Development of a multiplex immunocapture-RT-PCR for simultaneous detection of BMYV and BChV in plants and single aphids.
A multiplex immunocapture-reverse transcription-polymerase chain reaction protocol (mIC-RT-PCR) was successfully developed to improve the detection of Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV) in plants and aphids in single and mixed infections. Viral particles from plant and aphid extracts were enriched by antibody-capture and lysed by heating to release the viral RNA. During the RT-PCR step, 5' end sequences specific to each virus were amplified and the products analysed by gel electrophoresis; the PCR products corresponding to BMYV and BChV were 440 and 348bp respectively. The test was evaluated on single aphids carrying BMYV, BChV or both viruses and the results demonstrated that the mIC-RT-PCR is specific and sensitive. Its sensitivity was found to be 10(5) times higher than the TAS-ELISA routinely used for detecting BMYV and BChV and 10(4) times better than RT-PCR when both viruses were present. Eliminating the antibody-capture step to simplify the technique did not affect the sensitivity of the test and a procedure using microtitre plates was developed to allow simultaneous processing of large numbers of samples.
Broom's Barn Research Centre, Higham, Bury St. Edmunds, Suffolk IP28 6NP, UK. felicita.vigano@bbsrc.ac.uk
Felicita
F
Viganó
Mark
M
Stevens
D000818:Animals; D001042:Aphids / Q000821:virology*; D027461:Beta vulgaris / Q000821:virology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D052679:Luteoviridae / Q000302:isolation & purification*; D010942:Plant Viruses / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity