An improved electrochemiluminescence polymerase chain reaction method for highly sensitive detection of plant viruses.
Recently, we have reported an electrochemiluminescence polymerase chain reaction (ECL-PCR) method for detection of genetically modified organisms. The ECL-PCR method was further improved in the current study by introducing a multi-purpose nucleic acid sequence that was specific to the tris(bipyridine) ruthenium (TBR) labeled probe, into the 5' terminal of the primers. The method was applied to detect plant viruses. Conserved sequence of the plant viruses was amplified by PCR. The product was hybridized with a biotin labeled probe and a TBR labeled probe. The hybridization product was separated by streptavidin-coated magnetic beads, and detected by measuring the ECL signals of the TBR labeled. Under the optimized conditions, the experiment results show that the detection limit is 50 fmol of PCR products, and the signal-to-noise ratio is in excess of 14.6. The method was used to detect banana streak virus, banana bunchy top virus, and papaya leaf curl virus. The experiment results show that this method could reliably identity viruses infected plant samples. The improved ECL-PCR approach has higher sensitivity and lower cost than previous approach. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.
Tang
Ya-bing
YB
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou, China.
Xing
Da
D
Zhu
De-bin
DB
Liu
Jin-feng
JF
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000302:isolation & purification; D004563:Electrochemistry / Q000379:methods*; D049449:Luminescence; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010942:Plant Viruses / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes