Simultaneous detection and typing of plum pox potyvirus (PPV) isolates by heminested-PCR and PCR-ELISA.
Two techniques for simultaneous detection and typing of plum pox potyvirus (PPV) isolates belonging to the D or M serotypes, heminested PCR (H-PCR) and PCR-ELISA, have been developed. Ten PPV isolates typed using PPV-D and PPV-M specific monoclonal antibodies by ELISA-DASI were used to validate these two methods. The results obtained show a complete coincidence of the nucleic acid-based techniques with the serological data. When serial dilutions of infected plant extracts were assayed, H-PCR and PCR-ELISA were found to be 100 times more sensitive than the more conventional immunocapture-PCR (IC-PCR) assay. Testing of 228 PPV-infected fruit tree samples coming from different hosts and locations indicated that so far only PPV type D appears to be present in Spain and in Chile. Coupled with print-capture sample preparation (Olmos et al., Nucl. Acids Res. 24, 2192-2193, 1996) the increased sensitivity provided by heminested-PCR allowed the detection of PPV targets of D and M types, in wingless individuals of the aphid vector Aphis gossypii.
Olmos
A
A
Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias, Moncada, Spain.
Cambra
M
M
Dasi
M A
MA
Candresse
T
T
Esteban
O
O
Gorris
M T
MT
Asensio
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids / Q000821:virology; D001483:Base Sequence; D015342:DNA Probes; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005638:Fruit / Q000821:virology; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D017801:Plum Pox Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000379:methods; D014026:Nicotiana / Q000821:virology
D015342:DNA Probes