All the procedures involving human participants are to be conducted in accordance with ethical standards and relevant guidelines, including the ethical principles for medical research involving human subjects established by the World Medical Association Declaration of Helsinki. This study was approved by the human research ethics committee under license protocol number CAAE: 80247417.4.0000.5190. Informed consent of all patients included in this study was waived by the Fiocruz-PE Institutional Review Board (IRB) for diagnostic samples.
NOTE: The PLUM device will be hereafter referred to as a 'portable plate reader'.
1. Computational design of nucleic acid sequence-based amplification primers
2. Computational design of toehold switches
Parameter | Definition |
Name | The desired names of the output toehold switch sequences. |
Outer sequence | Full NASBA transcript produced from amplification. |
Inner sequence | The outer sequence excluding the primer binding sites. It matches the outer sequence but excludes the portions of the transcripts that bind to the forward and reverse primers. |
Temperature | The temperature used by the algorithms to compute the RNA structures. |
Output name | The name of the output gene (e.g. lacZ, gfp). |
Output sequence | The sequence of the output gene. |
Table 1: The definition of each parameter used in the toehold switchdesign software.
3. Construction of toehold switches by PCR
NOTE: These steps describe the construction of LacZ toehold switches by overlap extension PCR. Here, the DNA oligo is used as a forward primer and the T7 terminator is used as a reverse primer. We use the pCOLADuet-LacZ plasmid as a template for the lacZ gene (addgene: 75006). Any other DNA templates that contain the corresponding sequence can be used as templates, provided that the T7 terminator is included in the final construct.
Component | Volume | Concentration |
5X Q5 Reaction Buffer | 10 µL | 1x |
10 mM dNTPs | 1 µL | 200 µM |
10 mM Forward Primer (Synthetic Switch DNA FW) | 2.5 µL | 0.5 µM |
10 mM Reverse Primer (T7 terminator RV) | 2.5 µL | 0.5 µM |
Template DNA (pCOLADuet-LacZ) | variable | <1 ng |
Q5 High-Fidelity DNA Polymerase | 0.5 µL | 0.02 U/µL |
Nuclease-free water | to 50 µL | – |
Table 2: The PCR components used to construct toehold switches.
Step | Temperature | Time | |
Initial Denaturation | 98 °C | 30 s | |
35 Cycles | Denaturation | 98 °C | 10 s |
Annealing | 60 °C | 20 s | |
Extension | 72 °C | 1.45 min | |
Final extension | 72 °C | 5 min | |
Hold | 4 °C | – |
Table 3: Cycling conditions used during construction of toehold switches by PCR.
4. Preparation of synthetic RNA target (Trigger)
5. In vitro transcription of selected trigger sequences
Component | Volume | Concentration |
10X Reaction Buffer | 1.5 µL | 0.75x |
25 mM NTP mix | 6 µL | 7.5 mM |
Template trigger DNA | X µL | 1 µg |
T7 RNA Polymerase Mix | 1.5 µL | – |
Nuclease-free water | X µL | To 20 µL |
Table 4: In vitro transcription (IVT) of selected trigger sequences.
6. Initial screening of the switches
NOTE: This section describes the steps associated with setting up cell-free, paper-based toehold switch reactions, and how to screen for high-performing toehold switches. The BSA blocked filter paper used in step 6.10 should be prepared in advance as described in the Supplementary Protocol.
Component | Volume | Final Concentration Per Reaction |
Solution A | 2.38 µL | 40% |
Solution B | 1.78 µL | 30% |
RNase Inhibitor | 0.03 µL | 0.5% v/v |
CPRG (25 mg/mL) | 0.14 µL | 0.6 mg/mL |
Toehold Switch | X µL | 33 nM |
Target RNA | X µL | 1 µM |
Nuclease-free water | to 5.94 µL | – |
Total volume: | 5.94 µL |
Table 5: PURExpress cell-free transcription-translation reaction components.
7. Identifying high-performing toehold switches
NOTE: This section describes how to analyze data from step 6 in order to select the best performing toehold switches to move forward with.
8. Nucleic acid sequence-based amplification primer screening and sensitivity
NOTE: In the following steps, first a screen for functional isothermal amplification primers is done, and then their sensitivity is assessed by determining the number of target RNA copies per µL of synthetic RNA that a given toehold switch can reliably detect when coupled with isothermal amplification. Following isothermal amplification, perform cell-free reactions to identify successful nucleic acid sequence-based amplification primer sets. However, it may be more cost-effective to run polyacrylamide or agarose gels on nucleic acid sequence-based amplification reactions to first narrow the pool of candidate primer sets. In that case, nucleic acid sequence-based amplification primer sets that generate a band on the gel at the appropriate amplicon size can be shortlisted for subsequent cell-free screening.
Component | Volume per reaction | Final Concentration |
NASBA Reaction Buffer | 1.67 µL | 1x |
NASBA Nucleotide Mix | 0.833 µL | 1x |
25 µM Forward Primer | 0.1 µL | 0.5 µM |
25 µM Reverse Primer | 0.1 µL | 0.5 µM |
RNase Inhibitor (40 U/µL) | 0.05 µL | 0.4 U/µL |
Target RNA | 1 µL | |
NASBA Enzyme Mix | 1.25 µL | 1x |
Total volume | 5 µL |
Table 6: NASBA reaction components.
Step | Temperature | Time |
Denaturation | 65 °C | 2 min |
Equilibration | 41 °C | 10 min |
Hold | 41 °C | ∞ |
Incubation | 41 °C | 1 h |
Hold | 4 °C | – |
Table 7: Reaction conditions for the NASBA.
Component | Volume | Final Concentration Per Reaction |
Solution A | 2.38 µL | 40% |
Solution B | 1.78 µL | 30% |
RNase Inhibitor | 0.03 µL | 0.5% v/v |
CPRG (25 mg/mL) | 0.14 µL | 0.6 mg/mL |
Toehold Switch | X µL | 33 nM |
Target RNA (if applicable) | X µL | 1 µM |
NASBA (if applicable) | 0.85 µL | 1:7 |
Nuclease-free water | to 5.94 µL | – |
Total volume: | 5.94 µL |
Table 8: Paper-based cell-free reaction components.
9. Patient samples collection and viral RNA extraction
NOTE: This section describes the protocol to collect patient samples and to extract the RNA using an RNA purification kit. The protocol below is used to obtain serum from peripheral blood. The samples used in this study were collected from patients presenting fever, exanthema, arthralgia, or other related symptoms of arbovirus infection in Pernambuco state, Brazil.
10. Portable plate reader device
11. RT-qPCR for Zika virus detection
NOTE: This section outlines the steps to perform the RT-qPCR for Zika virus detection from patient samples (see Supplementary Protocol).
Component | Volume | Concentration |
2X QuantiNova Probe RT-PCR Master Mix | 5 µL | 1 X |
100 µM Forward Primer | 0.08 µL | 0.8 µM |
100 µM Reverse Primer | 0.08 µL | 0.8 µM |
25 µM Probe | 0.04 µL | 0.1 µM |
QuantiNova ROX Reference Dye | 0.05 µL | 1 X |
QuantiNova Probe RT Mix | 0.1 µL | 1 X |
Template RNA | 3.5 µL | – |
Nuclease-free water | to 10 µL | – |
Table 9: RT-qPCR components to amplify Zika virus RNA based on Centers for Disease Control and Prevention–CDC USA protocol to detect Zika virus from patient samples31.
Step | Temperature | Time | |
Reverse transcription | 45 °C | 15 min | |
PCR initial activation step | 95 °C | 5 min | |
45 Cycles | Denaturation | 95 °C | 5 s |
Combined annealing/extension | 60 °C | 45 s |
Table 10: Cycling conditions for RT-qPCR.
Following the computational design pipeline, the construction of three toehold switches was performed by PCR. The PCR products were analyzed using agarose gel electrophoresis (Figure 2). The presence of a clear band around 3,000 bp, roughly the size of the lacZ gene coupled to a toehold switch, typically indicates a successful reaction. Alternatively, a lane without a band, multiple bands, or a band of the incorrect size, indicates a failed PCR. In the case of a failed PCR, the reaction conditions and/or primer sequences should be optimized.
The assembled toehold switches were screened to assess each sensor against its respective in vitro transcribed trigger RNA (Figure 3). While all three sensors displayed an increased OD570 absorbance, switch 27B (Figure 3A) had the most rapid on-rate. Switches 33B and, to a lesser extent, 47B showed an increased OD570 absorbance in the absence of target trigger RNA, indicating that these switches possess some background activity or leakiness (Figure 3B,C)-a trait not wanted in a candidate sensor as it can reduce specificity. To more clearly identify the sensor with the highest ON/OFF signal ratio, the fold change of OD570 absorbance was calculated (see supplemental information section 5) and plotted (Figure 4). From this analysis, it is clear that switch 27B is the sensor that has the best performance with an ON/OFF ratio of around 60.
The sensitivity of the top-performing toehold switch (27B) was then evaluated by determining the lowest RNA concentration required to activate the toehold switch when coupled with a NASBA reaction. The graph illustrates that the top-performing Zika sensors can detect RNA at concentrations as low as 1.24 molecules per µL (equivalent to ~2 aM; Figure 5).
After switch 27B was identified and validated, the sensor materials were distributed to the team members in Recife in the Pernambuco state of Brazil. In Brazil, the clinical diagnostic accuracy of the Zika virus diagnostic platform was assessed using Zika virus patient samples, in parallel with RT-qPCR for comparison. To validate the paper-based Zika diagnostic platform, the portable plate reader was used, which is capable of incubating and reading the colorimetric output of paper-based sensors. The color change from yellow to purple is used to identify a positive sample, while a negative sample remains yellow (Figure 6). An additional option for visualizing the results generated by the portable plate reader (Figure 8) is to plot the colorimetric response for each paper-based reaction over time. Samples were tested in triplicate and those that exceeded the threshold (red line set to 1) were considered positive, while samples below the threshold were considered negative (Figure 6 and Figure 7).
Finally, to evaluate and compare the clinical performance of the Zika toehold switch sensor with the current gold standard method for diagnosing Zika virus infection, all patient samples were tested in parallel with RT-qPCR. The amplification plot of two representative patient samples was tested in triplicate for the detection of Zika virus by RT-qPCR (Figure 9). The samples are considered positive when the cycle threshold (Ct) value is ≤38; the red line indicates a positive sample and the blue line indicates a negative sample for Zika virus.
Figure 2: Agarose gel electrophoresis to assess the quality of PCR products. PCR products are analyzed on a 1% agarose gel in 1X TAE, run at 80 V for 90 min. A single clear band typically indicates a successful reaction. Lane 1: 1 kb DNA ladder; Lanes 2-4: 27B switch DNA, 33B trigger DNA, and 47B trigger DNA, respectively.The numbers on the left-hand side represent band size in bp. Please click here to view a larger version of this figure.
Figure 3: Prototyping three toehold switches for paper-based Zika sensors. The performance of three paper-based RNA toehold switch sensors was measured at 37 °C for over 130 min. Each graph contains two traces, one represents the switch only control, while the other represents the switch and trigger. The three graphs represent data acquired using toehold switch sensors 27B (A), 33B (B), and 47B (C). Error bars represent the standard error of the mean (SEM) from three replicates. Please click here to view a larger version of this figure.
Figure 4: Top-performing sensors are identified by calculating the fold change in absorbance at 570 nm. Fold change (or maximum ON/OFF ratio) is calculated by measuring the ratio of absorbance (OD570) at 130 min between the switch only control, and the switch plus trigger CFPS assay. Error bars represent SEM from three replicates. Please click here to view a larger version of this figure.
Figure 5: Sensitivity assessment of top-performing switch. In vitro transcribed Zika RNA is titrated into NASBA reactions. After a 1 h incubation, the reactions were added to cell-free PURExpress reactions on paper discs at a ratio of 1:7. The fold change after 130 min at 37 °C is plotted. This figure has been reproduced from24. Please click here to view a larger version of this figure.
Figure 6: Portable plate reader capture page loaded with captured image data. This figure shows a sample picture of the final image captured by the portable plate reader during a data collection run. The original date/time stamp is visible at the top of the image. Yellow color indicates control or negative reactions, and the purple color indicates a positive reaction. Please click here to view a larger version of this figure.
Figure 7: Data analysis mode. On the left, users select the data sets they would like to plot; the graphs are then displayed on the right with unique colors for each sample or control set. The dashed red line serves as a threshold for determining positive and negative samples. Samples tested in triplicate that exceed the threshold are considered positive, while samples below the threshold are deemed negative. Error bars represent standard deviation (SD) from three replicates. Ctrl 1 to Ctrl 5 indicates controls. Please click here to view a larger version of this figure.
Figure 8. PLUM, a portable plate reader. This portable plate reader acts as a lab-in-a-box and serves as a temperature-controlled plate reader to incubate and monitor colorimetric reactions. This portable device can provide quantitative and high-throughput measurements of the paper-based Zika sensors on-site. Please click here to view a larger version of this figure.
Figure 9: RT-qPCR plot of the amplification of two patient samples tested in triplicate for the detection of the Zika virus. Samples are considered positive when the cycle threshold (Ct) value is ≤38. The dotted red line serves as a threshold for determining positive and negative samples. The red trace indicates a positive sample, and the blue trace indicates a negative sample. ΔRn (delta Rn) value represents the normalized magnitude of the fluorescence signal detected by the RT-qPCR instrument for all the samples tested. Please click here to view a larger version of this figure.
Supplementary Protocol File. Please click here to download this File.
Supplemental Figures. Please click here to download these figures.
384 well plate covers – aluminum | Sarstedt | 95.1995 | Used to cover the 384-well plates before they are inserted into the PLUM reader |
384 well plate covers – transparent | Sarstedt | 95.1994 | Used to cover the 384-well plates before they are inserted into the BioTek plate reader |
384 well plates | VWR | CA11006-180 | 2 mm paper-based diagnostics are placed into the wells of these plates for quantification |
Agarose | BioShop Canada | AGA001.500 | Gel electrophoresis |
BSA | BioShop Canada | ALB001.500 | Blocking agent for the Whatman filter paper |
Cell free reactions | New England Biolabs | E6800L | PURExpress |
CPRG | Roche | 10884308001 | Chlorophenol red-b-D-galactopyranoside |
Disposable Sterile Biopsy Punches | Integra Miltex | 23233-31 | Used to create 2 mm paper discs that fit into a 384-well plate |
DNAse I | Thermo Scientific | K2981 | Digests template DNA following incubation of the in vitro transcription reaction |
DNAse I Kit | Thermo Scientific | 74104 | DNase I Kit For removing template DNA from IVT RNA |
dNTPs | New England Biolabs | N0446S | Used for PCRs |
electrophoresis system | Bio-Rad | 1704487 | Used to run the agarose gels |
Gel imaging station | Bio-Rad | 1708265 | ChemiDoc XRS+ Imaging System |
IVT kit | New England Biolabs | E2040S | Used for in vitro transcribing template (trigger) RNA for switch screening |
Nanodrop One | Thermo Scientific | ND-ONE-W | Used for determining nucleic acid concentrations |
NASBA kit | Life Sciences Advanced Technologies | NWK-1 | Isothermal amplification reaction components |
Nuclease free H20 | invitrogen | 10977015 | Added to reaction mixes |
PAGE electrophoresis system | Biorad | 1658001FC | Used to cast and run polyacrylamide gels |
pCOLADuet-LacZ DNA | Addgene | 75006 | https://www.addgene.org/75006/ |
Phusion polymerase/reactioin buffer | New England Biolabs | M0530L | Used for PCRs |
Plate reader | BioTek | BioTek NEO2 | Multi Mode Plate Reader, Synergy Neo2 |
Primers | Integrated DNA Technologies | Custom oligo synthesis | |
Q5 polymerase/reaction buffer | New England Biolabs | M0491L | Used for PCRs |
Qiagen PCR Puriifcation Kit | QIAGEN | 27106 | QIAprep Spin Miniprep Kit |
RNA loading dye | New England Biolabs | B0363S | 2X RNA loading dye |
RNA Purification Kit | QIAGEN | EN0521 | QIAamp Viral RNA extraction kit |
RNase inhibitor | New England Biolabs | M0314S | Used to prevent contamination of RNases A, B, and C |
RT-qPCR kit | QIAGEN | 208352 | QuantiNova Probe RT-PCR Kit |
SYBR Gold | Invitrogen S11494 | S11494 | PAGE gel stain for nucleic acids |
TAE Buffer | BioShop Canada | TAE222.4 | Gel electrophoresis buffer |
Thermal Cycler | Applied Biosystems | 4484073 | Used for temperature cycling and incubating reactions |
Whatman 42 filter paper | GE Healthcare | 1442-042 | Used to imbed molecular components for paper-based diagnostics |
Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.
Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.
Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.