Detection and differentiation of respiratory syncytial virus subgroups A and B with colorimetric toehold switch sensors in a paper-based cell-free system
Detection and differentiation of respiratory syncytial virus subgroups A and B with colorimetric toehold switch sensors in a paper-based cell-free system
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DOI：
10.1016/j.bios.2021.113173
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发表时间：
2021-03-25
影响因子：
12.6
通讯作者：
Wang, Jufang
中科院分区：
文献类型：
--
作者：
Cao, Mengcen;Sun, Qiuli;Wang, Jufang
呼吸道合胞病毒（Respiratory syncytial virus，RSV）感染是临床上最常见的威胁人类生命安全的传染病。本文中，我们提供了一种在纸基无细胞系统中用比色支点开关传感器检测和区分RSV亚群A（RSVA）和B（RSVB）的灵敏且特异的方法。在这种方法中，我们应用了立足点开关，一种基于RNA的核糖开关，来调节？半乳糖苷酶（lacZ）基因。在存在靶触发RNA的情况下，支点开关传感器被激活，并且表达的LacZ水解显色底物以产生可以在无细胞系统中用肉眼直接观察到的比色结果。此外，还使用基于核酸序列的扩增（NASBA）通过扩增靶触发RNA来提高灵敏度。在最佳条件下，我们的方法产生了一个可见的结果，用于检测RSVA和RSVB的检测限分别为52 aM和91 aM，分别。该方法的交叉反应与其他密切相关的呼吸道病毒，包括人类冠状病毒HKU 1（HCoV-HKU 1）和严重急性呼吸道综合征冠状病毒2（SARS-CoV-2）进行了验证。此外，我们使用纸质载体材料，可以稳定储存我们的检测元件，并在实验室外快速检测。该方法无需专业操作人员和精密仪器，即可灵敏、特异地区分RSVA和RSVB，并产生可见的比色结果。基于上述优点，该方法在资源贫乏地区和床旁检测（POCT）场景中用作简单且便携的检测器。
Respiratory syncytial virus (RSV) infection is the most common clinical infectious disease threatening the safety of human life. Herein, we provided a sensitive and specific method for detection and differentiation of RSV subgroups A (RSVA) and B (RSVB) with colorimetric toehold switch sensors in a paper-based cell-free system. In this method, we applied the toehold switch, an RNA-based riboswitch, to regulate the translation level of ?-galactosidase (lacZ) gene. In the presence of target trigger RNA, the toehold switch sensor was activated and the expressed LacZ hydrolyzed chromogenic substrates to produce a colorimetric result that can be observed directly with the naked eye in a cell-free system. In addition, nucleic acid sequence-based amplification (NASBA) was used to improve the sensitivity by amplifying target trigger RNAs. Under optimal conditions, our method produced a visible result for the detection of RSVA and RSVB with the detection limit of 52 aM and 91 aM, respectively. The cross-reaction of this method was validated with other closely related respiratory viruses, including human coronavirus HKU1 (HCoV-HKU1), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Furthermore, we used the paper-based carrier material that allows stable storage of our detection elements and rapid detection outside laboratory. In conclusion, this method can sensitively and specifically differentiate RSVA and RSVB and generate a visible colorimetric result without specialized operators and sophisticated equipment. Based on these advantages above, this method serves as a simple and portable detector in resource-poor areas and point-of-care testing (POCT) scenarios.