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. 2024 Dec 21;14(12):637.
doi: 10.3390/bios14120637.

A Novel Approach Using LuxSit-i Enhanced Toehold Switches for the Rapid Detection of Vibrio parahaemolyticus

Xiaodan Kang  1   2 , Chen Zhao  1 , Shuting Chen  1   2 , Shuran Yang  1 , Xi Zhang  1 , Bin Xue  1 , Chenyu Li  1 , Shang Wang  1 , Xiaobo Yang  1 , Chao Li  1 , Zhigang Qiu  1 , Jingfeng Wang  1 , Zhiqiang Shen  1
Affiliations

A Novel Approach Using LuxSit-i Enhanced Toehold Switches for the Rapid Detection of Vibrio parahaemolyticus

Xiaodan Kang et al. Biosensors (Basel). .

Abstract

Vibrio parahaemolyticus (V. parahaemolyticus) is a significant concern, as it can cause severe infections and hemolytic trauma. Given its prevalence in seawater and coastal seafood, it poses a substantial risk as a foodborne pathogen. Biosensor-based detection technology has been continuously evolving, and toehold switches have emerged as a promising area within it, especially in the detection of RNA viruses. Here, we have developed a cell-free toehold switch sensor for V. parahaemolyticus detection. Traditional toehold switch detection methods usually use green fluorescent protein (GFP) or enzyme LacZ as the output signal, with an incubation time as long as 2 h, and are also mainly applied to the detection of RNA viruses. In this study, we introduced a novel, artificially designed luciferase (LuxSit-i) as an output signal and constructed toehold switches with two different output signals (sfGFP, LuxSit-i), aimed at reducing the incubation time of toehold switches. Moreover, to further improve the detection process, we separately utilize recombinase polymerase amplification (RPA) and nucleic acid sequence-based amplification (NASBA) to amplify dead and live bacterial suspensions for detection and attempt to distinguish between dead and live bacteria. This study provided a convenient, rapid, and accurate method for the on-site detection of V. parahaemolyticus, especially beneficial for resource-limited settings. By eliminating the requirement for specialized facilities and personnel, this system has the potential to be a valuable tool in improving public health responses, especially in developing regions.

Keywords: LuxSit-i; Vibrio parahaemolyticus; on-site detection; toehold switch.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
A schematic of the toehold switch sensor. RBS: ribosome binding sites. AUG: start codon.
Figure 2
Figure 2
Screen depicting trigger/switch pairs in vivo. (a) ON/OFF sfGFP fluorescence ratios of in vivo expression (VPS-G). (b) ON/OFF chemiluminescence ratios of in vivo expression (VPS-L).
Figure 3
Figure 3
The optimization of the reaction time and concentration of switches in a cell-free system. (a) ON/OFF sfGFP fluorescence ratios were monitored as a function of time (VPS-G). (b) ON/OFF chemiluminescence ratios were monitored as a function of time (VPS-L). (c) ON/OFF sfGFP fluorescence ratios under the different switch concentrations (VPS-G). (d) ON/OFF chemiluminescence ratios under the different switch concentrations (VPS-L).
Figure 4
Figure 4
The sensitivity of the cell-free toehold switch sensors combined with RPA. (a) ON/OFF sfGFP fluorescence ratios of different concentrations of the trigger (VPS-G). (b) The corresponding image for the different triggers of VPS-G. (c) ON/OFF chemiluminescence ratios of different concentrations of the trigger (VPS-L). (d) The corresponding image for the different triggers of VPS-L. (e) ON/OFF sfGFP fluorescence ratios of different concentrations of the trigger with RPA (VPS-G). (f) ON/OFF chemiluminescence ratios of different concentrations of the trigger with RPA (VPS-L).
Figure 5
Figure 5
The sensitivity of the cell-free toehold switch sensors combined with NASBA. (a) ON/OFF sfGFP fluorescence ratios of different concentrations of the trigger RNA (VPS-G). (b) The corresponding image for the different trigger RNA of VPS-G. (c) ON/OFF chemiluminescence ratios of different concentrations of the trigger RNA (VPS-L). (d) The corresponding image for the different trigger RNA of VPS-L. (e) ON/OFF sfGFP fluorescence ratios of different concentrations of the trigger RNA with NASBA (VPS-G). (f) ON/OFF chemiluminescence ratios of different concentrations of the trigger RNA with NASBA (VPS-L).
Figure 6
Figure 6
Toehold switch sensor performance on V. parahaemolyticus. (a) ON/OFF fluorescence ratios of VPS-G for the detection of both dead and live V. parahaemolyticus with RPA. (b) ON/OFF chemiluminescence ratios of VPS-L for the detection of both dead and live V. parahaemolyticus with RPA. (c) ON/OFF fluorescence ratios of VPS-G for the detection of both dead and live V. parahaemolyticus with NASBA. (d) ON/OFF chemiluminescence ratios of VPS-L for the detection of both dead and live V. parahaemolyticus with NASBA.
Figure 7
Figure 7
The specificity of the cell-free toehold switch sensor for V. parahaemolyticus. (a) ON/OFF sfGFP fluorescence ratios of the different strains (VPS-G). (b) ON/OFF chemiluminescence ratios of the different strains (VPS-L). (c) ON/OFF sfGFP fluorescence ratios of the different mixed strains (VPS-G). (d) ON/OFF chemiluminescence ratios of the different mixed strains (VPS-L). Mix represents all test samples/bacteria mixture.
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