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

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。

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

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.