采用等温链置换扩增结合纳米金测流层析试纸条检测登革热病毒

doi:10.3785/j.issn.1008-9292.2018.08.13

浙江大学学报（医学版）

2018, Vol. 47

Issue (4): 405-412 DOI: 10.3785/j.issn.1008-9292.2018.08.13

原著

采用等温链置换扩增结合纳米金测流层析试纸条检测登革热病毒

赵锦1(

),夏海雄1,刘雨杰1,RavinderPal2,范安然1,何志旭1,*(

),曾令文3,*(

)

1. 贵州医科大学基础医学院免疫教研室组织工程与干细胞实验中心, 贵州贵阳 550004
2. 贵州医科大学海外教育学院, 贵州贵阳 550004
3. 中国科学院广州生物与医药健康研究院干细胞与分子诊断室, 广东广州 510530

Application of gold nanoparticles-based lateral flow strip for dengue virus detection based on strand-displacement amplification

ZHAO Jin1(

),XIA Haixiong1,LIU Yujie1,Ravinder Pal2,FAN Anran1,HE Zhixu1,*(

),ZENG Lingwen3,*(

)

1. Tissue Engineering and Stem Cell Research Center, School of Basic Medicine, Guizhou Medical University, Guiyang 550004, China
2. Overseas Education Institute, Guizhou Medical University, Guiyang 550004, China
3. South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

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摘要：

目的: 探讨等温链置换扩增结合纳米金测流层析试纸条用于登革热病毒（DENV）快速检测的可行性。方法: 采用磁珠富集法提取病毒总RNA，并逆转录为cDNA用于等温链置换扩增检测体系；等温链置换扩增用于检测DENV，琼脂糖凝胶电泳用于分析等温链置换扩增产物及其检测的敏感度；采用柠檬酸三钠还原法制备用于试纸条的纳米金颗粒；利用核酸碱基互补配对原理设计纳米金测流层析试纸条的检测线与控制线，建立DENV的检测体系。结果: 等温链置换扩增的敏感度为10 fmol/L。基于等温链置换扩增的纳米金测流层析试纸条检测DENV的敏感度也为10 fmol/L，取其检测样品浓度的对数值进行线性分析，检测浓度范围为10~10¹² fmol/L，符合线性回归方程，线性相关系数（R²）为0.98。基于等温链置换扩增的纳米金测流层析试纸条检测DENV的特异性较高，与其他对照组无交叉。结论: 建立了基于等温链置换扩增反应的纳米金试纸条用于DENV的检测方法，检测结果肉眼可见，敏感度高，可用于DENV的快速检测。

关键词： 登革热/诊断; DNA引物; 序列分析; 核酸扩增技术; 纳米技术; 金/诊断应用; 生物技术

Abstract:

Objective: To investigate the rapid and accurate method for the detection of dengue virus (DENV) by using nicking enzyme assisted strand-displacement amplification (SDA) combined with gold nanoparticles-based lateral flow strip. Methods: Total RNA of the virus was extracted by using magnetic beads method and transcribed to cDNA for SDA detection system. Nicking enzyme-assisted method was used for detecting DENV, and agarose gel electrophoresis was used for analyzing the sensitivity of SDA amplification products. A gold nanoparticles-based lateral flow strip was developed based on the principle of nucleic acid base complementary pairing to design the test line and control line. The gold particles were prepared by using sodium citrate reduction method for gold nanoparticles-based lateral flow strip construction. Results: The sensitivity of the SDA method was 10 fmol/L, and the sensitivity of gold nanoparticles-based lateral flow strip based on SDA method was also 10 fmol/L. In a linear range from 10 fmol/L to 10¹² fmol/L, the corresponding linear correlation coefficient (R²) of DENV was 0.98. The specificity of nanoparticles-based lateral flow strip based on SDA for DENV detection was high, which was no crossing with other control groups. Conclusion: A gold nanoparticles-based lateral flow strip based on SDA method for DENV detection has been established, which is convenient, fast, and the result is visible to naked eyes.

Key words: Dengue/diagnosis DNA primers Sequence analysis Nucleic acid amplification techniques Nanotechnology Gold/diagnostic use Biotechnology

收稿日期: 2018-03-20 出版日期: 2018-12-04

R446.9

基金资助: 广东省科技计划(2014B020212011)

通讯作者: 何志旭,曾令文 E-mail: 1019751340@qq.com;hzx@gmc.edu.cn;zeng_lingwen@gibh.ac.cn

作者简介: 赵锦(1992-), 女, 硕士研究生, 主要从事出血热病毒检测、食品安全检测以及诱导多能性干细胞用于神经退行性疾病的治疗研究; E-mail:1019751340@qq.com; https://orcid.org/0000-0003-1442-6873

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引用本文:

赵锦,夏海雄,刘雨杰,RavinderPal,范安然,何志旭,曾令文. 采用等温链置换扩增结合纳米金测流层析试纸条检测登革热病毒[J]. 浙江大学学报（医学版）, 2018, 47(4): 405-412.

ZHAO Jin,XIA Haixiong,LIU Yujie,Ravinder Pal,FAN Anran,HE Zhixu,ZENG Lingwen. Application of gold nanoparticles-based lateral flow strip for dengue virus detection based on strand-displacement amplification. J Zhejiang Univ (Med Sci), 2018, 47(4): 405-412.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.08.13 或 http://www.zjujournals.com/med/CN/Y2018/V47/I4/405

表 1 登革热病毒引物及探针设计

表 2 各病毒阳性质粒构建保守序列的选择

图 1 等温链置换扩增最佳反应时间的确定

图 2 紫外光分光光度仪分析胶体金溶液

图 3 纳米金溶液酸碱度测定

图 4 最佳纳米金-DNA喷金量的确定

图 5 纳米金测流层析试纸条结构图

图 6 最佳缓冲液的确定

图 7 等温链置换扩增检测登革热病毒的敏感性

图 8 基于等温链置换扩增的纳米金测流层析试纸条检测登革热病毒的敏感性分析

图 9 基于等温链置换扩增的纳米金测流层析试纸条检测登革热病毒的特异性分析

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