Okuda M, Hanada K
Kyushu National Agricultural Experiment Station, Nishigoshi-Machi, 861-1192, Kumamoto, Japan.
J Virol Methods. 2001 Aug;96(2):149-56. doi: 10.1016/s0166-0934(01)00321-4.
RT-PCR procedures for detection of multiple species of tospovirus from plant tissues were investigated. Downstream primers were designated from the 3' untranslated sequences of the S RNA. An upstream primer was designated from the degenerated sequences of the nucleocapsid protein. Approximately 450 bp DNA fragments were detected when Tomato spotted wilt virus (TSWV)- or Impatiens necrotic spot virus (INSV)- infected tissues were examined. Approximately 350 bp DNA fragments were detected when Watermelon silver mottle virus (WSMoV)- or Melon yellow spot virus (MYSV)-infected tissues were examined. Template RNA was extracted using CF 11 cellulose powder, and nonspecific amplification became unnoticeable when double-stranded RNA was used. The amplified fragments of WSMoV were differentiated from those of MYSV by AluI or TaqI digestion. The amplified fragments of TSWV were differentiated from those of INSV by DraI or HindIII digestion. An alstroemeria plant that was infected with an unidentified tospovirus was also examined, and a 350 bp fragment that was 97% identical to Iris yellow spot virus was detected. This method, therefore, detected at least five distinct Tospovirus species.
对用于从植物组织中检测多种番茄斑萎病毒属病毒的RT-PCR程序进行了研究。下游引物是根据S RNA的3'非翻译序列设计的。上游引物是根据核衣壳蛋白的简并序列设计的。当检测感染番茄斑萎病毒（TSWV）或凤仙坏死斑病毒（INSV）的组织时，检测到约450 bp的DNA片段。当检测感染西瓜银斑驳病毒（WSMoV）或甜瓜黄斑病毒（MYSV）的组织时，检测到约350 bp的DNA片段。使用CF 11纤维素粉末提取模板RNA，当使用双链RNA时，非特异性扩增变得不明显。WSMoV的扩增片段通过AluI或TaqI消化与MYSV的扩增片段区分开来。TSWV的扩增片段通过DraI或HindIII消化与INSV的扩增片段区分开来。还检测了一株感染未鉴定番茄斑萎病毒属病毒的六出花植株，并检测到一个与鸢尾黄斑病毒97%相同的350 bp片段。因此，该方法检测到至少五种不同的番茄斑萎病毒属病毒。
