Name: two-step reverse transcription droplet digital pcr protocols for sars-cov-2 detection and quantification
Uploaded: 2021-03-31T13:30:00
Description: Watch this Scientific Journal Video about Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification at JoVE.com

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)
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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) ...

These protocols can be used to develop in-house multiplex ddPCR assays for detection of SARS-CoV-2 using a two-color ddPCR system. This is useful for people who want to develop their own kits for the detection of SARS-CoV-2 and other pathogens. Compared to the gold standard RT-qPCR, RT-ddPCR can be used to quantify multiple SARS-CoV-2 genes without the need of a standard curve.Since ddPCR is an emerging technique, visual demonstration is important to help current and future users understand and easily develop multiplex assays for SARS-CoV-2 detection and other pathogens. For cDNA synthesis, add two microliters of 5X reverse transcription master mix, five microliters of the RNA sample of interest, and three microliters of RNase-free double distilled water into a 100 or 200 microliter PCR tube to a final volume of 10 microliters as solution, and place the tube on ice. When off the reagents have been added, load the sample into a thermal cycler and set the instrument to run a reverse transcription at 37 degrees Celsius for 15 minutes, a heat inactivation of the reverse transcription for 5 seconds at 85 degrees Celsius, and a final infinite hold at four degrees Celsius.Before performing droplet digital PCR, add 19.8 microliters of freshly prepared master mix to the appropriate number of wells of a nuclease free 96-well droplet digital PCR plate. Add 2.2 microliters of cDNA to each well of master mix to a final volume of 22 microliters of reaction mix per well and seal the plate with a disposable PCR plate sealer. Then, vortex the plate for 15 to 30 seconds in centrifuge for 10 to 15 seconds to collect the well contents at the bottom of the plate.For automated droplet generation on the automated droplet generator touchscreen, click configure sample plate and select the columns in which the samples are located on sample plate. Click okay and open the automated droplet generator door. Load the sample and droplet generation plates and the other materials necessary for the experiment into the respective locations within the instrument and close the door.When the touchscreen turns green, indicating that all of the materials have been loaded into their correct positions, select oil for probes and start droplet generation. Wait for the droplet generation to run to completion according to the time indicated on the screen. When the screen says Droplets ready, open the door to remove the plate containing the droplets and use a plate sealer for five seconds at 180 degrees Celsius to seal the plate with a pierceable foil seal.Within 30 minutes of the droplet generation, place the sealed droplet plate into a 96 deep well thermal cycler. Set the sample volume to 40 microliters and the lid temperature to 105 degrees Celsius. Then, PCR amplify the droplets.For droplet reading, after the amplification, transfer the thermal cycled 96-well plate into a droplet reader and open the accompanying software on a computer connected to the droplet reader. From the setup mode, select New and double click on any well to open the well editor dialogue box. Select the wells to be read and set experiment to absolute quantification, supermix to droplet digital PCR supermix for probes.Target one type to channel 1 unknown and target two type to channel 2 unknown. Assign the sample names based on the plate layout and click apply and okay. After saving the template, click Run.When prompted by the software, select the FAM/HEX color. At the end of data acquisition, remove the plate from the reader. Before analyzing the data, check the data from all of the wells to obtain the total number of droplets.And set a cutoff to accept a minimum number of droplets to be counted as positive or negative results. For simplex and duplex assay analysis, double click on the QLP file to be analyzed and select Analyze. Use the 1D amplitude and 2D amplitude threshold tools to distinguish between the positive and negative droplets for each well in the correct channel, using the node template control and positive control samples as a guide.Then, export the results as a CSV file for additional analysis. For a triplex probe mix assay, open the QLP file and select the wells to be analyzed in the plate editor tab. Set the experiment type to direct quantification and the assay to probe mix triplex, and enter the target name.Then click Apply. Use the graph tools to assign specific colors to different target clusters as directed by the select to assign cluster window popup suggestions. When all of the target colors have been assigned, the quantification data will be displayed in the well data window.Export the data as a CSV file for downstream analysis. For a quadruplex assay, set the experiment type to direct quantification and the assay to amplitude multiplex. Enter the target name and click Apply.Use the graph tools to assign specific colors to different target clusters as directed by the select to assign cluster window popup suggestions. When all of the target colors have been assigned, the quantification data will be displayed in the well data window. Export the data as a CSV file for additional analysis.As observed in this representative temperature gradient analysis, higher kneeling temperatures could not clearly distinguish positive droplets from negative droplets when a duplex assay was run. However, with a decrease in a kneeling temperature, an optimal separation between positive and negative droplets was achieved. Using a two color RT droplet digital PCR detection system, it is possible to detect one, two, three, and four SARS-CoV-2 targets within a single sample.A no template control sample can be used to locate negative droplets to help set the negative threshold in simplex assays. In duplex assays, the analysis can be performed in individual channels or in the 2D amplitude. Although higher order multiplex assays data analysis is not straightforward, these assays are useful, because they allow up to eight droplet clusters to be assessed in triplex probe mix assays and up to 16 droplet clusters for quadruplex amplitude-based assays.Depending on the assay to be developed, make sure the correct target primer and probe concentrations are added to the master mix. Using this procedure, one may alternate the targets to develop novel RT-ddPCR assays. This assays can be used for different research purposes or for the diagnosis of emerging infectious diseases.Since its development, this technique has been used for the accurate determination of viral nucleic acid copies in different standards, the human body, and environmental samples.

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