Huang Sisi, Wang Shihui, Wang Tianlong, Song Hongwei, Guo Yan, Xiong Xiong, Wang Libin
College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China.
College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China.
Foods. 2024 Nov 26;13(23):3816. doi: 10.3390/foods13233816.
The self-quenching fluorogenic probe facilitates precise identification of LAMP (loop-mediated isothermal amplification) amplicons, unaffected by non-specific products resulting from primer dimers. However, low quenching efficiency by surrounding nucleobases leads to high background signal, posing significant challenges for visual inspection with the naked eye. The present study aims to identify an oligonucleotide sequence that is complementary to the self-quenching fluorogenic probe, and to employ the fluorescence super-quenching mechanism of double-stranded DNA to establish a visualization system for the LAMP assay. The results indicated that the incorporation of a sequence fully complementary to the probe could significantly reduce the system's background fluorescence ( < 0.05). When the melting temperature exceeds room temperature, truncating the complementary sequence from the 3' end does not compromise the probe's quenching efficiency. The LAMP visualization system, using a 10-13-base complementary sequence of the loop primer-based probe, could effectively minimize background fluorescence and yield straightforward visual results post-reaction. Applied to rainbow trout and Atlantic salmon detection, the system detected 1 pg DNA in a closed-tube format. In conclusion, a suitable complementary sequence can reduce the background fluorescence of the self-quenching fluorogenic probe. Employing this sequence alongside the self-quenching fluorogenic probe to develop a low-background fluorescence LAMP system demonstrates great potential for successful visual detection and holds considerable promotional merit.
自猝灭荧光探针有助于精确鉴定环介导等温扩增（LAMP）扩增子，不受引物二聚体产生的非特异性产物影响。然而，周围核碱基的猝灭效率低导致背景信号高，给肉眼目视检测带来重大挑战。本研究旨在鉴定与自猝灭荧光探针互补的寡核苷酸序列，并利用双链DNA的荧光超猝灭机制建立LAMP检测的可视化系统。结果表明，掺入与探针完全互补的序列可显著降低系统的背景荧光（<0.05）。当解链温度超过室温时，从3'端截断互补序列不会损害探针的猝灭效率。使用基于环引物的探针的10 - 13个碱基的互补序列的LAMP可视化系统可有效最小化背景荧光，并在反应后产生直接的视觉结果。应用于虹鳟鱼和大西洋鲑鱼检测时，该系统以封闭管形式检测到1 pg DNA。总之，合适的互补序列可降低自猝灭荧光探针的背景荧光。将该序列与自猝灭荧光探针一起用于开发低背景荧光LAMP系统，在成功进行视觉检测方面显示出巨大潜力，具有相当大的推广价值。
