Detection and differentiation of respiratory syncytial virus subgroups A and B with colorimetric toehold switch sensors in a paper-based cell-free system.

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 ß-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.

A simple and low-cost paper-based colorimetric method for detecting and distinguishing the GII.4 and GII.17 genotypes of norovirus.

In modern times, viruses still threaten people's lives. Among them, norovirus was the main pathogenic factor in the cause of gastroenteritis and foodborne illness, of which the GII.4 and GII.17 genotypes are prevalent in China and most parts of the world. A simple and low-cost platform for rapid and accurate norovirus detection remains a major challenge. After the cell-free system and paper-based chromogenic system were optimized, a rapid and specific norovirus detection method was established based on norovirus-specific sequences in combination with toehold switch elements. The development of a visible color change during detection eliminates the need for any complicated instruments. We validated this strategy and its specificity in differentiating GII.4, GII.17, Zika virus, and human coronavirus HKU1. The results showed that the optimized detection system not only provided a simple and rapid detection method for the sufficient differentiation of the two norovirus genotypes but also showed high specificity and no cross-reactivity with other viruses. Using nucleic acid isothermal amplification, this assay showed a limit of detection of 0.5 pM for the GII.4 genotype and 2.6 fM for the GII.17 genotype in reactions that could be observed directly with the naked eye. Our results suggested that this paper-based colorimetric method could serve as a simple and low-cost visual detection method for pathogens in clinical samples, especially in remote or rural areas.