Development of a highly sensitive nested RT-PCR method for Beet necrotic yellow vein virus detection
Development of a highly sensitive nested RT-PCR method for Beet necrotic yellow vein virus detection
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DOI：
10.1016/s0166-0934(01)00308-1
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发表时间：
2001-06-01
影响因子：
3.1
通讯作者：
Henry, CM
中科院分区：
文献类型：
--
作者：
Morris, J;Clover, GRG;Henry, CM
建立了甜菜坏死黄脉病毒（Beet necrotic yellow vein virus，BNYVV）的逆转录聚合酶链反应（RT-PCR）和巢式聚合酶链反应（nPCR）检测方法。RT-PCR使用由（亨利等人. J. Virol.方法54（1995）15），但对方法进行了改进，包括简化提取方法、使用标准试剂和采用一步法。这些变化均未损害灵敏度或特异性。RT-PCR也可用于扩增免疫捕获的病毒，但其灵敏度略低于纯化RNA的扩增。在nPCR中，使用引物进行第二轮扩增。产生一种特异的326碱基对产物。RT-PCR和nPCR检测了从欧洲收集的21株分离株。美洲和亚洲（包括A. B和P致病型）。这两种检测都没有产生PCR产物，使用从健康甜菜或感染甜菜土传病毒的甜菜根中提取的总RNA。然而. nPCR的灵敏度是标准RT-PCR的1000倍。标准RT-PCR和nPCR的可靠性证明了使用一系列的品种从受感染的领域网站。对于需要提高灵敏度超过标准RT-PCR的应用，建议使用nPCR检测。用于早期检测来自诱饵测试土壤的感染以及检疫和繁殖目的。(C)2001 Elsevier Science B，V.保留所有权利。
A diagnostic test incorporating reverse-transcription polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (nPCR) was developed for the detection of Beet necrotic yellow vein virus (BNYVV). The RT-PCR used the primers designed by (Henry et al.. J. Virol. Methods 54 (1995)15) but refinements were made to the protocol including simplification of the extraction method, the use of standard reagents and adoption of a one-step procedure. None of these changes impaired sensitivity or specificity. The RT-PCR could also be used to amplify immunocaptured virus but this was slightly less sensitive than amplification from purified RNA. In nPCR, a second round of amplification was performed using primers. which produce a specific 326 base-pair product. Both RT-PCR and nPCR detected a range of 21 isolates collected From Europe. America and Asia (including A. B and P pathotypes) isolated from either sugar beet or Chenopodium quinoa. Neither assay produced PCR products using total RNA extracted from the roots of healthy sugar beet or beet infected with Beet soil-borne virus. However. the sensitivity of the nPCR was 1000 times greater than the standard RT-PCR. The reliability of the standard RT-PCR and nPCR was demonstrated using a range of cultivars collected from an infected field site. The use of the nPCR assay is recommended for applications where its improved sensitivity over standard RT-PCR is necessary. for trample in the early detection of infection from bait-test soils and for quarantine and breeding purposes. (C) 2001 Elsevier Science B,V. All rights reserved.