Single-step, high-specificity detection of single nucleotide mutation by primer-activatable loop-mediated isothermal amplification (PA-LAMP)
Single-step, high-specificity detection of single nucleotide mutation by primer-activatable loop-mediated isothermal amplification (PA-LAMP)
复制标题
通过引物激活环介导的等温扩增 (PA-LAMP) 单步、高特异性检测单核苷酸突变
DOI：
10.1016/j.aca.2018.10.068
复制
发表时间：
2019-03-07
影响因子：
6.2
通讯作者：
Jiang, Jian-Hui
中科院分区：
文献类型：
--
作者：
Du, Wen-Fang;Ge, Jian-Hui;Jiang, Jian-Hui
环介导等温扩增 (LAMP) 是即时护理 (POC) 情况下核酸检测的有用平台，但开发用于特异性检测单核苷酸突变 (SNM) 的单步、闭管 LAMP 反应仍然是一个挑战。我们开发了一种新型引物激活 LAMP (PA-LAMP) 策略，可使用单步、闭管反应实现高度特异性和灵敏的 SNM 检测。该策略设计了带有核糖核苷酸插入的末端封闭的内部引物，通过核糖核酸酶（RNase）H2对完美匹配的靶标进行特异性切割和激活，从而实现突变基因的高效扩增。它显示了突变靶标在 220 aM 至 22 pM 线性范围内的动态响应，最低可检测浓度为 22 aM。它还在识别大量野生型突变体方面表现出非常高的特异性，辨别率高达 10,000 左右。已成功应用于肿瘤细胞基因组DNA突变检测。 PA-LAMP 策略提供了一个有用、便携且经济实惠的 POC 平台，用于临床应用中基因突变的高灵敏度和特异性检测。 (c) 2018 Elsevier B.V. 保留所有权利。
Loop-mediated isothermal amplification (LAMP) is a useful platform for nucleic acids detection in pointof- care (POC) situations, and development of single-step, close-tube LAMP reactions for specific detection of single nucleotide mutations (SNMs) remains a challenge. We develop a novel primer-activatable LAMP (PA-LAMP) strategy that enables highly specific and sensitive SNM detection using single-step, close-tube reactions. This strategy designs a terminal-blocked inner primer with a ribonucleotide insertion, which is cleaved and activated specifically to perfectly matched targets by ribonuclease (RNase) H2, to realize efficient amplification of mutant genes. It has shown dynamic responses of mutant target in a linear range from 220 aM to 22 pM with a lowest detectable concentration of 22 aM. It also demonstrates very high specificity in identifying the mutant in a large excess of the wild-type with a discrimination ratio as high as similar to 10,000. It has been successfully applied to mutation detection of genomic DNA in tumor cells. The PA-LAMP strategy provides a useful, portable and affordable POC platform for highly sensitive and specific detection of genetic mutations in clinical applications. (c) 2018 Elsevier B.V. All rights reserved.