To establish a rapid identification method for the authenticity of honey botanical origin,based on an isothermal amplification approach for honey pollen gene detection, combined with a time-resolved fluorescent microsphere lateral flow strip (TR-LFS).
A rapid DNA extraction method for honey pollen was constructed to obtain the effective maturase K (matK) target gene template of Robinia pseudoacacia within 20 min.Denaturation bubble-mediated strand exchange amplification (SEA) was employed to achieve exponential amplification of the target sequence under a constant temperature of approximately 65 °C. To enhance amplification efficiency, single strand DNA-binding protein (SSB) was added to inhibit non-specific annealing of single strands,significantly improving primer hybridization efficiency and amplification specificity. When target pollen DNA was present in the honey, the SEA amplification generated double-stranded products labeled at both ends, which were subsequently detected visually via the TR-LFS based on fluorescence.
The fluorescence signal intensity of the TR-LFS allowed for visual determination of honey botanical origin authenticity. Using Robinia pseudoacacia honey as a model, the naked-eye detection sensitivity of this method for the target content reached 1% (V:V), and no cross-reaction was observed with other botanical honey sources.
The detection method established in this study significantly enhances the amplification efficiency of SEA technology through the introduction of SSB.Combined with the high sensitivity and stable fluorescence output characteristics of TR-LFS, and eliminating the need for complex thermal cycling equipment, this method is suitable for on-site rapid detection, providing reliable technical support for authenticating honey floral sources.
| 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 |