西番莲中夜来香花叶病毒实时荧光定量PCR检测方法的建立及应用

夜来香花叶病毒(telosma mosaic virus, TeMV)是对西番莲危害较大的一种病毒病原。根据病毒末端结合蛋白(VPg)序列设计引物, 建立了以Vpg-334F/506R为特异性引物, 退火温度54℃, 引物浓度0.6 μmol/L的SYBR Green Ⅰ实时荧光定量PCR检测方法。该方法可特异性扩增TeMV基因组6 483~6 675 nt区域, 所得标准曲线扩增效率为102.77%, 决定系数为0.996 1, 最低检测浓度为2.370×10 2 拷贝/μL, 灵敏度是普通PCR的1 000倍。应用该方法对接种TeMV的西番莲进行检测, 发现接种3 d后可在叶片中检测到TeMV, 定量分析不同温度下TeMV在叶片中的积累, 发现26~28℃下病毒积累速度最快, 且植株症状表现与病毒积累量密切相关。对赣南地区采集的76份西番莲田间样品进行检测, 共检出71份阳性样品, 检出率为93.4%。综上, 本研究建立的实时荧光定量PCR方法特异性强, 灵敏度高, 适于TeMV的快速检测。

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 2.370×10 2 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.