Real-time assay for quantitative detection of non-persistently transmitted Plum pox virus RNA targets in single aphids.
A TaqMan real-time RT-PCR was developed to detect and quantify RNA-targets from the non-circulative, non-persistently transmitted Plum pox virus (PPV) in individual fresh or aphids captured previously and squashed on paper. Reliable quantitation ranged from 40 up to 4 x 10(8) copies of control transcripts. This technique was applied successfully to plant material and to individual PPV vector (Myzus persicae) and non-vector of PPV (Aphis nerii) aphid species demonstrating acquisition of viral targets by both vector and non-vector aphids. The number of viruliferous aphids detected by real-time RT-PCR and nested RT-PCR in a single closed tube was similar in parallel assays, nevertheless the sensitivity provided by real-time RT-PCR was 100 times higher than nested RT-PCR and 1000 times higher than DASI-ELISA and conventional RT-PCR. The quantities of PPV-RNA targets detected in a single aphid ranged from 40 to more than 2 x 10(3) units. The combined system (immobilization of targets on paper by squash capture and real-time RT-PCR) allows, for the first time, reliable quantitation of PPV targets acquired by individual aphid species and constitute an excellent tool for understanding better PPV epidemiology.
Olmos
Antonio
A
Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera km 5, 46113 Moncada, Valencia, Spain.
Bertolini
Edson
E
Gil
Maite
M
Cambra
Mariano
M
D000818:Animals; D001042:Aphids / Q000821:virology*; D010935:Plant Diseases / Q000821:virology; D010936:Plant Extracts / Q000032:analysis; D017801:Plum Pox Virus / Q000235:genetics / Q000302:isolation & purification*; D027861:Prunus / Q000821:virology; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity; D014026:Nicotiana / Q000821:virology
D010936:Plant Extracts; D012367:RNA, Viral