摘要
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普遍发生。为实现对该病毒的精准检测测报,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,建立ZyVX实时荧光定量PCR方法,并利用该方法对贵州火龙果主产区罗甸、望谟、册亨、镇宁等4个县的田间40份疑似病毒病病样进行检测。结果表明,获得的靶标序列157 bp,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检测方法,ZyVX的标准曲线方程为y=-3.3433 x+43.198(R^(2)=0.9983),扩增效率为99.11%,对标准品的检测极限浓度为8×10^(2)拷贝/μL,其灵敏度是普通PCR的100倍。田间病样平均检出率为82.5%,与普通PCR检测结果一致。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。
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 pitaya fruit producing areas in Guizhou.This study established a real-time fluorescent quantitative PCR method to achieve accurate prediction of ZyVX.Based on the conserved sequence of the coat protein(CP)from multiple ZyVX isolates,specific primers were designed to establish a real-time fluorescence quantitative PCR(RT-qPCR)method for detection ZyVX.This method was subsequently applied to test 40 suspected viral disease samples collected from field surveys in four major pitaya-producing counties of Guizhou Province,including Luodian,Wangmo,Ceheng,and Zhenning.The results showed that the target sequences is 157 bp.A SYBR Green I real-time fluorescence quantitative PCR(RT-qPCR)detection method was established under the condition of primer concentration of 150 nmol/L and annealing temperature of 61℃.The standard curve equation of ZyVX was y=-3.3433 x+43.198(R^(2)=0.9983),with an amplification efficiency of 99.11%.The limit of detection(LOD)for the standard sample was 8×10^(2)copies/μL,demonstrating 100-fold higher sensitivity than conventional PCR.The average detection rate for field-collected diseased samples was 82.5%,which was consistent with conventional PCR detection results.This method had high sensitivity and strong reliability,and could be used for rapid and accurate diagnosis of ZyVX in plants and transmission vectors,providing technical support for monitoring,scientific prevention and control of ZyVX,with broad application prospects.
作者
柏自琴
郑乾明
解璞
罗会
郑伟
王正媛
欧子艳
奉皇书
BAI Ziqin;ZHENG Qianming;XIE Pu;LUO Hui;ZHENG Wei;WANG Zhengyuan;OU Ziyan;FENG Huangshu(Institute of Pomological Sciences,Guizhou Academy of Agricultural Sciences,Guiyang,Guizhou,550000,China;Luodian County Agriculture and Rural Bureau,Luodian,Guizhou,550100,China)
出处
《中国南方果树》
北大核心
2025年第4期51-55,61,共6页
South China Fruits
基金
国家自然科学基金(32060674)
黔农科种质资源[2023]05号项目资助。
