A simple and reliable protocol for the detection of apple stem grooving virus by RT-PCR and in a multiplex PCR assay.
Primers were identified which amplify specifically a 499 bp fragment in the coat protein coding region of apple stem grooving virus (ASGV) genome. These primers were used in various RT-polymerase chain reaction (PCR) analyses for the detection of ASGV in Chenopodium quinoa, Nicotiana occidentalis, and in species of Malus and Pyrus. Isolates of ASGV in Malus and Pyrus from locations in Canada, China, Israel, Japan, Nepal, Pakistan, South Africa, and the U.S.A. were reliably detected in leaf and bark (budwood) tissue. Storage of the tissues at -80 degrees C for more than 4 months did not affect the reliability of detection by immunocapture (IC) RT-PCR. Triton-X was not necessary for the detection of ASGV by IC/RT-PCR, and it was also possible to combine the antibody incubation and virus sap incubation into a single step without any obvious loss in sensitivity. A Tube Capture (TC) RT-PCR procedure was developed that eliminated the need for antibody binding of the virus. Phosphate buffered saline with 2% PVP was identified as the most effective sample-grinding buffer for the detection of ASGV by IC/RT-PCR and TC/RT-PCR. TC/RT-PCR facilitated the simultaneous detection (multiplex PCR) of ASGV and cherry mottle leaf virus.
James
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Centre for Plant Health, Canadian Food Inspection Agency, Sidney, BC. jamesd@em.agr.ca
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D010942:Plant Viruses / Q000235:genetics* / Q000302:isolation & purification*; D012328:RNA Viruses / Q000235:genetics* / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019685:Rosales / Q000821:virology*; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D012367:RNA, Viral