This work was supported by King Abdullah University of Science and Technology through core funding and the National Term Grand Challenge (NTGC) to S.M.H.

1. Taq polymerase expression and purification
<ol>
	<li>Construct a plasmid with a cleavable hexa-histidine tag at the C-terminus of the enzyme.</li>
	<li>Transform 50 ng of the expression vector into E. coli BL21-(DE3) strain following the standard protocol38.</li>
	<li>Inoculate the transformed cells in four 6 L flasks each containing 2 L of 2YT media broth at 37 &#176;C with shaking at 170 rpm until the OD 600 of 0.8 or cell number 6.4 x 108 is reached.</li>
	<li>Induce Taq polymerase expression with 0.5 mM of isopropyl-&#946;-d-thiogalactopyranoside (IPTG) and further incubate at 16 &#176;C for 18 h with shaking.</li>
	<li>Harvest the cells by spinning them down at 4 &#176;C at 7808 x g for 10 min. Resuspend the pellets in 200 mL of an ice-cold Taq polymerase lysis buffer (Table 1) in 5 mL/g of cell pellet.</li>
	<li>Incubate the cells with lysozyme (2 mg/mL of lysis buffer) and protease inhibitors for 45 min and then pass the lysate through a cell disruptor at 30 kPsi and centrifuge at 22,040 x g for 30 min at 4 &#176;C to separate the cell debris.</li>
	<li>Heat the supernatant for 15 min at 85 &#176;C. Spin down at 95,834 x g for 1 h at 4 &#176;C. Collect the supernatant and filter it on ice using 0.45 &#181;m filter.</li>
	<li>Perform protein purification using Fast Protein Liquid Chromatography (FPLC) by first passing the sample through a Ni-NTA HP 5 mL column at 4 mL/min flow rate using Taq polymerase buffer A (Table 2; Figure 1A).</li>
	<li>Wash with 10 column volumes (CV) of Taq polymerase buffer A and 4% of Taq polymerase buffer B (Table 2). Elute the bound protein with a linear gradient using Taq polymerase Buffer B over 20 CV in a 100 mL bottle.</li>
	<li>Pass the eluent in the 100 mL bottle through a cation exchange 5 mL column at 4 mL/min using Taq polymerase buffer C (Table 2; Figure 1A).</li>
	<li>Wash with 20 CV of 100% Taq polymerase buffer C and 5% Taq polymerase buffer D (Table 2).</li>
	<li>Elute with a linear gradient using Taq polymerase buffer D (Table 2) starting from 5% to 100%.</li>
	<li>Check the eluted fractions by performing SDS-PAGE gel electrophoresis 39 followed by gel staining 40 as previously described. Briefly, take 10 &#181;L of each fraction and add an equal volume of 2x SDS loading dye. Denature the sample at 90oC for 10 min. Load the samples onto 10% SDS-PAGE gel and run the gel for 25 min at 200 V. Stain the gel using Coomassie Brilliant Blue and then de-stain.</li>
	<li>Collect all the fractions that contain purified Taq polymerase and dialyze against taq polymerase storage buffer (Table 3) at 4 &#176;C. Briefly, prepare 2 L of the storage buffer and immerse the dialysis cassette in the buffer for 2 min to hydrate. Inject the collected fractions using a needle into the dialysis cassette and leave it overnight in the dialysis buffer at 200 rpm on the stirrer.</li>
	<li>After dialysis, measure the protein concentration using a spectrophotometer, make 10 &#181;L aliquots, snap freeze in liquid nitrogen and store at -80 &#176;C. 
	&#8203;NOTE: Filter all buffers for purification using a 0.45 &#181;m filter before loading them into the FPLC system.</li>
</ol>
2. MMLV-RT expression in insect cell expression system and purification
<ol>
	<li>Bacmid DNA generation and isolation
	<ol>
		<li>Clone the sequence of MMLV-RT with a C-terminus cleavable TEV-8xHis-Strep tag as previously described41.</li>
		<li>Mix 100 ng of the expression vector into 50 &#956;L of DH10Bac cells and mix gently by tapping the tube.</li>
		<li>Incubate the cells on ice for 15 min. Heat-shock the cells for 1 min at 42 &#176;C.</li>
		<li>Immediately transfer on ice and add 400 &#956;L of S.O.C medium. Incubate at 37 &#176;C with shaking for 4 h.</li>
		<li>Plate 10 &#956;L and 15 &#956;L of the mixture on LB Agar plates containing three antibiotics; 50 &#956;g/mL Kanamycin, 10 &#956;g/mL Tetracycline and 7 &#956;g/mL Gentamicin along with 40 &#956;g/mL IPTG and 100 &#956;g/mL X-Gal for blue-white selection.</li>
		<li>Incubate the plates at 37 &#176;C for 48 h. Pick several white colonies and one blue colony as a control, and re-streak them on fresh LB agar plates containing the above antibiotics. Incubate the plates overnight at 37 &#176;C.</li>
		<li>After confirming the white phenotype, pick a couple of colonies and inoculate them into 10 mL of LB media containing 50 &#956;g/mL Kanamycin, 10 &#956;g/mL Tetracycline, and 7 &#956;g/mL Gentamicin in 50 mL tubes.</li>
		<li>Incubate the culture at 37 &#176;C with shaking at 170 rpm overnight. Pellet down the cells by spinning them down at 22,040 x g for 10 min.</li>
		<li>Decant the supernatant and resuspend the pellet in 250 &#181;L of resuspension solution from miniprep kit (this solution needs to be handled in accordance with the manufacturer&#39;s instructions), then transfer the solution to a 1.5 mL microcentrifuge tube.</li>
		<li>Add 250 &#181;L of lysis solution, gently mix and incubate for 3 min. Add 350 &#181;L of neutralizing solution, invert 2x-3x and centrifuge at 22,040 x g for 10 min.</li>
		<li>Transfer the supernatant to a fresh tube and add an equal volume of ice-cold isopropanol and incubate for 30 min at -20 &#176;C.</li>
		<li>Pellet down the precipitated DNA by spinning at 22,040 x g for 10 min. Discard the supernatant and wash the pellet with 700 &#181;L of 70% ice-cold ethanol, 2x.</li>
		<li>Remove the supernatant with a pipette without touching the pellet. Let the pellet air dry in the laminar flow hood for 5-10 min or until no liquid is seen in the tube. Dissolve the pellet in 100 &#181;L of EB buffer.</li>
	</ol>
	</li>
	<li>Bacmid transfection and virus amplification
	<ol>
		<li>P1 virus preparation
		<ol>
			<li>In a 6-well tissue culture plate, seed ~ 9 x 105 cells/well in 2 mL of insect cell culture medium.</li>
			<li>Incubate the plate for ~30 min at room temperature to allow the cells to attach.</li>
			<li>Prepare Bacmid/Fugene mix as follows: mix 1 &#181;g of Bacmid DNA and 300 &#181;L of insect cells media in one tube. In another tube, mix 8 &#181;L of transfection reagent and 300 &#181;L of insect cells media. Mix both mixtures by gentle pipetting and leave for ~30 min at room temperature for the bacmid/transfection reagent complex to form.</li>
			<li>Add bacmid complex mix (~210 &#181;L) to each well dropwise and seal the plate with a transparent film.</li>
			<li>Incubate the plate at 27 &#176;C for 3-4 days.</li>
			<li>Take the medium which that contains the virus, add FBS (final concentration 2%), filter using 0.45 &#181;m filter and store at 4&#176;C in the dark as P1 virus stock.</li>
		</ol>
		</li>
		<li>P2 virus preparation
		<ol>
			<li>Transfect 50 mL of insect cells at 2 x 106 cells/mL with 3 mL of P1 virus in a culturing flask.</li>
			<li>Incubate at 27 &#176;C with shaking at 100 rpm for 4-6 days until the percentage of dead cells reaches 25%-30%.</li>
			<li>Centrifuge at 300 x g for 10 min and collect the supernatant that contains the virus.</li>
			<li>Add FBS to a final concentration of 2%, filter through 0.45 &#181;m filter and aliquot 1 mL each and store at -80 &#176;C as P2 virus stock.</li>
		</ol>
		</li>
		<li>P3 virus preparation
		<ol>
			<li>Transfect 100 mL of insect cells at 2 x 106 cells/mL with the 2 mL of P2 virus in a culturing flask.</li>
			<li>Incubate at 27 &#176;C with shaking at 100 rpm for 3-4 days until the percentage of dead cells reaches 15%-20%.</li>
			<li>Centrifuge the cells at 300 x g for 10 min and collect the supernatant that contains the virus.</li>
			<li>Add FBS to a final concentration of 2%. Filter through a 0.45 &#181;m filter. It can be stored in the dark at 4 &#176;C for 1 month.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Expression and purification of MMLV-RT in insect cells
	<ol>
		<li>Add 5 mL of P3 virus to fresh insect cells at a density of 2 x 106 cells/mL (700 mL/2L flask) in total 6 flasks.</li>
		<li>Harvest the cells after 55-60 h of post-transfection by spinning at 7808 x g for 10 min.</li>
		<li>Resuspend the cell pellets in 200 mL of MMLV-RT lysis buffer (Table 4). Pass the lysate through a cell disruptor at 15 kPsi and centrifuge at 22,040 x g for 30 min at 4 &#176;C.</li>
		<li>Transfer the supernatant into new tubes and spin down again at 95,834 x g at 4 &#176;C for 1 h. Collect the supernatant and filter it on ice using a 0.45 &#181;m filter.</li>
		<li>Start protein purification using FPLC by first passing the sample through Ni-NTA Excel 5 mL column (Figure 1B) at 4 mL/min flow rate by using MMLV-RT buffer A (Table 5).</li>
		<li>Wash the column with 10 CV of MMLV-RT buffer A and 4% of MMLV-RT buffer B (Table 5) and elute the protein with the linear gradient of MMLV-RT buffer B over 20 CV in a 100 mL bottle.</li>
		<li>Pass the eluted sample through the Strep 5 mL column (Figure 1B) equilibrated with MMLV-RT buffer C (Table 5).</li>
		<li>Wash the column with 10 CV of 100% MMLV-RT buffer C and elute the protein with a linear gradient with 20 CV of buffer D (Table 5).</li>
		<li>Check the eluted fractions by performing SDS-PAGE as done is steps 1.13.-1.14. and collect the fractions that contain purified MMLV-RT to be dialyzed against MMLV-RT storage buffer (Table 6) at 4 &#176;C.</li>
		<li>Measure the protein concentration using Nanodrop, aliquot, snap freeze in liquid nitrogen and store at -80 &#176;C. 
		&#8203;NOTE: Filter all buffers for purification using a 0.45 &#181;m filter before loading them into the FPLC system.</li>
	</ol>
	</li>
</ol>
3. Preparation of in-house multiplex R3T one-step RT-qPCR kit components
<ol>
	<li>Buffer mix preparation
	<ol>
		<li>Prepare the 2x buffer for the RT-qPCR reaction that contains the reagents previously shown in37. Prepare all the reagents in DNase/RNase-Free water and the buffer mix stored in a -20&#160;&#176;C&#160;freezer.</li>
	</ol>
	</li>
	<li>Primer and Probe Sets and primers mixture preparation 
	NOTE: The probes and primers used are listed in Table 7. The Influenza and SARS-CoV-2 multiplexed kit contains 3 probes and 4 forward and reverse primer sets. The probes are labeled at the 5&#8242; ends using reporters 6-carboxyfluorescein (FAM) for InfA, Texas Red-XN for SARS-CoV-2 (N gene) and Yakima Yellow for InfB. The MERS-CoV and SARS-CoV-2 multiplexed kit contains 3 probes and 3 forward and reverse primer sets. The probes are also labeled at the 5&#8242; ends using reporters Texas Red-XN for MERS-CoV (ORF1a gene), VIC for MERS-CoV (UpE gene) and 6-carboxyfluorescein (FAM) SARS-CoV-2 (N gene).
	<ol>
		<li>Prepare a primer mix containing all the primers and probes listed in Table 7 with a final concentration of 6.7 &#181;M for each forward and reverse primer and 1.25 &#181;M for each probe in a 1.5 mL tube. Store the primer mix in the -20&#160;&#176;C&#160;freezer. Use elution buffer to make up the required volume.</li>
	</ol>
	</li>
	<li>Enzyme mix preparation
	<ol>
		<li>Prepare Taq polymerase (30 U/&#181;L) and MMLV-RT (60 ng/&#181;L) enzyme mix in the Taq polymerase storage buffer as shown in Table 3 to make up the required volume. Perform this step on ice and store the enzyme mix at -20 &#176;C.</li>
	</ol>
	</li>
	<li>RNA template
	<ol>
		<li>Resuspend synthetic RNAs of SARS-CoV-2, Influenza A H1N1, Influenza A H3N2, Influenza B and MERS-CoV separately in 100 &#181;L of 1x Tris-EDTA buffer (10 mM Tri-Cl and 1 mM EDTA (pH 8.0) to make a stock of 1 x 106 RNA copies/&#181;L.</li>
		<li>Mix synthetic RNAs for RT-qPCR in the following configurations: 1) SARS-CoV-2, Influenza A and Influenza B by adding 5 &#181;L of each virus stock to 50 &#181;L volume to make 1 x 105 RNA copies/&#181;L; 2) SARS-CoV-2, MERS-CoV by adding 5 &#181;L of each virus stock to 50 &#181;L volume to make 1 x 105 RNA copies/&#181;L. Make 10-fold serial dilutions of each synthetic RNA mixture ranging from 1 x 105 RNA copies/&#181;L to 10 RNA copies/&#181;L. 
		&#8203;NOTE: Make sure to use ice-cold DNase/RNase-Free water to prepare the serial dilution of the templates.</li>
	</ol>
	</li>
</ol>
4. In-house multiplexed SARS-CoV-2, Influenza A, Influenza B and SARS-CoV-2, MERS-CoV one-step RT-qPCR test
NOTE: Disinfect workstation surfaces and use a 96-well plate template to plan the PCR plate layout.
<ol>
	<li>Preparation of 96-well plate
	<ol>
		<li>Thaw 2x buffer and primer mix. Keep enzyme mix on ice.</li>
		<li>To each well, add 10 &#181;L of 2x buffer mix, 1.5 &#181;L of the primer mix, 1 &#181;L of enzyme mix, 6.5 &#181;L of DNase/RNase-Free Water, and 1 &#181;L of corresponding RNA mixture that needs to be tested. The final volume in each well is 20 &#181;L. Please refer to the prepared layout for help with this step. 
		NOTE: It is recommended to make a master mix based on the number of reactions that contain all the reagents except the RNA sample to avoid pipetting bias. In addition, perform the reactions in duplicate or triplicate to avoid technical errors.</li>
		<li>Seal the plate with an adhesive PCR plate seal and centrifuge briefly for 1 min to collect all liquid at the bottom of the plate. Ensure that each well has the same volume of liquid and is free of bubbles before transferring the samples to the qPCR machine. 
		NOTE: All the PCR reactions need to be set up on ice.</li>
	</ol>
	</li>
	<li>PCR program
	<ol>
		<li>Open the real-time qPCR machine program and choose the following experimental properties: 
		The block type: Fast 96-well (0.1 mL) 
		Experiment setup: Standard curve 
		Reagents: TaqMan reagents 
		&#8203;Run properties: Standard.</li>
		<li>Define all the gene targets and their reporter dye as presented in Table 7. In addition, define sample names for each reaction to be tested for easier analysis of the amplification curves.</li>
		<li>Assign targets and samples to the program&#39;s plate layout based on the 96-well plate.</li>
		<li>Set the following cycle program in the PCR instrument as follows: 
		55&#160;&#176;C for 10 min 
		40&#160;cycles: 94&#160;&#176;C for 1 min 
		94 &#176;C for 10 s 
		68 &#176;C for 10 s 
		&#8203;68&#160;&#176;C for 20 s; here acquire fluorescence.</li>
		<li>Transfer the plate to the real-time qPCR machine and place it in the holder ensuring the correct orientation of the plate and start the run.</li>
		<li>Choose the file location to save the experimental data.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>It is essential to first examine the amplification plots produced by the qPCR program. Analyze the limit of detection to assess the minimum RNA concentration that can be detected.</li>
		<li>Plot the log of RNA copy number on the x-axis and the corresponding average Ct value on the y-axis to assess the sensitivity of the assay. The slope and the R2 indicate the reliability and efficiency of the reaction.</li>
	</ol>
	</li>
</ol>

Multiplex RT-qPCR Assay for Respiratory Virus Detection

The authors declare that they have no competing financial interests.

There is a heavy economic burden on the healthcare system worldwide resulting from the high infection and mortality rates due to the spread of common respiratory viruses such as SARS-CoV-2, Influenza A/B and MERS-CoV variants12,19,20. Motivated by the sense of responsibility towards alleviating this burden, we realized the need for a quick, precise and accessible diagnostic assay such as RT-qPCR to distinguish between these comm...

The pandemic of the ongoing coronavirus disease 2019 (COVID-19) is caused by the novel coronavirus known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Due to SAR-CoV-2&#39;s strong contagiousness and capacity for rapid transmission, the COVID-19 pandemic emerged in Wuhan City, China, and spread quickly throughout the world. This eventually led to the start of respiratory distress signs and even death2,3,4. COVID-19 has been declared a pandemic in more than 213 countries, expecting a steep increase in the number of confirmed cases, as evidenced by the papers published by different research studies3,5. COVID-19 is transmitted primarily by small respiratory droplets that infected individuals release into the environment and then get exposed to vulnerable individuals through inhalation or close contact with contaminated surfaces. When these droplets come into contact with the mucosa of the eyes, mouth, or nose, a person may become infected6. Statistics released by the World Health Organization (WHO) show that there have been more than 76 million confirmed cases of the pandemic worldwide, with a staggering 7 million deaths7. Thus, the United Nations classified the pandemic caused by COVID-19 disease as a disaster because of its direct impact on the lives of billions of people around the globe and had far-reaching economic, environmental, and social effects.
Public health initiatives including thorough testing, early detection, contact tracing, and case isolation have all been shown to be crucial in keeping this pandemic under control8,9,10,11. The winter months will increase the circulation of other respiratory viruses like Influenza A and B with COVID-19-like symptoms making it difficult to identify, track down, and isolate COVID-19 instances early on. Every year, Influenza A and B outbreak starts in the late fall or early January with a predictable seasonality12. Numerous epidemiological traits are shared by SARS-CoV-2 and Influenza viruses. Besides, sharing similarities in the susceptible populations which include children, the elderly, immunocompromised, and individuals with chronic comorbidities such as asthma, chronic obstructive pulmonary disease, cardiac and renal failure, or diabetes12,13. These viruses not only share vulnerable populations but also transmission routes of contact and respiratory droplets14. It is anticipated that patients may likely contract more than one of these respiratory viruses as flu season approaches14. For that, the screening of SARS-CoV-2 and the Influenza viruses needs to be done on symptomatic patients before they are isolated. Running separate tests for the three viruses (SARS-CoV-2, Influenza A, and Influenza B) is not possible due to the global lack of resources for nucleic acid extraction and diagnostics. In order to screen them all in one reaction, a method or test needs to be developed.
Middle East respiratory syndrome (MERS)-CoV is a human coronavirus (CoV) family member. The first MERS-CoV virus isolates came from a hospitalized patient in Saudi Arabia who had died in September 2012 due to acute respiratory issues15. There is evidence that suggests that a prominent reservoir host for MERS-CoV is dromedary camels. It has been proven that viruses from infected dromedary camels are zoonotic and thus can infect humans16,17. Humans infected with this virus can spread it to others through close contact18. As of January 26th,&#160;2018, there had been 2143 laboratory-confirmed cases of MERS-CoV infection including 750 deaths globally19. The most typical MERS-CoV symptoms are coughing, fever, and shortness of breath. MERS-CoV infections have also been reported to exhibit pneumonia, diarrhea and gastrointestinal sickness symptoms20. Currently, no commercial vaccine or specific treatment for MERS-CoV is available. Therefore, prompt and precise diagnosis is essential for preventing the widespread MERS-CoV outbreaks and differentiating MERS-CoV from SARS-CoV-2 disease.
To date, many approaches have been proposed to detect these viruses such as multiplex RT-PCR21,22,23,24,25, CRISPR/Cas1226,27, CRISPR/Cas928, and CRISPR/Cas329, lateral flow immunoassay30, paper-based biomolecular sensors31, SHERLOCK testing in one pot32, DNA aptamer33, loop-mediated isothermal amplification (LAMP)19,34, etc. Each of the aforementioned methods has unique benefits and drawbacks in terms of sensitivity and specificity. Among these methods, the nucleic acid amplification-based test: multiplex qRT-PCR, is the most common and is considered to be the gold standard for the diagnosis of SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV.
In this study, we designed and assessed various primer combinations and probes for the effective, accurate, and simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV utilizing standard twist synthetic viral RNAs. The multiplexed assays developed for either MERS-CoV or SARS-CoV-2 target genes are recommended by the World Health Organization (WHO). These genes generally encode proteins and complexes that contribute to the formation of a replication/transcription complex (RTC)35 such as the region within the open reading frame 1a (ORF1a) that is used for MERS-CoV assay. In addition, structural proteins are encoded by the genes utilized in diagnostic assays such as the upstream region of envelope gene (upE) and nucleocapsid gene (N) which are used for MERS-CoV and SARS-Cov-2 assays, respectively35,36. We used in-house R3T one-step RT-qPCR kit to establish the RT-qPCR for the detection of viruses37. Virus detection, sensitivity, specificity, and dynamic range of our R3T one-step RT-qPCR kit and primer sets were tested and evaluated using 10-fold serial dilutions of the standard twist synthetic RNAs. The lowest practical detection limit was approximately 10 transcripts copies per reaction. As a result, the in-house R3T one-step RT-qPCR kit and primer/probe sets can be successfully used and implemented for routine simultaneous diagnosis of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;m filter cups</td>
			<td>Thermo Scientific</td>
			<td>291-4545</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Glycine SDS running buffer</td>
			<td>Novex</td>
			<td>LC2675</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culturing plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Fisher Scientific</td>
			<td>A701-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Corning</td>
			<td>61-238-RH</td>
			<td></td>
		</tr>
		<tr>
			<td>Cation exchange (HiTrap SP HP) 5 mL</td>
			<td>Cytiva</td>
			<td>17-1152-01</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Biotin, 98+%</td>
			<td>Thermo Scientific</td>
			<td>A14207.60</td>
			<td></td>
		</tr>
		<tr>
			<td>DH10Bac competent cells</td>
			<td>Fisher Scientific</td>
			<td>10361012</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (Snakeskin 10,000 MWC)</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Thermo Scientific</td>
			<td>R0862</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase/Rnase Free Distilled Water</td>
			<td>Ambion</td>
			<td>AM9930</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Thermo Scientific</td>
			<td>R0192</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli BL21(DE3) competent cells</td>
			<td>Invitrogen</td>
			<td>C600003</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP120-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution Buffer</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>ESF 921 insect cell culture medium (Insect cells media)</td>
			<td>Expression Systems</td>
			<td>96-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Solution</td>
			<td>Gibco</td>
			<td>A38400-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene (transfection reagent)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Fisher Scientific</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516-500</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma Aldrich</td>
			<td>I8896-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma Aldrich</td>
			<td>56750-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A (H1N1) synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103001</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A (H3N2)&#160; synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103002</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza B synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103003</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gold Biotechnology</td>
			<td>I3481C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Gibco</td>
			<td>11815-032</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth media</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma Aldrich</td>
			<td>L6876-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>13152-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>MERS-CoV synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103015</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-well Reaction plates with Barcode (0.1 mL)</td>
			<td>Applied Biosystems</td>
			<td>10310855</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini- PROTEAN TGX Precast Gel</td>
			<td>Bio-Rad</td>
			<td>456-1093</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Excel (HisTrap Excel) 5 mL</td>
			<td>Cytiva</td>
			<td>17-3712-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA HP (HisTrap HP) 5 mL</td>
			<td>Cytiva</td>
			<td>17-5248-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Adhesice Covers (PCR Compatible,DNA/Rnase/PCR Inhibitors Free</td>
			<td>Applied Biosystems</td>
			<td>4311971</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and Probes</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Mini tablets EDTA-Free</td>
			<td>Thermo Scientific</td>
			<td>A32955</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein marker</td>
			<td>Fermentas</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>RT-qPCR machine (QuantStudio 7 Flex)</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C medium</td>
			<td>Fisher Scientific</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-+A2:C442 synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>102024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 insect cells</td>
			<td>Gibco</td>
			<td>A35243</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>StrepTrap XT 5 mL</td>
			<td>Cytiva</td>
			<td>29401323</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>IBI Scientific</td>
			<td>IB02200</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base Molecular Biology Grade</td>
			<td>Promega</td>
			<td>H5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Affymetrix</td>
			<td>22676</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P1379-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Invitrogen</td>
			<td>B1690</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In recent years, there have been significant advances in the diagnostic approach for detecting common respiratory viruses using PCR approaches21,22,23,24,25. However, despite these advancements, the multiplexed approach, which allows for detecting multiple viruses in a single test, has not been widely implemented, particularly in the RT-qPCR platform. This met...

Watch this Scientific Journal Video about Advancements in Multiplex Detection of Respiratory Viruses at JoVE.com

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.

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 ...

development-multiplex-real-time-rt-qpcr-assays-for-detection-sars-cov

Research

JoVE Journal

Biology

980 Views.  King Abdullah University of Science and Technology (KAUST). 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. 

Video: Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses 

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...

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.

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) ...