Liu Jianghua, Wang Yu, Zhang Xinyue, Huang Mingquan, Li Guoliang
School of Food Science and Engineering, Shaanxi University of Science and Technology, Xi'an, 710021, China.
Laboratory of Food Quality and Safety, Beijing Technology and Business University, Beijing, 100048, China.
Mikrochim Acta. 2025 Feb 25;192(3):187. doi: 10.1007/s00604-024-06932-x.
A novel direct lysis method combined with amplification-free CRISPR/Cas12a-SERS genosensor was for the first time developed to rapidly and sensitively identify meat adulteration. Notably, polystyrene (PS) microspheres, with distinct shrinking and swelling properties, were dexterously employed to encapsulate biological-silent Raman reporter 4-mercaptobenzonitrile (4-MBN) and act as a controlled-release signal probe. Target DNA activated the trans-cleavage activity of CRISPR/Cas12a towards ssDNA linked with PS microsphere to liberate the signal probe, which was able to release numerous Raman reporters after treatment with THF solution, resulting in high signal amplification. Through this platform, trace target DNA was deftly transformed into a sensitive Raman signal and could be on-site determined through a portable Raman equipment. Under optimized conditions, this strategy displayed good linearity in the range 1-450 ng/μL (R = 0.9943) and favorable sensitivity with limit of detection as low as 0.23 ng/μL without any pre-amplification. Moreover, it exhibited good applicability to on-site identification of commercial meat samples in complicated food matrix. In addition, DNA extraction by direct lysis and amplification-free detection realized ultrafast meat adulteration determination within 35 min from sampling to result. This method possessed great potential in rapid and on-site accurate determination of meat authenticity.
首次开发了一种结合无扩增CRISPR/Cas12a-表面增强拉曼光谱（SERS）基因传感器的新型直接裂解方法，用于快速、灵敏地识别肉类掺假。值得注意的是，具有明显收缩和膨胀特性的聚苯乙烯（PS）微球被巧妙地用于封装生物沉默拉曼报告分子4-巯基苯甲腈（4-MBN），并作为控释信号探针。靶DNA激活CRISPR/Cas12a对与PS微球相连的单链DNA的反式切割活性，以释放信号探针，该信号探针在用四氢呋喃（THF）溶液处理后能够释放大量拉曼报告分子，从而实现高信号放大。通过该平台，微量靶DNA被巧妙地转化为灵敏的拉曼信号，并可通过便携式拉曼设备进行现场测定。在优化条件下，该策略在1-450 ng/μL范围内表现出良好的线性（R = 0.9943），灵敏度良好，检测限低至0.23 ng/μL，无需任何预扩增。此外，它对复杂食品基质中商业肉类样品的现场鉴定具有良好的适用性。此外，通过直接裂解进行DNA提取和无扩增检测实现了从采样到结果在35分钟内超快速的肉类掺假测定。该方法在快速、现场准确测定肉类真实性方面具有巨大潜力。
