Detection of potato leafroll virus in single aphids by the reverse transcription polymerase chain reaction and its potential epidemiological application.
A reverse transcription and polymerase chain reaction (RT-PCR) system was developed using two 20-mer primers located in the potato leafroll virus (PLRV) capsid gene. A 336-bp PCR product was detected from aphids (Myzus persicae) which had been fed on PLRV-infected plants. The PCR band was specific to PLRV as determined by Southern blots and detection by a PLRV-specific probe. As little as 5 min exposure of aphids to PLRV-infected leaves resulted in the presence of PLRV-specific bands in 13% of aphids. However, the percentage of PLRV-positive aphids increased with longer exposure to infected sources and reached 90% after 3-4 days of feeding. PLRV can be detected from a single viruliferous aphid or a single viruliferous aphid combined with up to 29 non-viruliferous aphids. PLRV can be detected from freshly collected aphids, those stored at -70 degrees C, or those stored in 70% ethanol at room temperature for extended periods. This method is applicable to assess the viruliferous nature of aphids caught in yellow-pan traps during the growing season or stored for over a year.
Singh
R P
RP
Agriculture & Agri-Food Canada, Research Centre, Fredericton, New Brunswick, Canada.
Kurz
J
J
Boiteau
G
G
Bernard
G
G
D000818:Animals; D001042:Aphids / Q000821:virology*; D001483:Base Sequence; D017931:DNA Primers; D004812:Epidemiologic Methods; D005069:Evaluation Studies as Topic; D005240:Feasibility Studies; D017793:Luteovirus / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010935:Plant Diseases; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis; D011198:Solanum tuberosum / Q000821:virology; D013997:Time Factors; D014158:Transcription, Genetic
D017931:DNA Primers; D012367:RNA, Viral