Use of reverse transcription loop-mediated isothermal amplification for the detection of Plum pox virus.
A one step, accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) procedure was developed for the detection of Plum pox virus (PPV). The six primers required for accelerated RT-LAMP were designed using a conserved region in the C-terminus of the coat protein coding region of PPV. RT-LAMP was used to detect isolates of five strains of PPV including the strains D, M, EA, C, and W. The virus was detected reliably in both infected herbaceous and woody hosts. RT-LAMP was compared to real-time RT-PCR with SYBR Green I and melting curve analysis, using serial dilutions of total RNA extracts. Similar sensitivities were observed, except that real-time RT-PCR was more consistent at lower template concentrations. The purity of the FIP and BIP primers affected the efficiency of the reaction, and incubation time and template concentration affected the ladder-like pattern observed after agarose gel electrophoresis. Although PPV could be detected after 30min of incubation at 63 degrees C, a longer incubation time was required for lower concentrations of the target. RT-LAMP is a very sensitive, low cost diagnostic tool that should be of value in more accurate determination of the distribution of PPV. This should assist in preventing further spread of this devastating virus.
Varga
Aniko
A
Sidney Laboratory, Centre for Plant Health, Canadian Food Inspection Agency, 8801 East Saanich Road, Sidney, British Columbia V8L 1H3, Canada.
James
Delano
D
D001483:Base Sequence; D052160:Benzothiazoles; D036022:Capsid Proteins / Q000235:genetics; D017931:DNA Primers; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D016679:Genome, Viral; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals; D017801:Plum Pox Virus / Q000235:genetics / Q000302:isolation & purification*; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013194:Staining and Labeling; D044366:Transition Temperature
D052160:Benzothiazoles; D036022:Capsid Proteins; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I