单链DNA结合蛋白辅助增强型变性泡介导链交换扩增法快速鉴定蜂蜜花源成分

Rapid identification of floral origin in honey by single strand DNA-binding protein-enhanced denaturation bubble-mediated strand exchange amplification

中文关键词:  蜂蜜真实性  分子扩增  单链结合蛋白  试纸条  现场快速检测。

英文关键词:honey authenticity  molecular amplification  single strand DNA-binding protein  lateral flow strip  on-site rapid detection

中文摘要:

      目的 建立一种基于蜂蜜花粉基因检测的等温扩增方法, 结合时间分辨荧光微球试纸条(time-resolved fluorescent microspheres lateral flow strip, TR-LFS), 实现对蜂蜜花源真实性的快速鉴别。方法 通过构建蜂蜜中花粉DNA快速提取方法, 在20 min内获得有效的洋槐成熟酶K (maturase K, matK)目标基因模板。采用变性泡介导的链交换扩增(denaturation bubble-mediated strand exchange amplification, SEA), 在约65 ℃恒温条件下实现目标序列的指数扩增。为提高扩增效率, 加入单链结合蛋白(single strand DNA-binding protein, SSB)以抑制单链非特异性退火, 显著提高了引物杂交效率与扩增特异性。当蜂蜜中存在目标花粉DNA时, SEA生成两端标记的双链产物, 该产物通过TR-LFS实现可视化荧光检测。结果 利用TR-LFS的荧光信号强度, 可直观判定蜂蜜花源的真实性。以洋槐蜂蜜为例, 本方法对目标物含量的肉眼检测灵敏度可达1% (V:V)且与同其他花源蜂蜜无交叉反应。结论 本研究建立的检测方法通过引入SSB显著提升了SEA技术的扩增效率, 结合TR-LFS高灵敏与稳定的荧光输出特性, 无需复杂热循环设备, 适用于现场快速检测, 为蜂蜜花源的真实性鉴别提供了可靠的技术支持。

英文摘要:

      Objective 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). Methods 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. Results 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. Conclusion 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.