Establishment and application of a real-time fluorescence quantitative PCR method for detection of telosma mosaic virus in Passiflora edulis
Telosma mosaic virus(TeMV)is a devastating virus infecting Passiflora edulis populations.In this study,the specific primer pair,Vpg-334F/506R,was designed based on the conserved region of the viral protein genome-linked(VPg)gene of TeMV,and a method of the SYBR Green Ⅰ-based real-time fluorescent quantitative PCR(qPCR)assay was established with optimal annealing temperature of 54℃ and primer concentration of 0.6 μmol/L,respectively.This method specifically amplified the nucleotide sequence region of 6 483-6 675 nt in the genome of TeMV.The cycle threshold of the qPCR standard curve exhibited a positive linear relationship with template concentrations,and the amplification efficiency and R2 value were 102.77％and 0.996 1,respectively.This method could detect a minimum concentration of 2.370X102 copies/μL DNA,which was 1 000 folds of conventional PCR detection.Monitoring the virus accumulation at different temperatures after inoculation revealed that TeMV could be detected at day 3 after inoculation,and that accumulated in leaves could be more readily detected at 26-28℃ than at other temperatures.The symptoms were highly correlated with the content of virus accumulation.Finally,the developed assay was used to detect TeMV in 76 passion fruit samples,showing that the detection rate of TeMV was 93.4％.Thus,this assay can be served as a powerful tool for detecting TeMV and is valuable for further research on TeMV.
万方数据
维普
