Sun Yuanyuan, Han Bingjie, Sun Fangfang
Department of Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University Zhengzhou 450052 Henan Province P. R. China
School of Chemistry and Chemical Engineering, Shaanxi Normal University Xi'an 710062 Shaanxi Province P. R. China.
RSC Adv. 2021 May 10;11(28):17058-17063. doi: 10.1039/d1ra00851j. eCollection 2021 May 6.
Specific and accurate detection of single nucleotide variants (SNVs) plays significant roles in pathogenic gene research and clinical applications. However, the sensitive but ultra-specific detection of rare variants in biological samples still remains challenging. Herein, we report a novel, robust and practical SNV assay by integrating the outstanding features of high selectivity of an artificial mismatched probe, and the powerful loop-mediated isothermal amplification. In this strategy, we rationally introduce artificial mismatched bases into the 3'-terminal regions of the probe located in the ligation region to reduce the risk of nonspecific ligation, which can dramatically improve the specificity for the SNV assay. The proposed method can discern as little as 0.01% mutant DNA in the high background of wild-type DNA with high sensitivity (10 aM). In virtue of its outstanding performance, the artificial mismatched probe may also be employed and expanded in various DNA and RNA genetic analyses with ligase-assisted approaches, showing great potential in biomedical research, clinical diagnostics, and bioanalysis.
单核苷酸变异（SNV）的特异性和准确检测在致病基因研究和临床应用中发挥着重要作用。然而，生物样本中罕见变异的灵敏且超特异性检测仍然具有挑战性。在此，我们通过整合人工错配探针的高选择性这一突出特性与强大的环介导等温扩增技术，报告了一种新颖、稳健且实用的SNV检测方法。在该策略中，我们合理地将人工错配碱基引入位于连接区域的探针的3'末端区域，以降低非特异性连接的风险，这能够显著提高SNV检测的特异性。所提出的方法在野生型DNA的高背景下能够以高灵敏度（10 aM）识别低至0.01%的突变DNA。凭借其出色的性能，人工错配探针还可用于各种DNA和RNA遗传分析，并通过连接酶辅助方法进行扩展，在生物医学研究、临床诊断和生物分析中显示出巨大潜力。
