TITLE: Screening and Detection SARS-CoV-2 RNA from Buffy Coats
AUTHORS: Mahesh Bachu, Bikash Mishra, Marie-Dominique  Ah Kioon, Chaudhary  Vidyanath

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This SOP has been developed to minimize or eliminate employee occupational exposure to SARS-CoV-2 virus from blood samples of healthy volunteers obtained from the New York Center (NYBC) or from outpatients at HSS. Both are screened for symptoms and potential exposure at the point of care but are not tested for COVID-19.  The CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel is intended for the qualitative detection of nucleic acid from the 2019-nCoV in upper and lower respiratory specimens (such as nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage, and nasopharyngeal wash/aspirate or nasal aspirate). This protocol is a modified version of the CDC protcol meant to screen for Novel Coronavirus  (2019-nCoV) in RNA isolated from whole blood. The  Real-Time RT-PCR  is for the identification of 2019-nCoV RNA from buffy coats obtained from supposedly healthy donors from New York Blood Center (NYBC). The 2019-nCoV RNA is generally detectable in upper and lower respiratory specimens during infection while rarely detected in circulating blood. Positive results are indicative of active infection with 2019-nCoV and blood should be discarded as per the . Negative results do not preclude 2019-nCoV infection and should not be used as the sole basis of management decisions. The product contains oligonucleotide primers and dual-labeled hydrolysis probes (TaqMan®) and control material used in rRT-PCR for </span><span style = "font-style:italic;">in vitro </span><span>qualitative detection of 2019-nCoV RNA. The panel is designed for specific detection of the 2019-nCoV (two primer/probe sets). An additional primer/probe set to detect the human RNase P gene (RP) in control samples. RNA is purified from whole blood in one-step Real-Time RT-PCR on an Applied Biosystems 7500 Real-Time PCR Instrument. Detection of viral RNA in whole blood aids in the safe operation of research labs working with human blood samples.</span></div></div>

[STEPS]
?. Transfer the buffy bag from a sealed NYBC container to a BSL2 hood. Decontaminate the surface of the bag by spraying generous amount of 70% Ethanol. Using a 21 or 22 gauge needle fitted on a 5-10 ml syringe carefully collect ~100 - 200 µl of blood by carefully puncturing the top of the tubing of buffy bag.
?. Erythrocyte lysis is a critical step in the recovery of RNA from whole blood and has to be performed carefully without generating aerosols. Using the same syringe aspirate 5 volumes of Lysis Buffer (L5) to every 1 volume of fresh whole blood . For example, add 500 μl of Lysis Buffer (L5) to 100 μl fresh whole blood sample. Using one-handed scoop method cap the syringe followed by incubation for 10 minutes in hood to allow hypotonic lysis of RBC.
?. NEEDLES SHOULD NOT BE RECAPPED, BENT, REMOVED OR OTHERWISE MANIPULATED BY HAND. One-Handed Scoop MethodPlace the cap on the desk or other flat surface with something firm to “push” the needle cap against.Holding the syringe with needle attached in one hand, slip the needle into the cap without using the other hand.Push the capped needle against a firm object to “seat” the cap onto the needle firmly using only one hand.
?. Transfer the contents from the syringe into a 1.5 ml sterile RNase-free microfuge tube. Incubate the contents on ice for 10 minutes. Within the BSL2 hood, vortex the tube briefly 2-3 times during the incubation step to allow complete lysis of erythrocytes. The solution turns translucent. Note: Guanidine isothiocyanate present in lysis buffer is a chaotropic agent that helps in lysis of the cells and virus particles and is capable of protecting the RNA from endogenous RNases.
?. Centrifuge the tube at 4°C at 400 x g for 10 minutes. Remove the supernatant completely and discard the supernatant. Do not discard the pellet as the pellet contains leukocytes.
?. Resuspend the leukocyte pellet in volumes of Lysis Buffer (L5). Mix well by vortexing briefly.  For example, use 200 μl Lysis Buffer (L5)/100 μl of whole blood from Step 1.
?. Centrifuge the tube at 4°C at 400 x g for 5 minutes. Remove the supernatant completely and discard the supernatant.  Do not discard the pellet as the pellet contains leukocytes. The leukocyte pellet should be white with no traces of red. If the pellet is significantly red, wash the pellet with Lysis Buffer (L5).
?. Resuspend the leukocyte pellet in 350 μl Lysis Buffer (L3). Mix well by vortexing briefly to completely resuspend the pellet ensuring the absence of any cell clumps.
?. Add 350 μl 70% ethanol to the tube and mix well by vortexing briefly.
?. Remove a Spin Cartridge in a Collection Tube from the package. Transfer the leukocyte lysate from Step 7, above, to the Spin Cartridge.
?. Centrifuge the Spin Cartridge at 8,000 × _g for 1 minute at room temperature.
?. Discard the flow through and place the Spin Cartridge into the collection tube.
?. Add 700 μl of Wash Buffer (W4) supplied in the kit to the Spin Cartridge.
?. Centrifuge Spin Cartridge at 8,000 × g for 30 seconds at room temperature.
?. Proceed to DNase I digestion if you need to remove genomic DNA or proceed directly to Step 16.
?. Optional (DNase Digestion): To remove genomic DNA from the samples, add 80 μl of DNase I solution (page 12 for a recipe) to the Spin Cartridge. Incubate at room temperature for 15 minutes.
?. Add 500 μl of Wash Buffer (W4) to the Spin Cartridge. If DNase I digestion is performed, incubate for 5 minutes at room temperature.
?. Centrifuge the Spin Cartridge at 8,000 × _g for 30 seconds at room temperature. Discard the flow through.
?. Add 500 μl of Wash Buffer (W5) with ethanol.
?. Centrifuge the Spin Cartridge at 8,000 × g for 30 seconds at room temperature. Repeat Step 18 once.
?. Discard the flow through and place the Spin Cartridge into the Wash Tube supplied with the kit and centrifuge the Spin Cartridge at 8,000 x g for 1 minute at room temperature to remove any residual Wash Buffer (W5).
?. Place the Spin Cartridge in a clean 1.7-ml Elution Tube supplied with the kit.
?. Add 20-50 μl of sterile, RNase-free water (supplied with the kit) to the center of the cartridge.
?. Incubate at room temperature for 1 minute. Centrifuge the Spin Cartridge at 8,000 × g for 1 minute at room temperature.
?. The elution tube contains your purified total RNA. Remove and discard the cartridge. Store the total RNA at -80°C or use total RNA for the desired downstream application.
?. The following is the schematic of  CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR:
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?. Reaction Master Mix and Plate Set UpNote: Plate set-up configuration can vary with the number of specimens and workday organization. NTCs and nCoVPCs must be included in each run.In the reagent set-up room clean hood, place rRT-PCR buffer, enzyme, and primer/probes on ice or cold-block. Keep cold during preparation and use.Mix buffer, enzyme, and primer/probes by inversion 5 times.Centrifuge reagents and primers/probes for 5 seconds to collect contents at the bottom of the tube, and then place the tube in a cold rack.Label one 1.5 mL microcentrifuge tube for each primer/probe set.Determine the number of reactions (N) to set up per assay. It is necessary to make excess reaction mix for the NTC, nCoVPC, HSC (if included in the RT-PCR run), and RP reactions and for pipetting error. Use the following guide to determine N: • If number of samples (n) including controls equals 1 through 14, then N = n + 1.If number of samples (n) including controls is 15 or greater, then N = n + 2For each primer/probe set, calculate the amount of each reagent to be added for each reaction mixture (N = # of reactions).
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?. Set up reaction strip tubes or plates in a 96-well cooler rack.
?. Dispense 15 μL of each master mix into the appropriate wells going across the row as shown below:
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?. Prior to moving to the nucleic acid handling area, prepare the No Template Control (NTC) reactions for column #1 in the assay preparation area.
?. Pipette 5 μL of nuclease-free water into the NTC sample wells (Figure 2, column 1). Securely cap NTC wells before proceeding.
?. Gently vortex nucleic acid sample tubes for approximately 5 seconds. Centrifuge for 5 seconds to collect contents at the bottom of the tube. After centrifugation, place extracted nucleic acid sample tubes in the cold rack.
?. Samples should be added to columns 2-11 (column 1 and 12 are for controls) to the specific assay that is being tested as illustrated in Figure 2. Carefully pipette 5.0 μL of the first sample into all the wells labeled for that sample (i.e. Sample “S1” down column #2). Keep other sample wells covered during addition. Change tips after each addition. Securely cap the column to which the sample has been added to prevent cross contamination and to ensure sample tracking.  Repeat the same steps for the remaining samples.
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?. If necessary, add 5 μL of Human Specimen Control (HSC) extracted sample to the HSC wells (Figure 2, column 11). Securely cap wells after addition. NOTE: Per CLIA regulations, HSC must be tested at least once per day.  Cover the entire reaction plate and move the reaction plate to the positive template control handling area.
?. Pipette 5 μL of nCoVPC RNA to the sample wells of column 12 (Figure 2). Securely cap wells after addition of the control RNA.NOTE: If using 8-tube strips, label the TAB of each strip to indicate sample position. DO NOT LABEL THE TOPS OF THE REACTION TUBES!Briefly centrifuge reaction tube strips for 10-15 seconds. After centrifugation return to cold rack. NOTE: If using 96-well plates, centrifuge plates for 30 seconds at 500 x g, 4°C.
?. Create a Run Template depending on the available real-time PCR instrument available in the lab and run the machine. Refer to the CDC- document for the detailed set up of the real-time PCR machine.
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