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| 西瓜细菌性果斑病菌现场快速双模荧光RPA检测方法的建立 | |
| 引用本文： | 王晨薇,汪小福,魏巍,陈笑芸,沈洁,徐俊锋,蔡健.西瓜细菌性果斑病菌现场快速双模荧光RPA检测方法的建立[J].浙江农业学报,2022,34(7):1519. |
| 作者姓名： | 王晨薇 汪小福 魏巍 陈笑芸 沈洁 徐俊锋 蔡健 |
| 作者单位： | 1.阜阳师范大学 生物与食品工程学院,安徽 阜阳 2360372.浙江省农业科学院 农产品质量安全危害因子与风险防控国家重点实验室,浙江 杭州 3100213.宁夏医科大学 临床医学院,宁夏 银川 750004 |
| 基金项目： | 浙江省自然科学基金(LGN22C140017);阜阳师范大学创新团队项目(TDYY20210001);安徽省林业科技创新项目(AULYCX-2021) |
| 摘 要： | 瓜类细菌性果斑病菌(bacrerial fruit of blotch,BFB)是中国检疫性病害,其传播非常迅猛,且目前没有商品化抗病品种。为建立西瓜果斑病菌现场快速荧光重组酶聚合酶扩增(recombinase polymerase amplification,RPA)检测方法,针对细菌性果斑病菌特异性序列设计并筛选出了RPA最佳的引物和探针组合,利用两种模式实时荧光监测RPA(real time RPA,RT-RPA)和终端荧光可视化检测RPA(end point RPA,EP-RPA)]对RPA的结果进行分析,并与实时荧光定量PCR(qRT-PCR)结果进行对比。结果显示,建立的细菌性果斑病菌双模荧光RPA检测方法特异性强,且其对病原物的检测灵敏度与qRT-PCR的灵敏度相当。在实际样品检测中,RT-RPA、EP-RPA和RT-PCR的检测结果一致性为100%。在检测时间上,EP-RPA的检测时间为20 min,RT-RPA平均检测时间约为5 min,qRT-PCR的平均检测时间约为44 min;RPA的检测速度最快,而且不依赖大型的仪器,方便快捷,适用于现场检测。该恒温快速检测方法的建立为植物病害的现场检测提供了新的技术支持。 |
| 关 键 词： | 瓜类细菌性果斑病 实时荧光监测RPA 终端荧光可视化检测RPA 可视化 |
| 收稿时间： | 2021-10-19 |
Establishment of on-site rapid dual-mode fluorescence RPA detection method for bacterialfruit of blotch |
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| WANG Chenwei,WANG Xiaofu,WEI Wei,CHEN Xiaoyun,SHEN Jie,XU Junfeng,CAI Jian.Establishment of on-site rapid dual-mode fluorescence RPA detection method for bacterialfruit of blotch[J].Acta Agriculturae Zhejiangensis,2022,34(7):1519. | |
| Authors: | WANG Chenwei WANG Xiaofu WEI Wei CHEN Xiaoyun SHEN Jie XU Junfeng CAI Jian |
| Affiliation: | 1. School of Biology and Food Engineering,Fuyang Normal University, Fuyang 236037, Anhui, China 2. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products,Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China 3. School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China |
| Abstract: | Bacterial fruit of blotch (BFB) is a quarantine disease in China, which spreads very rapidly and no commercial resistant varieties have been found at present. In order to establish rapid fluorescent recombinase polymerase amplification (RPA) detection method of BFB, the best combination of RPA primers and probe were designed and screened out according to the specific sequence of BFB, then RPA results was analyzed using two modes and compared with quantitative real-time PCR(qRT-PCR). One was real time RPA (RT-RPA), and the other was end point RPA (EP-RPA). The results showed that the established dual-mode fluorescence RPA method for detection of BFB had strong specificity, and its sensitivity for detection of pathogens was equivalent to that of qRT-PCR. The results of RT-RPA, EP-RPA and qRT-PCR were identical. The average detection time of EP-RPA, RT-RPA and qRT-PCR were about 20, 5 and 44 min,respectively. The detection speed of RPA was much faster, and the detection process did not rely on large instruments, which was convenient and fast. As a result, it was suitable for on-site detection. The establishment of this constant temperature rapid detection method provided a new technical support for the on-site detection of plant diseases. |
| Keywords: | bacterial fruit of blotch real time RPA end point RPA visualization |
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