| 柏自琴*, 罗 会*, 解 璞, 郑 伟, 刘 涛.火龙果中仙人掌X病毒实时荧光定量PCR检测体系的建立及应用[J].植物保护,2025,51(4):285-289. |
| 火龙果中仙人掌X病毒实时荧光定量PCR检测体系的建立及应用 |
| Establishment and application of a real-time fluorescence quantitative PCR assay for the detection of cactus virus X in pitaya |
| 投稿时间：2024-08-26 修订日期：2024-10-08 |
| DOI：10.16688/j.zwbh.2024443 |
| 中文关键词: 仙人掌X病毒 火龙果 实时荧光定量PCR 检测 |
| 英文关键词:cactus virus X pitaya real-time fluorescence quantitative PCR detection |
| 基金项目:黔农科种质资源［2023］05号 |
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| 中文摘要: |
| 仙人掌X病毒(cactus virus X, CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测, 本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R, 建立了引物浓度150 nmol/L, 退火温度62℃的SYBR Green I实时荧光定量PCR检测方法。该引物扩增效率为97.38%, R2为0.996 5, 对标准品的检测极限浓度为8×102拷贝/μL, 其灵敏度是普通PCR的100倍。对火龙果不同组织中CVX的相对表达量进行检测发现, 病毒在花丝中的含量最高, 之后依次为花萼、花瓣、嫩枝、老枝和根。对采自贵州黔南州的40份火龙果样品进行检测, 共检测出32份阳性样品。上述结果表明, 本研究建立的实时荧光定量PCR特异性强、灵敏度高, 为今后CVX的检测提供了重要的技术补充。 |
| 英文摘要: |
| Cactus virus X (CVX) poses a significant threat to global pitaya (dragon fruit) production. To enable accurate quantification of CVX, this study developed a real-time fluorescence quantitative PCR (RT-qPCR) assay using SYBR Green I. Specific primers (CVX-F/-R) targeting the conserved region of the CVX coat protein (CP) gene were designed. The optimized assay conditions included a primer concentration of 150 nmol/L and an annealing temperature 62℃. The amplification efficiency was 97.38%, with a correlation coefficient value (R2) of 0.996 5. The assay had a detection limit of 8×102 copies/μL, exhibiting a 100-fold greater sensitivity than conventional PCR assay. CVX titers in various pitaya tissues were quantified, with the highest viral load detected in filaments, followed by calyx, petals, young stems, mature stems and roots. Among 40 pitaya samples collected from Qiannan prefecture, Guizhou province, 32 tested positive for CVX using this RT-qPCR method. These results demonstrate that the developed RT-qPCR assay offers high sensitivity and strong specificity, providing an effective tool for CVX detection and epidemiological studies. |
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