Detection of Tospovirus species by RT-PCR of the N-gene and restriction enzyme digestions of the products.
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.
Dewey
R A
RA
Instituto de Bioquímica y Biología Molecular, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina.
Semorile
L C
LC
Grau
O
O
D001483:Base Sequence; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017865:Tospovirus / Q000235:genetics / Q000302:isolation & purification*; D014158:Transcription, Genetic
D017931:DNA Primers; D004262:DNA Restriction Enzymes; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C024400:GTYRAC-specific type II deoxyribonucleases