This research was funded by Megaproject of Infectious Disease Control from Ministry of Health of China, grant number 2017ZX10302301-005 and Sino-Africa Joint Research Center, grant number SAJC201605.

Ethical statement 
Wuhan Institute of Virology (WHIOV) is among the labs and institutes approved by China CDC of Wuhan city to conduct research on SARS-CoV-2 and detect COVID-19 from clinical samples. Research on developing new diagnostic techniques for COVID-19 using clinical samples has also been approved by the ethical committee of Wuhan Institute of Virology (2020FCA001).
1. Sample processing workflow (Figure 1A)
NOTE: Throughout the protocol, it is important to use separate rooms with dedicated pipettes for sample handling (extraction and storage), reagent/mastermix preparation and storage, reaction mix preparation (sample plus mastermix), and detection, to avoid cross contamination. The assays to be developed can be used in the detection of clinical samples or research samples. All samples should be treated as if they can transmit infectious agents even when using safe laboratory procedures. Sample processing steps should be done in a biosafety level 2 (BSL-2) laboratory following strict BSL-2 rules, including wearing of appropriate personal protective equipment (PPE).
<ol>
	<li>Sample inactivation
	<ol>
		<li>Take 1 mL of SARS-CoV-2 sample to a BSL-2 laboratory. Heat-inactivate samples at 65 &#176;C for 30 min. 
		NOTE: Samples may come from various sources, hence, one should make sure the volume to be inactivated is sufficient to ensure subsequent RNA extraction. Most extraction kits require a small sample volume of up to 200 &#181;L, hence, a sample volume of about 1 mL would be sufficient for inactivation. Surfactants, kits, and lysis buffers may also be used for direct inactivation and RNA extraction according to the labs own SOP.</li>
		<li>Take samples to a biosafety cabinet (BSC) and let them stand at room temperature for 10 min to allow potential aerosols to settle. Store samples in 4 &#176;C for up to 24 h if not processed immediately. 
		NOTE: No specific container is required for sample storage. Samples may be stored in the tubes or containers used during inactivation. For this work, most samples were stored in viral transport media (VTM) tubes or 1.5 mL tubes.</li>
	</ol>
	</li>
	<li>RNA extraction 
	NOTE: Many RNA extraction instruments and kits with specific protocols are available for ready use. Here, an automated procedure following the manufacturer&#39;s instructions is presented.
	<ol>
		<li>Take out the pre-filled 96-deep well orifice plate; invert it upside-down gently to mix the magnetic beads. After mixing, gently shake the plate on a flat surface to concentrate reagent and magnetic beads that may be on the wall to the plate&#39;s bottom.</li>
		<li>Carefully tear off the aluminum foil sealing film to avoid vibration of the plate and liquid spillage. Add 20 &#181;L of proteinase K and 200 &#181;L of the sample per well in rows 2 and 8 of the 96-well plate. Then, place the 96-well plate on the base of the 32-channel automatic nucleic acid extraction instrument. 
		NOTE: Run the program on the computer within 1 h after adding the sample.</li>
		<li>Insert the magnetic bar sleeve into the card slot of the 32-channel automatic nucleic acid extraction instrument. Select the program GF-FM502T5-TR&#34;1&#34;GF-FM502T5YH (quick version) and run it.</li>
		<li>After completion of the extraction, take out the nucleic acid samples in rows 6 and 12 (about 100 &#181;L/sample) and distribute in a clean nuclease-free 96-well plate. Store the samples at 4 &#176;C for up to 24 h until use or at -20 &#176;C for a longer time. 
		NOTE: It is recommended to use a 100 &#181;L multichannel pipette to transfer samples into a new 100-200 &#181;L plate as it can directly match the columns of the sample plate.</li>
	</ol>
	</li>
	<li>cDNA generation 
	NOTE: For samples stored for a long time, avoid multiple freeze/thaw cycles. Perform cDNA generation using a cDNA kit following the manufacturer&#39;s instructions.
	<ol>
		<li>In a 100 or 200 &#181;L PCR tube placed on ice/cooling block, add 2 &#181;L of 5x RT master mix (e.g., Perfect Real Time), 5 &#181;L of sample RNA, and 3 &#181;L of RNase free ddH2O per reaction (total volume 10 &#181;L).</li>
		<li>Place the PCR tube in a thermal cycler set to run at 37 &#176;C for 15 min (reverse-transcription), 85 &#176;C for 5 s (heat inactivation of reverse transcriptase), and 4 &#176;C for an infinite time. 
		&#8203;NOTE: Process the samples immediately, or store at 4 &#176;C for up to 24 h until use or at -20 &#176;C for up to 6 months.</li>
	</ol>
	</li>
</ol>
2. Optimization of ddPCR assay and workflow
NOTE: Optimize the assays before/after reading the droplets. Dependent on the results, they can be optimized at any point of the work to achieve better results. Below are some common factors to be considered when optimizing ddPCR experiments.
<ol>
	<li>Validate the ddPCR primers and probes (Table 1). 
	NOTE: The primers ORF1ab and N were adapted from China CDC17, the human endogenous control gene (RPP30) from Lu et al.18, and RBD2 from Nyaruaba et al.13. All primers and probes except RBD2 had already been ddPCR tested and optimized7,8,18.</li>
	<li>Run the ddPCR test sample controls comprising a positive control (a sample with all four targets), No Template Control (NTC; Nuclease free water or sample with no targets), and extraction/negative control (total nucleic acid extracted from a non-infectious cultured human cell or pooled samples from&#160;health volunteers). 
	NOTE: Standard control samples with known copies of target genes are preferred. However, in the absence of standards, use the available samples to define their controls or sample matrix. Run the controls together with test samples.</li>
	<li>Optimize the annealing temperature (Figure 2D) by inserting a temperature gradient of 55 &#176;C to 65 &#176;C in the annealing step of PCR (step 3.2). 
	NOTE: Annealing temperature optimization helps to find the best temperature where droplet separation is maximum (easy target identification) with minimal rain. After obtaining the results, narrow down the temperature range for better optimization, e.g., 55 &#176;C to 60 &#176;C.</li>
	<li>Optimize the primer and probe concentrations. If optimum separation between positive and negative droplets is not achieved in the assays, vary the primer (300-900 nM) and/or probe (100-400 nM) concentrations. 
	NOTE: Lowering primer/probe concentrations results in a lower target droplet amplitude and increasing it also increases the target&#39;s amplitude. This can be helpful in amplitude-based multiplexing.</li>
	<li>Test the analytical sensitivity considering different parameters including, limit of blank (LoB), limit of detection (LoD), specificity, and sensitivity using both standard and test samples, as preferred.</li>
</ol>
3. ddPCR workflow (Figure 1B) and assay development (Table 2)
NOTE: Like other ddPCR detection systems, this workflow also consists of four steps (Figure 1B), including reaction mix preparation, droplet generation, PCR amplification, and droplet reading.
<ol>
	<li>Reaction mix preparation 
	NOTE: Prepare all assays in a clean separate room and inside a BSC. Observe standard precautions, including use of clean gloves, clean pipettes, nuclease free water, and disinfectants. Aliquot reagents in separate tubes and store at -20 oC to avoid repeated freeze thawing cycles. Prepare assays as shown in Table 2. Primer and probe stock solutions should be stored in high concentrations of 100 &#181;M. The working solutions can be stored in aliquots of 20-40 &#181;M (diluted in nuclease free water).
	<ol>
		<li>Common steps in all assays 
		NOTE: Before preparing the assays, calculate the total volume of mastermix needed based on the number of samples. Here, each mastermix component volume was multiplied by 1.05 to account for any pipetting errors.
		<ol>
			<li>Thaw and equilibrate reaction materials to room temperature. Vortex the reactions components briefly (30 s) to ensure homogeneity, and briefly centrifuge to collect the contents at the bottom of the tube.</li>
			<li>Prepare a primer-probe (PP) mix per assay by mixing specific reaction volumes as detailed in Table 2 from the working solutions.</li>
			<li>Prepare the mastermix by mixing 11 &#181;L (1x) of 2x ddPCR supermix for probes (No dUTP), PP mix (dependent on assay as shown in Table 2 from the working solution) and nuclease free water to a final volume of 19.8 &#181;L per well. Distribute the mastermix into the wells of a nuclease-free 96-well ddPCR plate based on the number of samples. 
			&#8203;NOTE: For the sample plate, make sure all 8 wells in one column have the solution. If only a few wells are used, e.g., 5 samples, fill the other 3 wells with 22 &#181;L of nuclease free water or control buffer each.</li>
			<li>To get the final reaction mix volume&#160;(22 &#181;L), add 2.2 &#181;L of cDNA sample per well containing mastermix. Seal the plate with a disposable PCR plate sealer. 
			NOTE: Perform the sample additions in the designated room. Include the controls i.e., positive, NTC, and extraction. Additionally, if the RNA samples are of low concentration, reduce the volume of water in the mastermix and increase the sample volume accordingly up to 5.5 uL.</li>
			<li>Once the reaction mix is distributed per well, vortex briefly (15-30 s), and centrifuge (10-15 s) to collect the contents at the bottom of the plate. Proceed for droplet generation.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Automated droplet generation (Supplementary Figure 1) 
	NOTE: Different droplet generators can be used; however, this study was performed with an automated droplet generator (AutoDG). To avoid amplicon contamination, ensure the droplet generator and readers have dedicated space in separate areas. When loading consumables, it is recommended to start loading from the back of the instrument working toward the front to avoid moving hands over the consumables to avoid cross contamination.
	<ol>
		<li>Obtain all consumables needed to set up the AutoDG, including AutoDG oil for probes, DG32 Cartridges, pipette tips, cooling block, sample plate, droplet plate (new clean ddPCR plate), and a waste bin.</li>
		<li>On the AutoDG touch screen, touch Configure Sample Plate and select columns where samples are located on sample plate and press OK. 
		NOTE: The screen will turn yellow indicating LOAD where consumables should be loaded.</li>
		<li>Open the AutoDG door and load consumables in their respective places. 
		NOTE: The respective places where consumables should be loaded will have a yellow indicator that will later turn green if the consumables are loaded. This is similar to the touchscreen.</li>
		<li>Load the sample plate and droplet generation plate. 
		NOTE: The droplet generation plate should be placed on the cooling block. Ensure the cooling block is purple (ready to use) and not pink (should be kept in a freezer until the color turns back to purple).</li>
		<li>Close the AutoDG door, ensure the screen is green (consumables loaded), and select/ensure the oil type is&#160;Oil for Probes before pressing Start Droplet Generation.</li>
		<li>Wait for the droplets to be generated. Open the door once the screen shows Droplets Ready and remove the plate containing the droplets.</li>
		<li>Seal the plate with a pierceable foil seal using a plate sealer set to run at 185 oC for 5 s. Proceed to PCR amplification. 
		NOTE: PCR amplification should be started within 30 min after droplet generation.</li>
	</ol>
	</li>
	<li>PCR amplification
	<ol>
		<li>Insert the sealed droplet plate into a 96-deep well thermal cycler, set the sample volume to 40 &#181;L and lid temperature to 105 &#176;C.</li>
		<li>PCR amplify the droplets using the program: 95 &#176;C for 10 min (enzyme activation), 40 cycles of denaturation at 94 &#176;C for 30 s and 1 min annealing/extension at 57 &#176;C, enzyme deactivation at 98 &#176;C for 10 min, and holding step at 4 &#176;C indefinitely. After the PCR, read the droplets or store at 4 &#176;C for up to 24 h. 
		NOTE: Use a ramp rate of 2 &#176;C /s at all steps, as ramp rates may differ for different thermal cyclers. Hold the plate for at least 30 min at 4 &#176;C to allow droplet stabilization before reading the droplets.</li>
	</ol>
	</li>
	<li>Droplet reading
	<ol>
		<li>Transfer the thermal cycled 96-well plate into a droplet reader, and on a computer connected to the droplet reader, open/start the accompanying software.</li>
		<li>In the setup mode, select New and then double-click on any well to open the Well Editor dialog box. Select the wells to be read and choose Experiment: ABS, Supermix: ddPCR Supermix for Probes (no dUTP), Target 1 Type: Ch1 Unknown, Target 2 Type: Ch2 Unknown. 
		NOTE: Negative, blank, NTC, or positive can be selected from the target 1/2 type dropdown menu for the control samples.</li>
		<li>Assign the target or sample names based on plate layout and select Apply. When done, select OK, and save the created template. Click on Run and when prompted choose the right color (FAM/HEX). 
		NOTE: Priming the system before running is recommended before the first run of the day. Also, check whether all lights in the reader are green and if not, follow the recommended application on the tool tip status.</li>
		<li>After data acquisition is finished, remove the plate from the reader. Analyze data as required. 
		NOTE: Since preliminary results can be seen on the screen, running the positive samples in the first wells can be used for real-time quality check.</li>
	</ol>
	</li>
</ol>
4. Data analysis (Supplementary Figures 2 and 3)
<ol>
	<li>Check the data from all the wells for total number of droplets. If the droplet count is &#60;10,000, then discard the results and repeat the assay. Set a cut off to accept positive and negative results. E.g., A droplet count of up to 3 or more positive droplets can be considered as the cut off for positive results. 
	NOTE: Data for all assays, including simplex, duplex, triplex probe mix, and quadruplex assays are generated using the accompanying software. However, this software is only suitable for simplex and duplex assays. For high order multiplex assays (&#62;3 targets), an external software is needed.</li>
	<li>Simplex and duplex assays 
	NOTE: The external software can also analyze simplex and duplex data. However, using the accompanying software is easier for these assays, hence used in this article.
	<ol>
		<li>Double click on the .qlp file to be analyzed to open it and select Analyze.</li>
		<li>Use the threshold tools in the 1D Amplitude and 2D Amplitude to distinguish between positive and negative droplets for each well in the correct channel. The NTC and positive control samples can be used as a guidance for threshold setting. 
		&#8203;NOTE: FAM results are seen in Channel 1 while HEX/VIC in channel 2.</li>
		<li>After threshold setting, the results can be exported as a .csv file and further analyzed in Excel or recorded by reading directly on the results window.</li>
	</ol>
	</li>
	<li>Triplex probe mix (Supplementary Figure 2) and quadruplex assays (Supplementary Figure 2) 
	NOTE: Before analyzing the triplex probe mix and the quadruplex assay, download the external software. Refer to the minimal system requirements before installing.
	<ol>
		<li>Open the .qlp file by right clicking on it and choosing Open with the external software, or by opening the external software and clicking on the Browse option to locate the .qlp file in your folder, or by simply dragging the .qlp file and dropping it on the already open external software.</li>
		<li>In the plate editor tab, select the wells to be analyzed on the right side of the tab.
		<ol>
			<li>For triplex probe mix, select Direct Quantification (DQ) as experiment type, select Probe Mix Triplex as assay information and enter target names accordingly, and then click on Apply.</li>
			<li>For quadruplex, select Direct Quantification (DQ) as experiment type, select Amplitude Multiplex as assay information and enter target names accordingly, and then click on Apply.</li>
		</ol>
		</li>
		<li>On the left side of the 2D Amplitude tab, use the Graph Tools to assign specific colors to different target clusters following the Select to Assign Cluster window pop up suggestions. 
		NOTE: Preferred cluster mode can be applied based on the users&#39; preference.</li>
		<li>Once the target colors are assigned, quantification data can be read in the Well Data Window on the lower right. Export data to Excel/csv for further analysis using the triple-bar icon on the upper right hand of the well data table.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts to disclose.

Few resources are available on how to develop RT-ddPCR assays for SARS-CoV-2 detection. Though not used in this article, standard samples with known copies may be used to develop and optimize assays. In this work however, SARS-CoV-2 samples grown in Vero-E6 cells were spiked in a background of human genomic RNA and used as standard samples to develop the assays. Proper primer and probe sequences are essential when developing assays. Since most preliminary work on SARS-CoV-2 RT-ddPCR used the China CDC primer and probes t...

Polymerase chain reaction (PCR), a well-recognized technique, has undergone several transformations since its advent to become a powerful technique capable of providing answers to nucleic acid research. These transformations have been a constant improvement of the old technique. These transformations can be summarized into three generations1. The first generation is conventional PCR that relies on gel electrophoresis to quantify and detect amplified targets. The second generation is quantitative real time PCR (qPCR) that can detect samples in real time and rely on a standard curve to directly quantify targets in a sample. The third generation, digital PCR (dPCR), can perform both detection, and absolute quantification of nucleic acid targets without the need of a standard curve. dPCR has also been improved further from reaction chambers being separated by the wells of a wall into emulsions of oil, water, and stabilizing chemicals within the same well as seen in droplet-based digital PCR2. In droplet digital PCR (ddPCR), a sample is partitioned into thousands of nanoliter-sized droplets containing individual targets that will later be quantified using Poisson statistics2,3,4. This technique gives ddPCR an edge in quantifying low abundant targets when compared to the other generations of PCR.
Recently, multiple applications have highlighted the superiority of ddPCR over the commonly used qPCR when detecting and quantifying low abundant targets1,5,6. SARS-CoV-2 is no exception to these applications7,8,9,10,11,12. Since the outbreak of SARS-CoV-2, scientists have been working on all fronts to come up with solutions on how to diagnose the virus and detect it efficiently. The current gold standard still remains to be qPCR13. However, RT-ddPCR has been shown to be more accurate in detecting low abundant SARS-CoV-2 targets from both environmental and clinical samples when compared to RT-qPCR7,8,9,10,11,12. Most of the SARS-CoV-2 ddPCR published works depend on simplex assays with the multiplex ones depending on commercial assays. Hence, more should be done to explain how to develop multiplex RT-dPCR assays for SARS-CoV-2 detection.
In a proper assay design, multiplexing can be used to save on cost, increase sample throughput, and maximize on the number of targets that can be sensitively detected within a small sample. When multiplexing with ddPCR, one must take account of how many fluorophores can be detected in a particular system. Some ddPCR platforms can support up to three channels while others support only two channels. Hence, when multiplexing with two channels, one has to use different approaches, including higher order multiplexing to detect more than two targets14,15,16. In this work, a two color ddPCR detection system is used to show steps on how to develop different SARS-CoV-2 RT-ddPCR assays that can be adapted for different research applications.

<table><tbody><tr><td>32-channel fully automatic nucleic acid extractor Purifier 32</td><td>Genfine Biotech</td><td>FHT101-32</td><td>Automated extractor for RNA</td></tr><tr><td>AutoDG Oil for Probes</td><td>BioRad</td><td>12003017</td><td>QX200 AutoDG consumable</td></tr><tr><td>ddPCR 96-Well Plates</td><td>BioRad</td><td>12003185</td><td></td></tr><tr><td>ddPCR Supermix for Probes (No dUTP)</td><td>BioRad</td><td>1863024</td><td>Making ddPCR assay mastermix</td></tr><tr><td>DG32 AutoDG Cartridges</td><td>BioRad</td><td>1864108</td><td>QX200 AutoDG consumable</td></tr><tr><td>Electronic thermostatic water bath pot</td><td>Beijing Changfeng Instrument and Meter Company</td><td>XMTD-8000</td><td>Heat inactivation of samples</td></tr><tr><td>FineMag Rapid Bead Virus DNA/RNA Extraction Kit</td><td>Genfine Biotech</td><td>FMY502T5</td><td>Magnetic bead extraction of inactivated RNA samples</td></tr><tr><td>Pierceable Foil Heat Seals</td><td>BioRad</td><td>1814040</td><td></td></tr><tr><td>Pipet Tips for the AutoDG</td><td>BioRad</td><td>1864120</td><td>QX200 AutoDG consumable</td></tr><tr><td>Pipet Tip Waste Bins for the AutoDG</td><td>BioRad</td><td>1864125</td><td>QX200 AutoDG consumable</td></tr><tr><td>PrimeScript RT Master Mix (Perfect Real Time)</td><td>TaKaRa</td><td>RR036A</td><td>cDNA generation</td></tr><tr><td>PX1 PCR Plate Sealer</td><td>BioRad</td><td>1814000</td><td>Seal the droplet plate from AutoDG</td></tr><tr><td>QuantaSoft 1.7 Software</td><td>BioRad</td><td>10026368</td><td>Data acquisition and analysis</td></tr><tr><td>QuantaSoft Analysis Pro 1.0</td><td>BioRad</td><td>N/A</td><td>Data analysis</td></tr><tr><td>QX200 Automated Droplet Gererator (AutoDG)</td><td>BioRad</td><td>1864101</td><td>QX200 AutoDG consumable</td></tr><tr><td>QX200 Droplet Reader</td><td>BioRad</td><td>1864003</td><td>Droplet reading and data acquisition</td></tr><tr><td>T100 Thermal Cycler</td><td>BioRad</td><td>1861096</td><td>Droplet target amplification (PCR) and cDNA generation</td></tr></tbody></table>

two-step reverse transcription droplet digital pcr protocols for sars-cov-2 detection and quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

In a proof-of-concept study, the multiplex assays analytical performance was tested on clinical and research samples19. The performance of the multiplex assays was superior to that of an RT-PCR19. Since low numbers of droplets may indicate a problem during droplet generation, in this article a cutoff of 10,000 droplets per well was set based on empirical data.
A good separation between positive and negative droplets with minimal rain interference...

Watch this Scientific Journal Video about Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification at JoVE.com

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

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Research

JoVE Journal

Biology

4.5K Views.  Chinese Academy of Sciences. Deze protocollen kunnen worden gebruikt om interne multiplex ddPCR-assays te ontwikkelen voor detectie van SARS-CoV-2 met behulp van een tweekleurig ddPCR-systeem. Dit is handig voor mensen die hun eigen kits willen ontwikkelen voor de detectie van SARS-CoV-2 en andere ziekteverwekkers. Vergeleken met de gouden standaard RT-qPCR kan RT-ddPCR worden gebruikt om meerdere SARS-CoV-2-genen te kwantificeren zonder dat er een standaardcurve nodig is.Aangezien ddPCR een opkomende techniek is,...

Video: Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

The protocol is intended to serve as a blueprint for universities and other organizations considering large-scale testing for SARS-CoV-2 or developing preparedness plans for future viral outbreaks.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. ...

Genetics

Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples

Wastewater-based epidemiology has emerged as a promising and efficacious surveillance system for SARS-CoV-2 and other infectious diseases in many nations. The process typically involves wastewater concentration, nucleic acid extraction, amplification of selected genomic segments, and detection and quantification of the amplified genomic segment. This methodology can similarly be leveraged to detect and quantify infectious agents, such as SARS-CoV-2, in air samples. Initially, SARS-CoV-2 was presumed to spread primarily through close personal contact with droplets generated by an infected individual while speaking, sneezing, coughing, singing, or breathing. However, a growing number of studies have reported the presence of SARS-CoV-2 RNA in the air of healthcare facilities, establishing airborne transmission as a viable route for the virus. This study presents a composite of established protocols to facilitate environmental detection, quantification, and sequencing of viruses from both wastewater and air samples.

This protocol aims to quantify SARS-CoV-2 RNA in wastewater and air samples to be used for wastewater-based epidemiology studies and to assess the exposure risk to SARS-CoV-2 in indoor and outdoor aerosols. This protocol also describes a tiled amplicon long-template sequencing approach for SARS-CoV-2 whole genome characterization.

Wastewater-based epidemiology has emerged as a promising and efficacious surveillance system for SARS-CoV-2 and other infectious diseases in many nations. ...

Environment

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

Immunology and Infection

Quantifying Vaccine-Induced Neutralizing Antibodies in SARS-CoV-2 Variants

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

Standardized Protocol for Viral Genome Quantification Using Digital Droplet PCR

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/&#181;L), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/&#181;L and 10,000 copies/&#181;L, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

Author Spotlight: Optimizing Digital Droplet PCR Method for Accurate Adeno-Associated Viral Genome Quantification

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome titer.

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of ...

Bioengineering

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

Using Colorimetric and RT-LAMP Analysis to Detect SARS-CoV-2 

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.

Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP

Here we provide a complete protocol to standardize and implement the method for detecting the SARS-CoV-2 virus in human samples by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method, done within 60 min, could be adapted to any laboratory or point-of-care at a low cost and using inexpensive equipment.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern ...

Multiplex RT-qPCR Assay for Respiratory Virus Detection

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses 

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

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