Hopp H E, Hain L, Bravo Almonacid F, Tozzini A C, Orman B, Arese A I, Ceriani M F, Saladrigas M V, Celnik R, del Vas M
Instituto de Biología Molecular, CICV-INTA Castelar, Universidad de Buenos Aires, Argentina.
J Virol Methods. 1991 Jan;31(1):11-29. doi: 10.1016/0166-0934(91)90141-l.
cDNA clones of potato virus X (PVXcp strain), potato virus Y (PVYo strain), potato leaf roll virus (PLRV) and potato spindle tuber viroid (PSTV) were used separately or combined for the detection of the corresponding RNAs in extracts of infected plants. A general method for the rapid preparation of RNA extracts without use of organic solvents (i.e. phenol) was developed for this purpose. Plant extracts from a range of field, artificially inoculated germplasm genotypes, micro-propagated and protoplast samples, as well as vector insect extracts, were dot-blotted onto nylon or nitrocellulose membranes, subjected to sandwich nucleic acid hybridization with non-labelled specific single-stranded DNA probes followed by a biotin-labelled second step hybridization probe. Each probe was virus-specific but not strain-specific. Healthy or non-related plant extracts developed very faint or no signals. Sensitivity was tested by slot-blot hybridization. Detection levels were between 1.5 to 6 pg of viral nucleic acids and between 20 to 50 times more sensitive than standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The assay developed was tested with material that was prepared for processing in the field (combination of fresh sap with extraction solution) and tested under simple laboratory conditions for detection. It was also successfully employed for screening of germplasm for virus resistance, detection of pathogens in vector insects, plantlets grown in vitro and in more sophisticated quantitative determinations of viral replication in artificially inoculated plants and protoplasts.
马铃薯X病毒（PVXcp株系）、马铃薯Y病毒（PVYo株系）、马铃薯卷叶病毒（PLRV）和马铃薯纺锤块茎类病毒（PSTV）的cDNA克隆分别或联合用于检测受感染植物提取物中的相应RNA。为此开发了一种无需使用有机溶剂（即苯酚）快速制备RNA提取物的通用方法。将一系列田间、人工接种的种质基因型、微繁殖和原生质体样品的植物提取物以及载体昆虫提取物点样到尼龙或硝酸纤维素膜上，先用未标记的特异性单链DNA探针进行夹心核酸杂交，然后用生物素标记的第二步杂交探针进行杂交。每个探针都是病毒特异性的，但不是株系特异性的。健康或不相关的植物提取物产生的信号非常微弱或没有信号。通过狭缝杂交检测灵敏度。检测水平在1.5至6 pg病毒核酸之间，比标准双抗体夹心酶联免疫吸附测定（DAS-ELISA）灵敏20至50倍。所开发的检测方法用为田间处理制备的材料（新鲜汁液与提取液的混合物）进行测试，并在简单的实验室条件下进行检测。它还成功地用于筛选抗病毒种质、检测载体昆虫中的病原体、体外培养的幼苗，以及在人工接种的植物和原生质体中更精确地定量测定病毒复制。
