Development of universal primers for detection of potato carlaviruses by RT-PCR

文献类型： 外文期刊

第一作者： Nie, Xianzhou

作者： Nie, Xianzhou;Bai, Yanju;Molen, Teresa A.;Desjardins, David C.;Bai, Yanju

作者机构：

关键词： potato carlaviruses;RT-PCR;universal degenerate primers

期刊名称：JOURNAL OF VIROLOGICAL METHODS （ 影响因子：2.014； 五年影响因子：2.001 ）

ISSN： 0166-0934

年卷期： 2008 年 149 卷 2 期

页码：

收录情况： SCI

摘要： To facilitate efficient and accurate detection of potato-infecting carlaviruses, degenerated universal primers were designed based on conserved amino acid and nucleotide sequences. Two senseprimers, Car-F1 and Car-F2, were based on the amino acid sequences "SNNMA" and "GLGVPTE", respectively, in the coat protein. The reverse primer, Car-R, which was located at the border of the nucleic acid binding protein gene and the 3' untranslated region, and dT-B, which was derived from the oligo-dT targeting the poly(A) tail, were selected. Successful application of fragments within the predicted size range of carlaviruses was obtained using Car-F1 paired with either Car-R or dT-B from tested carlaviruses (Potato virus S, M and latent) by RT-PCR. The Car-F2 failed to yield clear-cut fragments within the predicted size range when paired with either Car-R or dT-B in RT-PCR. However, a less degenerated version of the primer, Car-F2b, resulted in amplicons within the predicted size range when paired with either Car-R or dT-B. Sequencing of the tentative carlavirus-fragments resulting from Car-F1/Car-R and Car-F2b/dT-B proved their carlavirus-origin, thus indicating the high specificity of these primers. The sensitivity of Car-F1/Car-R or Car-F2b/Car-R mediated RT-PCR for the detection of carlavirus-infected potato tubers were assessed using composite samples containing one carlavirus-infected-potato-tuber RNA sample with up to 49 virus-free-potato-tuber RNA samples under the optimal annealing temperature. The target carlaviruses were detected readily from all composites, demonstrating a high sensitivity. The method was further evaluated using presumed virus-free or carlavirus-infected potatoes of several cultivars, and reliable results were obtained. Crown Copyright (c) 2008 Published by Elsevier B.V. All rights reserved.

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