Materials
Whatman Grade 1 Qualitative Filter Paper was purchased from GE Healthcare Worldwide (UK). Microfluidic channels were wax printed by a Xerox ColorQube 8580 digital wax printer from Xerox (UK). Black Cast Acrylic was obtained from Stockline Plastics (UK) and was processed by a laser cutter from Laserscript (UK). A Bio-Rad C1000 Thermal Cycler, horizontal electrophoresis apparatus and a Gel Doc XR + Imager were from Bio-Rad Laboratories (UK). A hot plate, Digital Dry Bath, and UV torch (366 nm) were from Fisher Scientific (UK). A QuantStudio 3 Real-Time PCR System, TaqMan Fast Virus 1-Step Master Mix, Qubit 4 Fluorometer and MicroAmp Optical Adhesive Film were from Thermo Fisher Scientific (UK).
Brilliant III Ultra-Fast SYBR Green QPCR Master Mix was purchased from Agilent Technologies (UK), while GspSSD2.0 LF DNA Polymerase and AMV-Reverse Transcriptase were obtained from OptiGene (UK). EvaGreen was from Cambridge BioScience (UK), while calcein, manganese (II) chloride, syringes, syringe filters and Swinnex filter holders were from SIGMA (UK). Punchers were from Kai Europe GmbH (Germany). The RNeasy Mini Kit and Zymoclean Gel DNA Recovery Kit were from Qiagen (USA) and Zymo Research (Germany), respectively. The HiScribe T7 High Yield RNA Synthesis Kit and Monarch RNA Cleanup Kit were from New England Biolabs (UK). All primers, DNA fragments and plasmids were ordered from SIGMA (UK), Eurofins Genomics (Germany) and GENEWIZ (Germany).
Preparation Of Template Dna And Rna
Six sets of LAMP primers were selected for the ORF1ab36, S37 and N38 genes on SARS-CoV-2, as well as H1N139, H7N940 and influenza B41 genotypes. For initial optimization experiments, we designed 6 DNA fragments containing target sequences and primers with a T7 RNA polymerase promoter, Fig. 2. Details of these LAMP primers and PCR primers for in vitro transcription are provided in Table S1 and Table S2. Intact viral RNA was also extracted from samples using a RNeasy Mini Kit.
For in vitro transcribed RNA template preparation, the target sequence was first PCR-amplified with PCR primers containing the T7 RNA polymerase promoter. The PCR reaction was performed in a total volume of 20 µL, comprising 10 µL PCR Master Mix, 0.8 µL PCR primers (0.4 µm), 1 µL EvaGreen, 7.2 µL ddH2O and 1 µL DNA fragment (5 ng µL− 1). The PCR conditions consisted of an initial denaturation step at 95°C for 7 min, followed by 40 cycles, with denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min 30 s. The PCR amplicons were analyzed on 1.5% agarose gel stained with SYBR Safe DNA gel stain, then extracted and purified from agarose gel using a Zymoclean Gel DNA Recovery Kit to form an in vitro transcription template. Thereafter, the purified PCR amplicons were incubated at 37°C for 2 hours to synthesize artificial RNA using the HiScribe T7 High Yield RNA Synthesis Kit. Next, the synthesized RNA was treated with DNase I to remove the DNA template and purified using a Monarch RNA Cleanup Kit. Finally, the purified RNA was stored at -80°C for further use.
For quantification of the template nucleic acid, the copy number of each template nucleic acid was determined by their molecular weight. The Qubit 4 Fluorometer with Qubit 1X dsDNA HS Assay Kit or Qubit RNA HS Assay Kit (Thermo Fisher Scientific, UK) was used to measure the concentration of the template DNA or RNA, the DNA/RNA Copy Number Calculator (http://endmemo.com/bio/dnacopynum.php) was then used to calculate the copy number of the template nucleic acid. The target sequence detected by LAMP was first amplified by real-time PCR with LAMP outer primers to confirm the accuracy of target detection. Subsequently, 10-fold serial dilutions of template nucleic acid were performed for sensitivity testing and to produce a standard curve.
Rt-qpcr Assay
The real-time RT-qPCR assay was performed on a QuantStudio 3 Real-Time PCR System. The qPCR primers sets for ORF1ab42, S43 and N42 genes, as well as H1N144, H7N945 and influenza B46 genotypes, porcine reproductive and respiratory syndrome virus (PRRSV)47 and mitochondrial DNA (mtDNA)48 are detailed in Table S3. mtDNA was chosen as the target of the internal positive control while ddH2O was added to the internal negative control. All RT-qPCR reactions were performed in triplicate in a total volume of 20 µL, including 6 µL TaqMan Fast Virus 1-Step RT-PCR Master Mix, 1.25 µL PCR primers, 7.75 µL ddH2O and 5 µL sample. The RT-qPCR conditions consisted of a reverse transcription step at 50°C for 5 min, an initial denaturation step at 95°C for 20 s, followed by 50 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and an extension at 72°C for 30 s.
Rt-lamp Assay
The real-time RT-LAMP assay was performed using a thermocycler. The LAMP primer sets for ORF1ab36, S37, N38, H1N139, H7N940, FluB41, PRRSV49 and mtDNA50 are detailed in Table S1. All RT-LAMP reactions were performed in triplicate in a total volume of 20 µL, including 10 µL LAMP Master Mix, 3 µL LAMP primers (0.1 µm F3/B3, 0.8 µm FIP/BIP, 0.6 µm LF/LB), 2 µL calcein (25 µM), 1 µL MnCl2 (500 µM), 1 µL Gsp 2.0, 0.2 µL reverse transcriptase, 0.8 µL ddH2O and 2 µL sample. The RT-LAMP was carried out at a constant reaction temperature of 63℃ for 60 min. In addition, the RT-LAMP assay was performed in the digital dry bath. In an RT-LAMP assay, calcein was used as a colorimetric indicator51. Positive results can be determined visually from the color change of the reaction solution from yellow to green. Results can also be read by a hand-held UV torch or digitally collected using a mobile phone camera.
Design, Characterization, And Optimization Of Paper Device With High-throughput Detection
The paper device was first characterized with geometries optimized using a model pathogen, PRRSV, as a surrogate, see Figure S1. Subsequently, the design of the paper-based device for the detection of 3 targets was defined and is shown in Fig. 1a. The device contains three components, including a filter paper-based microfluidic device with wax-printed microfluidic channels, a plastic device sealed with a single-sided optical film and one glass fiber circular disc (4 mm in diameter) for absorbing nucleic acids from the sample. For the 5 reaction chambers in the plastic device, N represents the internal negative control, T1, T2 and T3 represent the targets, and P represents the internal positive control.
The unfolded paper device comprised a sample preparation zone and a detection zone and was mounted to a plastic plate to show the detection results. The paper unit for each panel had a footprint of 3 cm × 3 cm (3 × 24 cm when unfolded). The paper device was fabricated on the Whatman Grade 1 Qualitative Filter Paper in three steps (Fig. 1c). First, the structure of the device was designed in the software CorelDRAW, with factors such as operation convenience being taken into consideration during the design process. Then, the device was fabricated by printing hydrophobic wax on the filter paper, defining the microfluidic channels. Finally, the patterned filter paper was baked on a hot plate at 130°C for 5 min to melt the printed wax into the porous structure of the filter paper (preventing lateral flow by capillarity). The wax-penetrated filter paper was cut into individual devices for subsequent LAMP experiments.
Paper-based Device For Pathogen Detection
This work demonstrated a paper-based platform for multiplex detection of SARS-CoV-2 and influenza viruses in wastewater. Figure S10 illustrates the workflow of the platform for virus detection in wastewater, including wastewater concentration, paper-based nucleic acid extraction, device-based RT-LAMP reaction, and signal read-out. In this case, a volume of wastewater was first filtered through syringe filters (0.45 µm and 0.025 µm), followed by the addition of lysis buffer, after which the filtrate was incubated at room temperature for 10 minutes (the filter pores are large enough to allow the viruses to pass through52).
The paper device was assembled with a glass fiber within a 4 mm hole punched into the printed panel. The lysate was introduced onto the glass fiber, where nucleic acids were adsorbed (the excess liquid was absorbed by the hydrophilic disc present on the third panel of the paper device). Any debris, including cell membrane residues were rinsed off using washing buffer. The paper device was then folded for elution and nucleic acids were released from the glass fiber, reaching the printed hydrophilic channels. Circular spots, defined by punching, formed the detection zone, contained within sample chambers of the plastic plate (which also contained RT-LAMP reagents). An optical adhesive film was also used to cover the plastic plate to avoid reagent evaporation during isothermal amplification in a digital dry bath. Finally, the results were read using a hand-held UV torch and the fluorescence signal was captured using a mobile phone camera.
The whole sample-to-answer process took < 1.5 hours and only required basic experimental equipment and consumables (flasks and syringes). In contrast, the workflow for the reference methods, performed on wastewater samples, and involving ultrafiltration concentration, kit-based RNA extraction and RT-qPCR assay took ~ 4 hours. With the modification of operating procedures, process workflow and experimental reagents (e.g., change the pore size of the syringe filter or LAMP primers), this paper-based platform can be readily extended to detect other viruses and bacteria in various environmental water samples, establishing new avenues for low-cost, rapid, and user-friendly multiplex detection of pathogens.
Sars-cov-2 Detection In Hotels
Autosamplers were used for wastewater sampling in the four quarantine hotels around London Heathrow Airport in the summer of 2021. Sampling personnel were clothed in standard personal protective equipment (PPE) used for wastewater sampling, such as protective clothing, safety glasses, face masks, and gloves. The autosampler was programmed to collect composite wastewater samples (50 mL wastewater samples every 30 minutes) over a 24-hour period, i.e., the samples that flowed through the sampling point over a day. An aliquot of each sample collected by the autosampler was transferred to 1 L plastic bottles, stored in a thermostatic bag filled with ice packs for sample transport, and then transferred to the hotel basement for sample analysis, showing in Figure S11. The process for on-site SARS-CoV-2 detection is presented in the section Paper-based device for pathogen detection, and the analytical reagents and paper-based kits were transported to the testing hotel from Cranfield University.