Single-step immunocapture RT-PCR in the detection of raspberry bushy dwarf virus.
An immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) method for a highly sensitive analysis of raspberry bushy dwarf virus (RBDV) in infected plants is described. In the method, preliminary purification of virus particles or viral RNA from the plant material is not necessary. Viruses are enriched during the assay by antibodies bound in the PCR microplate wells, followed by lysis of the viral particles, and RT-PCR of the viral RNA. The reaction mixtures, including reverse transcriptase and DNA polymerase, have been selected so that both enzymes are active in the lysis and amplification conditions; by this way, it is possible to conduct the whole procedure in a single step. Using the method, four fragments from RNA-3 of RBDV have been amplified with various combinations of four primers. The procedure is sensitive enough to allow a simple detection of RBDV in in vitro cultured plants in which the detection of viruses by conventional immunological methods is difficult or even impossible.
Kokko
H I
HI
Department of Biochemistry and Biotechnology, University of Kuopio, Finland.
Kivineva
M
M
Kärenlampi
S O
SO
D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005638:Fruit / Q000821:virology*; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000032:analysis
C034477:RNA primers; D012367:RNA, Viral; D012313:RNA; D012194:RNA-Directed DNA Polymerase