Large scale evaluation of primers for diagnosis of rupestris stem pitting associated virus-1
Large scale evaluation of primers for diagnosis of rupestris stem pitting associated virus-1
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DOI：
10.1023/a:1008748924494
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发表时间：
2000-05-01
影响因子：
1.8
通讯作者：
Sequeira, OA
中科院分区：
文献类型：
--
作者：
Nolasco, G;Mansinho, A;Sequeira, OA
由于缺乏足够的免疫学试剂，无法利用酶联免疫吸附试验（ELISA）诊断葡萄蔓茎痘病。本研究比较了5对引物对大规模RT-PCR检测岩芥茎痘相关病毒-1的效果，并与生物索引法进行了对比。该病毒在35个葡萄牙葡萄藤品种的芽木中广泛分布，患病率为85%。由于假阴性率很高，生物测定法作为感染指标被证明是不可靠的。对5组引物进行了检测，并对它们的相对灵敏度和阴性预测值进行了比较。由于难以鉴定特异性扩增产物，因此排除了外壳蛋白基因特异性引物对。从其他四个引物对，那些特异性的推定聚合酶基因的解旋酶结构域具有最高的灵敏度和阴性预测值。然而，仅使用该引物对无法获得认证方案所需的测定的高置信度。在单独或多重RT-PCR反应中应使用额外的对。
The unavailability of adequate immunological reagents has prevented the use of ELISA for the diagnosis of rupestris stem pitting disorder of grapevines. In this work, the performance of five primer pairs for broad-scale detection of rupestris stem pitting associated virus-1 by RT-PCR using ds-RNA templates was compared and contrasted with biological indexing. The virus was widespread among the budwood of 35 Portuguese grapevine varieties assayed, with a prevalence of 85%. The biological assay proved to be unreliable as an index of infection due to the high number of false negatives. Five sets of primers were assayed and compared by means of their relative sensitivity and negative predictive value. The primer pair specific for the coat protein gene was excluded because of the difficulty in identifying the specific amplified product. From the other four primer pairs, those specific for the helicase domain of the putative polymerase gene had the highest sensitivity and negative predictive value. However, a high confidence in the assay, as desirable for a certification scheme, could not be obtained by the sole use of this primer pair. An additional pair should be used in a separate or in a multiplex RT-PCR reaction.