Xu Xinhui, Zhang Beibei, Gan Ping, Wu Jian, Dai Wei, Zhang Ling, Wang Jinke
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
Anal Biochem. 2017 Sep 15;533:26-33. doi: 10.1016/j.ab.2017.06.005. Epub 2017 Jun 10.
Positively-charged nylon membrane (NM) is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM, RCA and NIRF, named NIRF-based solid phase RCA on nylon membrane (NM-NIRF-sRCA). The detection system of this method only need two kinds of nucleic acid molecules: target-specific probes with a RCA primer (P) at their 3' end and a rolling circle (RC). The detection procedure consists of four steps: (1) immobilizing detected nucleic acids on NM by UV crosslinking; (2) hybridizing NM with specific probes and RC; (3) amplifying by a RCA reaction containing biotin-dUTP; (4) incubating NM with NIRF-labeled streptavidin and imaging with a NIRF imager. The method was fully testified by detecting oligonucleotides, L1 fragments of various HPV subtypes cloned in plasmid, and E.coli genomic DNA. This study thus provides a new facile method for detecting nucleic acid molecules.
带正电荷的尼龙膜（NM）是核酸检测中常用的固相支持物，因为它可以通过直接静电吸附方便地固定核酸材料，并通过简单的紫外线交联实现。滚环扩增（RCA）是一种广泛用于核酸检测的等温DNA扩增技术。近红外荧光（NIRF）是一种新型荧光技术，由于背景低而具有高灵敏度。本研究结合NM、RCA和NIRF的优点，开发了一种检测核酸分子的简单方法，称为基于近红外荧光的尼龙膜固相滚环扩增（NM-NIRF-sRCA）。该方法的检测系统仅需要两种核酸分子：3'端带有RCA引物（P）的靶标特异性探针和一个滚环（RC）。检测过程包括四个步骤：（1）通过紫外线交联将检测的核酸固定在NM上；（2）使NM与特异性探针和RC杂交；（3）通过含有生物素-dUTP的RCA反应进行扩增；（4）用近红外荧光标记的链霉亲和素孵育NM并用近红外荧光成像仪成像。通过检测寡核苷酸、克隆在质粒中的各种HPV亚型的L1片段和大肠杆菌基因组DNA，充分验证了该方法。因此，本研究提供了一种检测核酸分子的新的简便方法。
