Stevens M, Hull R, Smith H G
IACR-Broom's Barn, Edmunds, Suffolk, UK.
J Virol Methods. 1997 Oct;68(1):9-16. doi: 10.1016/s0166-0934(97)00103-1.
A reverse-transcription polymerase chain reaction (RT-PCR) was developed to detect beet yellows closterovirus (BYV) in plants and single aphids using primers spanning the conserved regions of the published sequences of the coat protein gene and open reading frames 7 and 8. Three total RNA extraction procedures were examined and all were found to produce RNA of sufficient quality for RT-PCR, although the RNA extraction kit supplied by Flowgen was found to be the most versatile for the extraction of BYV from individual aphids. When 60 aphids, which had fed on virus infected sugar beet were tested by RT-PCR, 55% of individuals were found to contain BYV. Two groups of 36 individual aphids were tested by TAS-ELISA using a specific BYV monoclonal antibody; 53% gave positive absorbance values when a substrate amplification system was used, but none was found to contain virus when the system was replaced with the substrate p-nitrophenyl phosphate. An immunocapture RT-PCR method was shown to detect BYV regardless of whether the RNA had been extracted from the trapped particles or the reverse transcription and PCR mixtures were added to the wells containing intact particles. The RT-PCR method is now used to determine the numbers of BYV carrying aphids migrating into sugar-beet crops.
开发了一种逆转录聚合酶链反应（RT-PCR），用于检测植物和单个蚜虫中的甜菜黄化线形病毒（BYV），所用引物跨越衣壳蛋白基因以及开放阅读框7和8的已发表序列的保守区域。研究了三种总RNA提取方法，发现所有方法都能产生质量足以用于RT-PCR的RNA，不过发现Flowgen提供的RNA提取试剂盒在从单个蚜虫中提取BYV方面用途最广。当对60只取食了感染病毒的甜菜的蚜虫进行RT-PCR检测时，发现55%的个体含有BYV。使用特异性BYV单克隆抗体通过TAS-ELISA对两组36只个体蚜虫进行检测；使用底物扩增系统时，53%的蚜虫给出了阳性吸光度值，但当该系统替换为对硝基苯磷酸底物时，未发现有蚜虫含有病毒。免疫捕获RT-PCR方法显示，无论RNA是从捕获的颗粒中提取，还是将逆转录和PCR混合物添加到含有完整颗粒的孔中，都能检测到BYV。现在，RT-PCR方法用于确定迁入甜菜作物的携带BYV的蚜虫数量。
