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中国精品科技期刊2020
程雅妮,孙林瑶,王晴,等. 双拖尾恒温扩增结合核酸杂交侧流层析试纸条高通量快速检测驴肉中的掺假动物源性成分J. 食品工业科技,2026,47(5):1−11. doi: 10.13386/j.issn1002-0306.2025040095.
引用本文: 程雅妮,孙林瑶,王晴,等. 双拖尾恒温扩增结合核酸杂交侧流层析试纸条高通量快速检测驴肉中的掺假动物源性成分J. 食品工业科技,2026,47(5):1−11. doi: 10.13386/j.issn1002-0306.2025040095.
CHENG Yani, SUN Linyao, WANG Qing, et al. Nucleic Acid Hybridization-Lateral Flow Strip Combined with Double Tailed Isothermal Amplification for High-Throughput Rapid Detection of Adulterated Animal-Derived Components in Donkey MeatJ. Science and Technology of Food Industry, 2026, 47(5): 1−11. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2025040095.
Citation: CHENG Yani, SUN Linyao, WANG Qing, et al. Nucleic Acid Hybridization-Lateral Flow Strip Combined with Double Tailed Isothermal Amplification for High-Throughput Rapid Detection of Adulterated Animal-Derived Components in Donkey MeatJ. Science and Technology of Food Industry, 2026, 47(5): 1−11. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2025040095.

双拖尾恒温扩增结合核酸杂交侧流层析试纸条高通量快速检测驴肉中的掺假动物源性成分

Nucleic Acid Hybridization-Lateral Flow Strip Combined with Double Tailed Isothermal Amplification for High-Throughput Rapid Detection of Adulterated Animal-Derived Components in Donkey Meat

  • 摘要: 目的:建立一种基于双拖尾重组聚合酶恒温扩增(recombinase polymerase amplification,RPA)结合核酸杂交侧流层析试纸条(nucleic acid hybridization-lateral flow strip,NAH-LFS)的多重可视化检测技术,用于同步鉴别驴肉及其制品中鸡、猪、马源性成分,满足快速检测、操作简便及低成本的需求。方法:首先制备不同掺假比例的样品并进行DNA提取。利用primer5软件设计针对鸡、猪、马线粒体细胞色素b(cytb)基因的特异性引物,引物包含3个功能区,即引物序列、spacer9间隔区和单链DNA标签序列(作为拖尾序列),spacer9可以阻止DNA聚合酶的作用使单链DNA标签序列在RPA扩增后仍保持单链状态。随后,制备胶体金纳米粒子并与识别探针偶联、构建核酸杂交侧流层析试纸条、优化RPA反应条件及试纸条的最佳工作条件,并对其检测灵敏度和特异性进行系统验证。最后,应用所建立的方法对40份市售驴肉样品进行检测。结果:RPA最佳反应温度为39 ℃,孵育时间是10 min,Mg2+(280 mmol/L)添加量为2.5 μL,鸡、猪、马引物(10 μmol/L)的加入量分别为0.7、0.7、1 μL。试纸条采用流速为33.75 cm/s型号的硝酸纤维素膜、流动缓冲液RB4(0.01 mol/L PBS pH8.0、0.05%吐温-20)时显色效果最佳。RPA-NAH-LFS可同时呈现T1(鸡源)、T2(猪源)、T3(马源)3条特征性检测线,总检测时间控制在15 min以内(含10 min扩增及5 min检测反应),对目标肉类的检测灵敏度达0.01%(w/w),对牛、羊等9种常见肉类无交叉反应。用RPA-NAH-LFS对40份市售驴肉样品检测结果显示,1份驴肉包子中检出猪肉和鸡肉成分,1份火锅驴肉卷中检出猪肉成分,检测结果与标准检测(DB42/T 1591-2020)结果完全一致。结论:RPA-NAH-LFS技术通过双拖尾引物设计与多重探针杂交策略,实现了肉类掺假成分的同步可视化检测。该方法兼具高灵敏度、强特异性和操作便捷性,特别适用于基层监管部门和现场快速筛查需求,为肉制品真实性鉴别提供了高效技术手段。

     

    Abstract: Objective: To develop a multiplex visual meat adulteration detection method, we conjugated dual-tailed recombinase polymerase amplification (RPA) with nucleic acid hybridization-lateral flow strip (NAH-LFS) for the simultaneous identification of chicken, pork, and horse derived components in donkey meat and its products. This RPA-NAH-LFS method met the requirements of meat adulteration detection for rapidity, simplicity, and low cost. Methods: DNA was extracted from the artificially prepared adulterated meat samples with different adulteration proportions using a commercial DNA extraction kit. Next, three primer pairs specific to chicken, pork, and horse DNA were designed with unique oligonucleotide tags and a spacer C9 to halt polymerase activity, enabling the RPA amplicons to possess a single-stranded DNA tail. Subsequently, colloidal gold nanoparticles were prepared and conjugated with recognition probes. NAH-LFS were then constructed. We optimized the RPA reaction conditions and the optimal working parameters for the LFS, followed by systematic validation of the sensitivity and specificity of the RPA-NAH-LFS. Finally, the established assay was applied to analyze 40 commercial donkey meat samples. Results: The optimal RPA conditions were determined as follows: reaction temperature of 39 °C, incubation time of 10 min, Mg2+ (280 mmol/L) addition volume of 2.5 μL, and primer addition volumes of 0.7 μL (chicken), 0.7 μL (pork), and 1.0 μL (horse). The NC membrane with a flow rate of 33.75 cm/s and the running buffer 4 (0.01 mol/L PBS, pH8.0, 0.05% Tween-20) were selected as optimal conditions. The LFS had three test lines targeting for chicken (T1), pork (T2), and horse (T3). The detection process was completed within 15 min, which included RPA reaction (10 min) and LFS detection (5 min). The method could detect as little as 0.01% (w/w) chicken, pork, or horse meat in simulated adulterated donkey meat. Cross-reactivity test with 9 meat species demonstrated the excellent specificity of the assay. Analysis of the 40 commercial meat products revealed pork adulteration present in one sample, and simultaneous chicken and pork adulteration in another sample. These results were 100% consistent with those obtained by the standard detection method (DB42/T 1591-2020). Conclusion: The RPA-NAH-LFS technique, utilizing the dual-tailed primer design and a multiplex probe hybridization strategy, enables the simultaneous visual detection of meat adulterants. This method has high sensitivity, strong specificity, and operational convenience, making it particularly suitable for rapid on-site screening by basic-level regulatory agencies. It provides an efficient technology for detecting the authenticity of meat products.

     

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