Detection of Tospovirus species by RT-PCR of the N-gene and restriction enzyme digestions of the products

J Virol Methods. 1996 Jan;56(1):19-26. doi: 10.1016/0166-0934(95)01896-4.

Abstract

Tomato spotted wilt is a serious disease that affects several economically important crops. From the epidemiological point of view and for the development of a successful plan for transgenic resistance plants, the four known Tospovirus species must be discriminated at the molecular level. A RT-PCR assay using primers complementary to the N gene was used to detect and differentiate fourteen Argentinian isolates of Tospovirus from different crops and geographical areas. Extracts were reverse transcribed using a thermo-resistant reverse transcriptase and PCR reactions were performed for 30 min in a capillar thermo-cycler. The products were digested with restriction enzymes and three of the four described species were identified. Additionally, the results were confirmed by DAS-ELISA. The method described here is rapid and reliable.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Primers
  • DNA Restriction Enzymes*
  • Deoxyribonucleases, Type II Site-Specific
  • Enzyme-Linked Immunosorbent Assay
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Tospovirus / genetics
  • Tospovirus / isolation & purification*
  • Transcription, Genetic

Substances

  • DNA Primers
  • DNA Restriction Enzymes
  • CCWGG-specific type II deoxyribonucleases
  • Deoxyribonucleases, Type II Site-Specific
  • GTYRAC-specific type II deoxyribonucleases