脱氧雪腐镰刀菌烯醇污染风险现场快速检测系统的建立

Establishment of an on-site rapid detection system for the risk of deoxynivalenol contamination

中文关键词:  呕吐毒素  镰刀菌  多酶恒温核酸快速扩增  现场检测

英文关键词:deoxynivalenol  Fusarium  multienzyme isothermal rapid amplification technique  on-site detection

中文摘要:

      目的 建立一种基于多酶恒温核酸快速扩增(multienzyme isothermal rapid amplification, MIRA)技术的脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)污染风险现场快速检测系统。方法 通过美国国家生物技术信息中心(National Center for Biotechnology Information, NCBI)基因序列查询比对, 获取了我国主要产DON镰刀菌(包括禾谷镰刀菌、亚洲镰刀菌、假禾谷镰刀菌和黄色镰刀菌)的Tri产毒基因簇高度同源序列, 设计特异性荧光探针并筛选引物, 建立了产DON镰刀菌的MIRA快速检测方法, 同时结合纸基DNA快速提取技术, 构建了一套现场快速检测系统。结果 该检测系统对质粒模版的检出限为1.95×101 copies/μL, 对基因组DNA的检出限为15 fg/μL, 对小麦灌浆期(穗)部样品的检测结果(共27个阳性)与实时荧光定量聚合酶链式反应技术(quantitative real-time polymerase chain reaction, qPCR)结果高度相关(r=0.820, P=0.000)。将MIRA检测的阈值线设定在第43个扫描点(起峰时间为7 min)时, 对89个小麦样品的高DON污染风险(DON>1000 μg/kg)与低DON污染风险(DON<1000 μg/kg)的综合判别准确率为87.15%。结论 该检测系统快速、经济、实用, 且试剂和仪器便携, 能够有效实现现场快速检测。这将有助于实施田间小麦DON污染风险早期预警, 指导干预, 防止或减少损失。

英文摘要:

      Objective To establish an on-site rapid detection system for the risk of deoxynivalenol (DON) contamination based on multienzyme isothermal rapid amplification (MIRA) technology. Methods Highly homologous sequences of Tri toxin-producing gene clusters from the major DON-producing Fusarium species (including Fusarium graminearum, Fusarium asiaticum, Fusarium pseudograminearum and Fusarium culmorum) in China were obtained through gene sequence query and comparison by the National Centre for Biotechnology Information (NCBI) of the United States of America. By designing specific fluorescent probes and screening primers, a MIRA detection method for DON-producing Fusarium was established, and an on-site rapid detection system was constructed by combining the rapid paper-based DNA extraction technique. Results The limit of detection of this assay system was 1.95×101 copies/μL for plasmid templates and 15 fg/μL for genomic DNA. The results of 27 positive wheat samples at the filling stage (spike) showed a highly significant correlation with those of the quantitative real-time polymerase chain reaction (qPCR) method (r=0.820, P=0.000). When the MIRA detection threshold line was set at the 43rd scan point (peak onset time of 7 min), the combined discrimination accuracy between high (DON>1000 μg/kg) and low (DON<1000 μg/kg) DON contamination risk for 89 wheat samples was 87.15%. Conclusion The detection system demonstrates speed, economy, practicality, and portability in terms of reagents and instrumentation, and is capable of effectively achieving the objective of rapid on-site detection. This will facilitate early warning of the risk of DON contamination in wheat production in the field, thereby guiding interventions to prevent or minimise losses.