槟榔隐症病毒1型TaqMan实时荧光定量PCR检测方法的建立

为了建立一种快速、精准且能定量分析槟榔隐症病毒1型（Areca palm velarivirus 1, APV1）的检测方法。本研究参照GenBank中已登录的APV1全基因组序列,在p21蛋白基因保守区域和p60蛋白基因保守区域之间设计1对特异性RT-PCR引物,并构建阳性重组质粒;在p60蛋白基因保守区域部分设计荧光定量PCR特异性引物和TaqMan-MGB探针,利用阳性重组质粒,构建标准曲线,并对该检测方法的敏感性、特异性、稳定性及其应用效果进行验证。结果显示：该方法对阳性质粒标准品检测的敏感性达3.0×10¹ copies/μL级别,是常规RT-PCR敏感性的100倍;标准曲线显示,C_t值与拷贝数的对数呈线性关系,其标准曲线方程为y=-3.2748x+42.957,扩增效率为102%,相关系数R²为0.9992;该方法对APV1的检测具有较好的特异性,其他槟榔病原物及内生菌不会对本方法产生非特异性干扰;批内与批间重复性试验结果显示,该方法对APV1的检测具有较好的重复性和稳定性;利用该方法对海南万宁、琼海、定安、文昌等4个市（县）采集的181份疑似样品进行检测,阳性率分别为91.95%、86.95%、89.65%、73.68%,其阳性的病毒拷贝数最高达1.59×10⁷copies/μL,表明海南部分地区APV1病毒载量较高、流行情况比较严重。

Development of a TaqMan Real-Time Fluorescent Quantitative PCR Method for Detection of Areca Palm Velarivirus 1

It is aimed to develop a rapid, precision and quantitative detection method for Areca palm velarivirus 1 (APV1). A pair of specificity RT-PCR primers were designed based on the APV1 complete genome sequence registered in GenBank to clone the sequence between the conservative region of p21 and the conservative region of p60, and a positive recombinant plasmid was constructed. Fluorescence quantitative PCR specificity primers and TaqMan-MGB probes were designed in the conservative region of p60 and a positive recombinant plasmid was constructed to establish a standard curve to verify the sensitivity, specificity, stability and application effect of the detection method. The sensitivity of this method to the detection of positive plasmid standards was up to 3.0×10 ¹ copies /μL, which was 100 times the sensitivity of conventional RT-PCR. The standard curve showed that the C_t value had a linear relationship with the logarithm of the copy number. The standard curve equation was y=-3.2748x+42.957, the amplification efficiency was 102%, and the correlation coefficient R² was 0.9992. The method has good specificity for the detection of APV1, and other areca pathogens and endophytes will not be affected by this method. The intra batch repeat test and inter batch repeat test results showed that this method had good repeatability and stability in the detection of APV1. This method was used to detect 181 suspected samples collected from 4 cities and counties in Hainan, including Wanning, Qionghai, Ding’an, and Wenchang. The positive rate was 91.95%, 86.95%, 89.65%, and 73.68%, respectively, and the number of positive virus copies was up to 1.59×10⁷ copies/μL. It shows that APV1 virus load is relatively high and the epidemic situation is relatively serious in some areas of Hainan.