Simultaneous TD/RT-PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers.
Cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) are the two most prevalent and economically important orchid viruses which have attained a world-wide distribution. We have designed one pair of common primers, which were able to detect, simultaneously, both viruses by a single polymerase chain reaction (PCR). Sequence alignment and primer analyses were employed in primer design. The effect of touch-down PCR, primer annealing temperature, number of thermal cycles, MgCl2 concentration and the degree of primer degeneracy, were investigated and applied to the optimisation of PCR conditions in the detection of both viruses in order to achieve equal amplification efficiencies. This is the first successful attempt at using a single pair of PCR primers to detect two viruses belonging to different taxonomic groups simultaneously.
School of Biological Sciences, National University of Singapore, Kent Ridge, Singapore.
M L
ML
Seoh
S M
SM
Wong
L
L
Zhang
D001483:Base Sequence; D017931:DNA Primers / Q000032:analysis; D006358:Hot Temperature; D015636:Magnesium Chloride; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017863:Potexvirus / Q000235:genetics / Q000302:isolation & purification*; D016415:Sequence Alignment; D017898:Tobamovirus / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D015636:Magnesium Chloride