RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template

RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template 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 Alu I or Taq I digestion. The amplified fragments of TSWV were differentiated from those of INSV by Dra I or Hin dIII 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Virological Methods Elsevier

RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template

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Publisher
Elsevier
Copyright
Copyright © 2001 Elsevier Science B.V.
ISSN
0166-0934
eISSN
1879-0984
DOI
10.1016/S0166-0934(01)00321-4
Publisher site
See Article on Publisher Site

Abstract

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 Alu I or Taq I digestion. The amplified fragments of TSWV were differentiated from those of INSV by Dra I or Hin dIII 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.

Journal

Journal of Virological MethodsElsevier

Published: Aug 1, 2001

References

  • Detection of Tospovirus species by RT-PCR of the N-gene and restriction enzyme digestions of the products
    Dewey, R.A.; Semorile, L.C.; Grau, O.
  • Watermelon bud necrosis tospovirus is a distinct virus species belonging to serogroup IV
    Jain, R.K; Pappu, S.S.; Pappu, H.R.; Reddy, M.K.; Vani, A.
  • Comparison of the S RNA segments among Japanese isolates and Taiwanese isolates of Watermelon silver mottle virus
    Okuda, M.; Taba, S.; Tsuda, S.; Hidaka, S.; Kameya-Iwaki, M.; Hanada, K.
  • Viral diseases causing the greatest economic losses to the tomato crop. I. The Tomato spotted wilt virus-a review
    Rosello, R.; Diez, M.J.; Nuez, F.

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