Pappu H R, Rosales I M, Druffel K L
Department of Plant Pathology, Washington State University, Pullman 99164-6430.
Plant Dis. 2008 Apr;92(4):588-594. doi: 10.1094/PDIS-92-4-0588.
Iris yellow spot virus (IYSV) has spread rapidly in the United States and has become an important economic constraint to the production of both bulb and seed onion crops. Symptoms caused by IYSV may be confused with those caused by other fungal and bacterial pathogens and virus-specific, reliable, sensitive, and rapid detection methods would improve the diagnosis. Antiserum was produced to Escherichia coli-expressed nucleocapsid protein of IYSV and an indirect format of the enzyme-linked immunosorbent assay (ELISA) was developed. IYSV could be detected in onion tissue at dilutions of up to 1:1,000. An IYSV-specific primer pair was designed and used in a real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for the rapid detection of IYSV. Compared with standard RT-PCR, real-time RT-PCR was more rapid and sensitive. A commercially available RNA extraction kit and a total nucleic acid extraction method were compared for the quality of the templates obtained for use in real-time RT-PCR and there was no difference in limits of detection. Availability of ELISA- and PCR-based rapid and sensitive detection methods would facilitate accurate virus diagnosis and aid in better understanding of the epidemiology of the disease and in development of management strategies.
鸢尾黄斑病毒（IYSV）已在美国迅速传播，并已成为鳞茎洋葱和种子洋葱作物生产的重要经济制约因素。IYSV引起的症状可能与其他真菌、细菌病原体引起的症状相混淆，而病毒特异性、可靠、灵敏且快速的检测方法将改善诊断。针对IYSV在大肠杆菌中表达的核衣壳蛋白制备了抗血清，并开发了间接酶联免疫吸附测定（ELISA）方法。在洋葱组织中，IYSV稀释至1:1000时仍可被检测到。设计了一对IYSV特异性引物，并将其用于实时逆转录聚合酶链反应（RT-PCR）测定以快速检测IYSV。与标准RT-PCR相比，实时RT-PCR更快速、灵敏。比较了市售RNA提取试剂盒和总核酸提取方法用于实时RT-PCR的模板质量，检测限没有差异。基于ELISA和PCR的快速灵敏检测方法的可用性将有助于准确的病毒诊断，并有助于更好地了解该疾病的流行病学以及制定管理策略。
