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>

<ol>
	<li>Nyaruaba, R., Mwaliko, C., Kering, K. K., Wei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+digital+PCR+applications+in+the+tuberculosis+world.">Droplet digital PCR applications in the tuberculosis world.</a> Tuberculosis. 117, 85-92 (2019).</li><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR+hits+its+stride.">Digital PCR hits its stride.</a> Nature Methods. 9 (6), 541-544 (2012).</li><li>Quan, P. L., Sauzade, M., Brouzes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dPCR:+a+technology+review.">dPCR: a technology review.</a> Sensors. 18 (4), 1271 (2018).</li><li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR.">Digital PCR.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (16), 9236 (1999).</li><li>Kuypers, J., Jerome, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+digital+PCR+for+clinical+microbiology.">Applications of digital PCR for clinical microbiology.</a> Journal of Clinical Microbiology. 55 (6), 1621 (2017).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+droplet+digital+PCR+to+detect+the+pathogens+of+infectious+diseases.">Application of droplet digital PCR to detect the pathogens of infectious diseases.</a> Bioscience Reports. 38 (6), (2018).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+comparisons+of+SARS-COV-2+detection+by+qRT-PCR+and+ddPCR+with+multiple+primer/probe+sets.">Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets.</a> Emerging Microbes &amp; Infections. 9 (1), 1175-1179 (2020).</li><li>Suo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ddPCR:+a+more+accurate+tool+for+SARS-CoV-2+detection+in+low+viral+load+specimens.">ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens.</a> Emerging Microbes &amp; Infections. 9 (1), 1259-1268 (2020).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerodynamic+analysis+of+SARS-CoV-2+in+two+Wuhan+hospitals.">Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals.</a> Nature. 582 (7813), 557-560 (2020).</li><li>Lv, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+SARS-CoV-2+RNA+residue+on+object+surfaces+in+nucleic+acid+testing+laboratory+using+droplet+digital+PCR.">Detection of SARS-CoV-2 RNA residue on object surfaces in nucleic acid testing laboratory using droplet digital PCR.</a> Science of The Total Environment. 742, 140370 (2020).</li><li>Yu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+detection+and+viral+load+analysis+of+SARS-CoV-2+in+infected+patients.">Quantitative detection and viral load analysis of SARS-CoV-2 in infected patients.</a> Clinical Infectious Diseases. 71 (15), 793-798 (2020).</li><li>Scutari, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+SARS-CoV-2+infection+associated+with+viral+dissemination+in+different+body+fluids+including+bile+in+two+patients+with+acute+cholecystitis.">Long-term SARS-CoV-2 infection associated with viral dissemination in different body fluids including bile in two patients with acute cholecystitis.</a> Life. 10 (11), 302 (2020).</li><li>Nyaruaba, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+field-deployable+RT-qPCR+workflow+for+COVID-19+detection.">Development of a field-deployable RT-qPCR workflow for COVID-19 detection.</a> Preprints. , (2020).</li><li>Whale, A. S., Huggett, J. F., Tzonev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+multiplexing+with+digital+PCR.">Fundamentals of multiplexing with digital PCR.</a> Biomolecular Detection and Quantification. 10, 15-23 (2016).</li><li>Dobnik, D., &#352;tebih, D., Blejec, A., Morisset, D., &#381;el, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+quantification+of+four+DNA+targets+in+one+reaction+with+Bio-Rad+droplet+digital+PCR+system+for+GMO+detection.">Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.</a> Scientific Reports. 6 (1), 35451 (2016).</li><li>Nyaruaba, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+single+dye+duplex+droplet+digital+PCR+assay+for+the+rapid+detection+and+quantification+of+mycobacterium+tuberculosis.">Development and evaluation of a single dye duplex droplet digital PCR assay for the rapid detection and quantification of mycobacterium tuberculosis.</a> Microorganisms. 8 (5), 701 (2020).</li><li>Lu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+detection+using+digital+PCR+for+COVID-19+diagnosis,+treatment+monitoring+and+criteria+for+discharge.">SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge.</a> medRxiv. , (2020).</li><li>Nyaruaba, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+multiplex+ddPCR+assays+for+SARS-CoV-2+detection+based+on+probe+mix+and+amplitude+based+multiplexing.">Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing.</a> Expert Review of Molecular Diagnostics. , 1-11 (2020).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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) ...

two-step-reverse-transcription-droplet-digital-pcr-protocols-for-sars

Microsoft Bing

Research

JoVE Journal

Biology

4.4K Views.  Chinese Academy of Sciences. 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.

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

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 ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

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 ...

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

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 ...

Improved Protein Quantification Using Two-Step Mitochondrial Isolation

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Author Spotlight: Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial ...

Quantification of Viral Vectors in Tears Using ddPCR for Bioshedding Studies

A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears

The use of viral vectors to treat genetic diseases has increased substantially in recent years, with over 2,000 studies registered to date. Adeno-associated viral (AAV) vectors have found particular success in the treatment of eye related diseases, as exemplified by the approval of voretigene neparvovec-rzyl. To bring new therapies to market, regulatory agencies typically request qualified or validated bioshedding studies to evaluate release of the vector into the environment. However, no official guidelines for the development of molecular based assays to support such shedding studies have been released by the United States Food and Drug Administration, leaving developers to determine best practices for themselves. The purpose of this protocol is to present a validatable protocol for the detection of AAV vectors in human tears by droplet digital polymerase chain reaction (ddPCR) in support of clinical bioshedding studies. This manuscript discusses current industry approaches to molecular assay validation and demonstrates that the method exceeds the target assay acceptance criteria currently proposed in white papers. Finally, steps critical in the performance of any ddPCR assay, regardless of application, are discussed.

Author Spotlight: Addressing Regulatory Gaps in Molecular Studies by Quantifying Viral Vectors in Complex Matrices 

Here, we present a protocol for the development and validation of good laboratory practices in the compliant detection of adeno-associated viral vectors in human tears by droplet digital polymerase chain reaction in support of clinical development of gene therapy vectors.