Shamloul A M, Hadidi A
Fruit Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705, USA.
J Virol Methods. 1999 Jul;80(2):145-55. doi: 10.1016/s0166-0934(99)00038-5.
A rapid and sensitive assay for the specific detection of plant viroids using reverse transcription-polymerase chain reaction (RT-PCR) -probe capture hybridization (RT-PCR-enzyme-linked immunosorbent assay (ELISA)) was developed. The assay was applied successfully for the detection of potato spindle tuber viroid, peach latent mosaic viroid, or apple scar skin viroid from viroid infected leaf tissue. Clarified sap extract from infected leaf tissue was treated first with GeneReleaser polymeric matrix to remove inhibitors of RT-PCR reactions. Viroid cDNA was then synthesized and amplified using viroid specific primers in RT-PCR assays and the amplified viroid cDNA (amplicon) was digoxigenin (DIG) -labelled during the amplification process. The amplicon was then detected in a colorimetric hybridization system in a microtiter plate using a biotinylated cDNA capture probe. This system combines the specificity of molecular hybridization, the ease of the colorimetric protocol, and is at least 100-fold more sensitive than gel electrophoretic analysis in detecting the amplified product. Viroid cRNA may replace viroid cDNA as the capture probe. The cRNA probe was several fold more sensitive than the cDNA probe for viroid detection. Six to seven hours are needed to complete the RT-PCR-ELISA for viroid detection from infected leaf tissue.
开发了一种利用逆转录-聚合酶链反应（RT-PCR）-探针捕获杂交（RT-PCR-酶联免疫吸附测定（ELISA））对植物类病毒进行特异性检测的快速灵敏检测方法。该检测方法已成功应用于从感染类病毒的叶片组织中检测马铃薯纺锤块茎类病毒、桃潜隐花叶类病毒或苹果锈果类病毒。首先用GeneReleaser聚合基质处理感染叶片组织的澄清汁液提取物，以去除RT-PCR反应的抑制剂。然后在RT-PCR检测中使用类病毒特异性引物合成并扩增产类病毒cDNA，扩增的类病毒cDNA（扩增子）在扩增过程中用洋地黄毒苷（DIG）标记。然后使用生物素化的cDNA捕获探针在微量滴定板的比色杂交系统中检测扩增子。该系统结合了分子杂交的特异性、比色检测方法的简便性，并且在检测扩增产物方面比凝胶电泳分析至少灵敏100倍。类病毒cRNA可替代类病毒cDNA作为捕获探针。cRNA探针在检测类病毒方面比cDNA探针灵敏几倍。从感染叶片组织中检测类病毒完成RT-PCR-ELISA需要6至7小时。
