Mixed-Dye-Based Label-Free and Sensitive Dual Fluorescence for the Product Detection of Nucleic Acid Isothermal Multiple-Self-Matching-Initiated Amplification
Mixed-Dye-Based Label-Free and Sensitive Dual Fluorescence for the Product Detection of Nucleic Acid Isothermal Multiple-Self-Matching-Initiated Amplification
复制标题
基于混合染料的无标记灵敏双荧光用于核酸等温多重自匹配启动扩增的产品检测
DOI：
10.1021/acs.analchem.5b02112
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发表时间：
2015-10-20
影响因子：
7.4
通讯作者：
Mu, Ying
中科院分区：
文献类型：
--
作者：
Ding, Xiong;Wu, Wenshuai;Mu, Ying
基于荧光的视觉检测和自然光下的颜色变化是等温核酸扩增（INAA）的两种有前途的产品检测，例如等温多重自匹配启动扩增（IMSA）作为即时检测技术。然而，目前使用的方法在应用中存在不足。对于前者，由于发射的单一荧光和强背景噪声，肉眼识别的荧光变化可能无法区分，这需要经验预设截止强度值。对于后者，自然光下的视觉检测灵敏度无法与基于荧光的视觉检测灵敏度相比。本文将羟基萘酚蓝（HNB）与SYBR绿色I（SG）偶联，获得无标记的双荧光，用于IMSA的目视产物检测。建立了混合染料的芯片外（管基）和芯片上（微流控芯片基）IMSA检测B型肝炎病毒的方法。结果表明，该双荧光可实现可分辨的荧光颜色变化，提高了视觉检测灵敏度，避免了预设的截止值。此外，混合染料在室温下保持稳定，并且与IMSA的试剂相容，而无需在扩增后打开管的易污染步骤。此外，这种耦合染料继承了HNB在自然光下实现颜色变化和SG实时检测的优点。总之，基于混合染料的双重荧光在即时检测应用中具有潜力，用于实现IMSA、环介导等温扩增（LAMP）或其他INAA的芯片外和芯片上产物检测。
Visual detections based on fluorescence and the color changes under natural light are two promising product detections for isothermal nucleic acid amplifications (INAAs) such as the isothermal multiple-self-matching-initiated amplification (IMSA) as point-of-care testing techniques. However, the currently used approaches have shortcomings in application. For the former, fluorescence changes recognized by naked eye may be indistinguishable because of single fluorescence emitted and strong background noise, which requires empirical preset of cutoff intensity values. For the latter, visual detection sensitivity under natural light is not comparable to that based on fluorescence. Herein, hydroxyl naphthol blue (HNB) and SYBR Green I (SG) were coupled to acquire a label-free dual fluorescence for the visual product detection of IMSA. The mixed-dye-loaded off-chip (tube-based) and on-chip (microfluidic chip-based) IMSAs for the detection of hepatitis B virus were conducted. The results demonstrated that this dual fluorescence could realize distinguishable fluorescent color changes to improve visual detection sensitivity and avoid the preset of cutoff values. Moreover, the mixed dye is stable when kept at room temperature and compatible with the IMSA's reagents without a contamination-prone step of opening tubes after amplification. Also, this coupled dye inherits the advantages of achieving color changes under natural light from HNB and real-time detection from SG. In conclusion, the mixed-dye-based dual fluorescence has a potential in the point-of-care testing application for realizing off-chip and on-chip product detection of IMSA, loop-mediated isothermal amplification (LAMP), or other INAAs.