| 柏自琴,郑乾明.蟹爪兰X病毒实时荧光定量PCR检测方法的建立及应用[J].中国南方果树,2025,54(4): |
| 蟹爪兰X病毒实时荧光定量PCR检测方法的建立及应用 |
| Establishment and Application of a Real-time fluorescence quantitative PCR Method for zygocactus virus X detection |
| 投稿时间：2025-02-21 修订日期：2025-04-30 |
| DOI： |
| 中文关键词: 蟹爪兰X病毒 火龙果 RT-qPCR 检测 |
| 英文关键词:zygocactus virus X pitaya RT-qPCR detection |
| 基金项目:国家自然科学基金项目（32060674）；黔农科种质资源[2023]05号 |
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| 摘要点击次数: 191 |
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| 中文摘要: |
| 【目的】蟹爪兰X病毒（zygocactus virus X，ZyVX）寄主广泛、发病率高、种传率高，在贵州火龙果主产区发生普遍，本研究将建立ZyVX的实时荧光定量PCR方法，实现对该病毒的精准测报。【方法】本研究根据多个ZyVX分离株外壳蛋白（CP）的保守序列设计特异性引物，获得157 bp的靶标序列，建立了引物浓度150 nmol/L，退火温度61℃的SYBR Green I实时荧光定量PCR（Real-time fluorescent quantitative polymerase chain reaction, RT-qPCR）检测方法，实现了对该病毒的精准定量检测。【结果】ZyVX的标准曲线方程为y=-3.343 3x + 43.198（R2=0.998 3），扩增效率为99.11%，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。利用该方法对贵州火龙果主产区罗甸、望谟、册亨、镇宁等4个县采集的田间40份疑似病毒病样品进行检测，共检测出33份阳性样品，平均检出率为82.5%，与普通PCR检测结果一致。【结论】该方法灵敏度高、可靠性强，可用于ZyVX在植株及传播介体体内的快速准确诊断，为开展该病毒的监测、科学防控及从源头遏制病毒传播提供技术支撑，应用前景广阔。 |
| 英文摘要: |
| 【Objective】Zygocactus virus X (ZyVX) has a wide range of hosts, a high incidence rate and a high seed transmission rate, which is common in the main producing areas of pitaya fruit in Guizhou. This study established a real-time fluorescent quantitative PCR method for ZyVX to achieve accurate prediction.【Method】This study designed specific primers based on the conserved sequence of the coat protein (CP) of various ZyVX isolates, obtained a 157 bp target sequence, and established a SYBR Green I real-time fluorescence quantitative PCR (RT-qPCR) detection method with primer concentration of 150 nmol/L and annealing temperature of 61℃, achieving accurate quantitative detection of ZyVX.【Results】The standard curve equation of ZyVX is y=-3.343 3x+43.198 (R2=0.998 3), with an amplification efficiency of 99.11%. The detection limit concentration for the standard sample is 8×102 copies/μL, and its sensitivity is 100 times that of RT-PCR. This method was used to detect 40 suspected virus disease samples collected from four counties in the main pitaya production areas of Guizhou, including Luodian, Wangmo, Ceheng, and Zhenning. A total of 33 positive samples were detected, with an average detection rate of 82.5%, which is consistent with the PCR detection results.【Conclusion】It indicate that this method has high sensitivity and strong reliability, and can be used for rapid and accurate diagnosis of ZyVX in plants and transmission vectors. It provides technical support for monitoring, scientific prevention and control of ZyVX, and containment of virus transmission from the source, and has broad application prospects. |
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