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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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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