PMID- 17686535 OWN - NLM STAT- MEDLINE DCOM- 20080125 LR - 20071107 IS - 0166-0934 (Print) IS - 0166-0934 (Linking) VI - 146 IP - 1-2 DP - 2007 Dec TI - Development of a multiplex immunocapture-RT-PCR for simultaneous detection of BMYV and BChV in plants and single aphids. PG - 196-201 AB - 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. FAU - Vigano, Felicita AU - Vigano F AD - Broom's Barn Research Centre, Higham, Bury St. Edmunds, Suffolk IP28 6NP, UK. felicita.vigano@bbsrc.ac.uk FAU - Stevens, Mark AU - Stevens M LA - eng PT - Comparative Study PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20070807 PL - Netherlands TA - J Virol Methods JT - Journal of virological methods JID - 8005839 SB - IM MH - Animals MH - Aphids/*virology MH - Beta vulgaris/*virology MH - Enzyme-Linked Immunosorbent Assay/*methods MH - Luteoviridae/*isolation & purification MH - Plant Viruses/*isolation & purification MH - Reverse Transcriptase Polymerase Chain Reaction/*methods MH - Sensitivity and Specificity EDAT- 2007/08/10 09:00 MHDA- 2008/01/26 09:00 CRDT- 2007/08/10 09:00 PHST- 2007/04/18 00:00 [received] PHST- 2007/06/21 00:00 [revised] PHST- 2007/06/27 00:00 [accepted] PHST- 2007/08/10 09:00 [pubmed] PHST- 2008/01/26 09:00 [medline] PHST- 2007/08/10 09:00 [entrez] AID - S0166-0934(07)00249-2 [pii] AID - 10.1016/j.jviromet.2007.06.018 [doi] PST - ppublish SO - J Virol Methods. 2007 Dec;146(1-2):196-201. doi: 10.1016/j.jviromet.2007.06.018. Epub 2007 Aug 7.