环介导等温扩增
核酸
核酸扩增试验
琼脂糖
逆转录环介导等温扩增
逆转录酶
化学
色谱法
聚合酶链反应
生物
DNA
生物化学
病毒学
基因
沙眼衣原体
作者
Thi Minh Nguyet Trinh,Nae Yoon Lee
标识
DOI:10.1016/j.jbiotec.2022.08.002
摘要
In this study, we applied a tube-based reverse transcription loop-mediated isothermal amplification technique using preloaded amplification and detection reagents for simple screening of viable vancomycin-resistant Enterococcus in a cordless manner. We adopted an mRNA-based approach to detect live Enterococcus in vancomycin-treated cultures. We used agarose to preload and store all reagents for amplification and detection inside the tube, which could achieve on-site isothermal nucleic acid amplification and detection in less than 1 h without using sophisticated instruments. Moreover, the use of a portable insulated water tumbler eliminated the need for electricity, which is usually important in nucleic acid amplification-based assays. The water tumbler acted as a heat source to supply a stable heat required for the amplification reaction, which could last up to 45 min. In addition, colorimetric detection was realized using pH-based methods. The detection was triggered by shaking the tube so that the amplified solution was reacted with phenolphthalein embedded in the tube cap. The introduced one-pot strategy has many advantages such as easy and cordless operation, low cost, disposability, and less chance of contamination because the amplification and detection occur in a closed system. The system could have a great impact on nucleic acid analyses in instrument-free and low-resource areas.
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