基于TaqMan探针实时荧光定量PCR检测兰花环斑病毒方法的建立

Development of a TaqMan-based real-time fluorescence quantitative PCR assay for CymRSV

摘要:

【背景】兰花环斑病毒(cymbidium ringspot virus, CymRSV)是一类重要的检疫性病毒，在世界范围内危害严重。【目的】建立一种特异性强、灵敏度高且能定量分析CymRSV携带情况的高效检测方法。【方法】通过比对5个CymRSV CP基因序列，利用高度保守区设计3对引物探针并优化筛选后，获得145 bp的靶标序列及对应的最优引物及探针组合。以该靶标序列为模板构建阳性重组质粒，建立标准曲线，并探究其灵敏度、特异性、稳定性和应用效果。【结果】建立的CymRSV实时荧光定量PCR (real-time fluorescence quantitative PCR, RT-qPCR)检测法对阳性质粒标准品的最低检出限可达1 copy，最低稳定检出限达5 copies/μL，是逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)灵敏度的10³−10⁴倍；标准曲线y=−3.332x+40.371显示，C_t值与拷贝数的对数线性关系良好，扩增效率为99.6%，相关系数R²为0.999；与其他5种常见病毒反应均无扩增曲线，检测特异性强；批组内与批组间重复性试验C_t值变异系数均小于0.6%，重复性和稳定性较好。利用该方法对4类不同种属的20个兰花样品进行检测，阳性对照在C_t为23.31出现扩增曲线，阴性对照及样品未出现扩增曲线。【结论】基于TaqMan探针的CymRSV实时荧光定量PCR检测方法的成功创建，可为开展兰花CymRSV病毒的精准检测与科学防控提供技术支撑。

Abstract:

[Background] Cymbidium ringspot virus (CymRSV) is an important phytosanitary virus, which is seriously harmful in the world. [Objective] To establish a specific and sensitive assay for the quantitative analysis of CymRSV. [Methods] According to the highly conserved region in five coat protein gene sequences of CymRSV, we designed three pairs of primers and three probes, and then obtained the target sequence (145 bp) and screened out the optimal primers and probe. We then used the target sequence as a template to construct a positive recombinant plasmid and established a standard curve, on the basis of which the sensitivity, specificity, stability, and performance of the established assay were explored. [Results] In this study, the established RT-qPCR detection method for CymRSV showed that the minimum limit of detection of 1 copy for the positive plasmid standards and the minimum limit of stable detection of 5 copies/μL, demonstrating the sensitivity 10³–10⁴ times that of RT-PCR. The standard curve was y=−3.332x+40.371 (R²=0.999), which showed a good linear relationship between C_t value and logarithm of copy number and the amplification efficiency was 99.6%. The assay showed good specificity as it generated no amplification curve for other 5 common viruses. The intra-group and inter-group coefficients of variation of C_t value were less than 0.6%, which suggested good repeatability and stability. Furthermore, the established assay was employed to detect 20 samples of 4 orchid species. The positive control showed an amplification curve at C_t of 23.31, and the negative control and the samples showed no amplification curves. [Conclusion] The successful establishment of CymRSV RT-qPCR detection method based on TaqMan probe can provide technical support for the accurate detection and scientific prevention and control of orchid CymRSV virus.