On-nylon membrane detection of nucleic acid molecules by rolling circle amplification
On-nylon membrane detection of nucleic acid molecules by rolling circle amplification
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尼龙膜上滚环扩增检测核酸分子
DOI：
10.1016/j.ab.2017.06.005
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发表时间：
2017-09-15
影响因子：
2.9
通讯作者：
Wang，Jinke
中科院分区：
文献类型：
--
作者：
Xu，Xinhui;Zhang，Beibei;Wang，Jinke
带正电荷的尼龙膜（NM）由于其通过直接静电粘附和简单的UV交联方便地固定核酸材料而成为核酸检测的通用固相支持物。滚环扩增（RCA）是一种广泛应用于核酸检测的等温DNA扩增技术。近红外荧光（NIRF）技术是一种新型的荧光检测技术，具有低背景、高灵敏度的特点。本研究将核磁共振（NM）、近红外共振（RCA）和近红外荧光（NIRF）的优点结合起来，建立了一种简便的核酸分子检测方法--尼龙膜上近红外荧光固相RCA（NM-NIRF-sRCA）。该方法的检测体系只需要两种核酸分子：3′端带有RCA引物的靶特异性探针（P）和滚环（RC）。检测过程包括四个步骤：（1）通过UV交联将检测到的核酸固定在NM上;（2）NM与特异性探针和RC杂交;（3）通过含有生物素-dUTP的RCA反应扩增;（4）将NM与NIRF标记的链霉亲和素孵育并用NIRF成像仪成像。通过对寡核苷酸、质粒中克隆的HPV各亚型L1片段和大肠杆菌基因组DNA的检测，充分验证了该方法的有效性。本研究为核酸分子的检测提供了一种新的简便方法。
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, andE.coligenomic DNA. This study thus provides a new facile method for detecting nucleic acid molecules.