevier B.V. All rights reserved.