key: cord-0815693-73cgapsx authors: Katchman, Benjamin A; Newland, Cory S; Rivas, Candy M; O’Brien, Kevin; Eggers, Rick; Wen, Fushi; Hogan, Mike title: Validation Study for PathogenDx Enviro(X)-Rv assay for the Detection of SARS-CoV-2 from Stainless Steel Environmental Surface Swabs Performance Tested Method(SM) 122003 date: 2021-04-11 journal: J AOAC Int DOI: 10.1093/jaoacint/qsab047 sha: 5de95a7dd8e4402409097a4103f53f1a76e28805 doc_id: 815693 cord_uid: 73cgapsx BACKGROUND: The PathogenDx Enviro(X)-Rv uses endpoint PCR + DNA microarray technology to detect SARS-CoV-2, the causative agent of COVID-19, from stainless steel environmental sample swabs. OBJECTIVE: : To validate the PathogenDx Enviro(X)-Rv assay as part of the Emergency Response Validation Performance Tested Method (SM) program. METHODS: : The PathogenDx Enviro(X)-Rv assay was evaluated for specificity using in silico analysis of ≥ 41 000 SARS-CoV-2 sequences and over 50 exclusivity organisms (both near neighbors and background organisms). The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the U.S. Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020). RESULTS: Results of the In silico analysis demonstrated the high specificity of the method in being able to detect target SARS-CoV-2 sequences and discriminate them from near neighbors and environmental background organisms. In the matrix study, the candidate method demonstrated a statistically significant difference when compared to the results of the CDC method utilized in this study, with the candidate method resulting in more positive replicates as it only requires 1 target to be present for a positive sample. CONCLUSIONS: The Enviro(X)-Rv assay rapidly and accurately detected SARS-CoV-2 RNA on environmental swabs from stainless steel surfaces at concentration of 2000 genomic copies per 2” × 2” test area. HIGHLIGHTS: The Enviro(X)-Rv assay employs dual PCR and hybridization techniques to provide highly accurate results when detecting SARS-CoV-2 from surfaces. Page 2 of 32 from stainless steel surfaces at concentration of 2000 genomic copies per 2" × 2" test area. Highlights: The Enviro X -Rv assay employs dual PCR and hybridization techniques to provide highly accurate results when detecting SARS-CoV-2 from surfaces. In late 2019, a novel coronavirus (SARS-CoV-2) was discovered that has resulted in a global pandemic and millions of confirmed human infections globally (1) . While easily spread person to person, there remains a high level of uncertainty about its ability to transmit on surfaces including food contact surfaces, which has resulted in the banning of certain imported products from manufacturing facilities where outbreaks have occurred (2). Reducing the risk associated with these surfaces includes both effective cleaning and disinfection and a robust environmental monitoring program, including rapid detection methods (3). Having access to this information can led to better infection prevention and improved control measures aimed at reducing surface transmission which has resulted in a need for rapid assays that can detect the virus from surfaces. The PathogenDx Enviro X -Rv assay is a test based on end-point reverse transcription polymerase chain reaction (RT-PCR) coupled to DNA microarray hybridization for the detection of multiple genes within SARS-CoV-1 and SARS-CoV-2 viruses. The Enviro X -Rv method requires a few hours longer to obtain results compared to quantitative RT-PCR (qRT-PCR), however, the advantage of end point PCR coupled to DNA microarrays in comparison to the more widely utilized qRT-PCR approach to viral detection is: (1) The ability to increase the assay sensitivity as demonstrated by the lower limit of detection of this assay as compared to the reported qRT-PCR assays approved by the FDA-EUA; (2) The ability to multiplex at a much greater capacity than qRT-PCR. The qRT-PCR method is generally not able to detect more than 5 unique targets in a single reaction. In qRT-PCR the primer and the probe are in the same reaction limiting the reactions capability. In comparison, microarrays can multiplex hundreds or thousands of unique targets due to the separation of the RT-PCR reaction (primers) from the DNA microarray detection (probes). The DNA microarray contains: (a) Enviro X -Rv Kit.-Enviro X -Rv SARS-CoV-2 Multiplex Assay-contains 5 (five) SARS-CoV-2 primer sets; 4 (four) SARS-CoV-2 probes targeting each N1, N2, and N3 genes; Enviro X -Rv Control-internal process control for nucleic acid extraction using a primer and probe sets as an internal positive control; Enviro X -Rv Swabs -WorldBio PUR-Blue TM Swabs in Hi-Cap Broth. (b) Enviro X -Rv SARS-CoV-2 Control -RNA control that contains targets specific to the SARS-CoV-2 genomic regions that are targeted by the assay. Viral and host nucleic acids are isolated and purified from surface swabs using the Zymo Research Quick-DNA/RNA™ Viral MagBead (R2140 or R2141) magnetic silica bead extraction kit. Subsequently, five microliters of the purified RNA product are reverse transcribed using RT-PCR System. Following RT-PCR, two microliters of that primary RT-PCR (amplified cDNA) product are then PCR amplified in a second, nested and biased, PCR reaction in which the PCR target is labeled with a Cy3 fluorophore for detection. The resulting PCR product is then ready for hybridization to the DNA microarrays without additional denaturation or purification. The DNA microarray is formatted in a standard 96-well SBS plate format, one microarray per well (i.e., 96 microarrays per plate) each microarray containing up to 144 synthetic ssDNA probes in a 12 × 12 array configuration. The array contains probes to identify genes in SARS-CoV-1, SARS-CoV-2, internal and external positive controls. The labeled PCR product is hybridized to the DNA microarray at room temperature, over the course of 1 hour, to determine if viral RNA is present in the surface sample. Following the hybridization, the arrays are scanned to determine the fluorescence intensity of each spot, using a Sensospot™ (Sensovation Inc.) scanner. The microarray results are uploaded to a secure server, quantified, and interpreted automatically using Augury™ software (PathogenDx Inc.) Data from the AOAC ERV PTM study indicated that a concentration of 2000 genomic copies per 2" × 2" test area resulted in 100% detection by the assay. (1) . (a) Probability of detection (POD) .-The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. POD is concentration dependent. Several POD measures can be calculated: POD R (reference method POD), POD C (confirmed candidate method POD), POD CP (candidate method presumptive result POD) and POD CC (candidate method confirmation result POD). (b) Difference of probabilities of detection (dPOD) .-Difference of probabilities of detection is the difference between any two POD values. If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level. (c) In silico.-The use of computer simulation to evaluate target and non-target sequences for molecular methods. (d) Microarray.-A laboratory tool used to detect the expression of thousands of genes at the same time. DNA microarrays are 96-well plates that are printed as a matrix of oligonucleotide probe "Spots" in defined positions, with each spot containing a known DNA sequence. (2) Buffer 1.-One bottle (4 mL). (3) Buffer 2.-One bottle (1.5 mL). (2) Collection Plate.-C2002. (3) 96-Well Block.-P1001. (4) Elution Plate.-C2003. (5) Cover Foil.-C2007. (6) Beta-mercaptoethanol. (7) Isopropanol, molecular grade. (8) Ethanol, molecular grade. (9) DNase/RNase Free Water. permitted. Primer Set 2 is light sensitive and must be stored away from light. All frozen reagents must be stored at -20 ± 5°C. They must NOT be stored at -80°C as this will cause degradation of reagents. PathogenDx microarrays are light and moisture sensitive and should be stored in the moisture barrier bag with desiccant packet provided with the kit. Buffer 1, and Buffer 2 can cause irritation upon contact, always wear gloves and eye protection when handling this product. Upon contact, rinse with water. See MSDS. In the post-hybridization protocol, centrifuge speed should not exceed 70 × g or slides may break. Refer to the Material Safety Data Sheets on the PathogenDx company website. Kit components from different lot numbers should not be mixed. PathogenDx Enviro X-RV Assay (a) Change pipette tips in between samples. (b) Prewarm Thermomixer heat block before initiating extraction (j) Always keep the microarray in the hybridization chamber to limit exposure to light. Stainless Steel Environmental Swabs (2" × 2" test area).-Label the outside of the swab container. Remove wet swab from vial by depressing the swab against the inside of the tube to ring out excess liquid. Swab the desired surface in an N or S shaped pattern, in 4 directions, if a flat surface, which should take between 10 to 15 s from start to finish (see Figure 1 ). Place the swab back into the sterile storage container. Close and seal the container. The swab is stable at room temperature until the user is ready for RNA extraction. Note: Alternatively, if the user has not prepared the samples within 5 days of receipt then the swabs can be stored at -20 or -80°C until use. In that case the swabs should be thawed on ice or at 4°C prior to analysis (2) To prepare the Extraction Control -Add 5 μL of positive control directly to a swab and follow the Sample Preparation directions above, continue as normal. (g) Transfer the plate/tube to a magnetic stand and allow to sit until the beads have pelleted. Aspirate and discard the supernatant and retain the pellet. (h) Add 500 μL MagBead DNA/RNA Wash 1 and vortex the tubes or pipet up and down to mix in plates. (i) Transfer the plate/tube to a magnetic stand and allow to sit until the beads have pelleted. Aspirate and discard the supernatant and retain the pellet. (j) Add 500 μL MagBead DNA/RNA Wash 2 and vortex the tubes or pipet up and down to mix in plates. (k) Transfer the plate/tube to a magnetic stand and allow to sit in the magnetic field until the beads have pelleted. Aspirate and discard the supernatant and retain the pellet. (l) Add 500 μL ethanol (95-100%) and vortex the tubes or pipet up and down to mix in plates. (m) Transfer the plate/tube to a magnetic stand and allow to sit until the beads have pelleted. Aspirate and discard the supernatant and retain the pellet. (n) Add 500 μL ethanol (95-100%) and vortex the tubes or pipet up and down to mix in plates. Then transfer the entire solution (beads and liquid) to a new tube/plate. (o) Transfer the plate/tube to a magnetic stand and allow to sit in the magnetic field until the beads have pelleted. Aspirate and discard the supernatant and retain the pellet. (p) Dry the beads for 10 min or until fully dry. Place the tubes/plates on a heat block at 55°C. Aseptically open the tubes and leave them cracked but not fully open to allow for drying. For the plates, without removing the sticker, place seal over the plate allowing drying to occur. There must be airflow into the tube or plate for drying to occur. (q) Once the beads are dry, they should turn from a glossy black to a dull brown. Elute the DNA/RNA from the beads by adding 50 μL DNase/RNase-Free water. Vortex the tubes or pipet up and down to mix in plates. Allow the tubes/plates to sit at room temperature for 2 min. (r) Transfer the plate/tube to a magnetic stand and allow to sit until the beads have pelleted. Aspirate the eluted DNA/RNA and transfer to a new tube/plate. (s) The DNA/RNA may be used immediately or stored frozen at -20°C. Avoid freeze/thaw cycles once the DNA/RNA is stored frozen. More than 6 freeze/thaw cycles may result in RNA degradation. PathogenDx Enviro X-RV assay.-(a) Mix and briefly centrifuge each component before use. Combine the following into a master mix, multiply per reaction as shown in Table 1 . (1) Determine the number of samples in the reaction and multiply each reagent to prepare the master mix, leaving out the Purified RNA Template from the master mix and add individually to each designated well. (2) Mix by pipetting and add 45 μL of the master mix per well. (3) Add 5 μL of the magnetic bead purified RNA template to each designated well making sure to (d) Place the plate in the thermal cycler and cover with a compression pad before closing the thermal cycler. (e) Input the Reverse Transcriptase and PCR cycling program as shown in Table 2 . (f) Proceed to Labeling PCR Amplification. (g) Plates may be stored @4°C for up to 2 weeks in not used immediately. (d) Briefly vortex Labeling PCR master mix and centrifuge at 1000 × g for 3-5 s. (e) Store all reagents at -20°C after use. Note: Reaction volumes have been scaled to account for a negative control and to account for pipetting losses and volume lost on reservoir/tube walls. If a reservoir is not used for multichannel pipetting, there will be extra volume remaining in the PCR Master Mix tube. (1) Always check pipette tip volumes before and after to ensure accuracy and release. (2) Warning: Add the Loci Enhancement PCR template into the Labeling PCR reaction outside of the PCR area to prevent contamination. (h) The Loci Enhancement PCR plate may be re-sealed and returned to 4°C. (i) Cap tubes, or seal plates with PCR film ensuring every well is completely sealed. (j) Centrifuge for 30 s. (k) Place tubes or plate into the thermal cycler with a pressure pad if necessary, before closing the thermal cycler lid. (l) Refer to Table 4 to run the Labeling PCR Program. (m) Labeling PCR product may be stored for 7 days at 4°C protected from light. DNA hybridization.-General guidelines to follow for hybridization: When pipetting with the multichannel onto the microarray plate, only dispense to the first stop. DO NOT depress the multichannel to the second stop, or full evacuation of the tips to avoid cross contamination. Caution: Avoid contact with the array surface of the plate during processing. Use slide edges or barcoded area for handling. The directions below are for 96 well plates. Please note that the bracketed volumes refer to the volume that should be added to the 96 well plates as opposed to the 12 well slides. (a) Before starting, thaw Buffer 2 at room temperature. (b) Cut paper towel to size to fit the bottom of the hybridization chamber provided. (c) Place the slides to be used in the Hybridization Chamber. Prepare the Pre-hybridization Buffer and Hybridization Buffers in clean tubes for the number of microarrays that will be hybridized as per Tables 5 and 6 . Vortex briefly to mix. (g) Aspirate the water wash and add 200 μL of Pre-hybridization Buffer to each well of the 96-well plate without touching the pipette tip to the array surface. Close the Hybridization Chamber box lid. (h) Allow Pre-hybridization Buffer to stay on the arrays for 5 min; do not remove slides from the Hybridization Chamber. (i) Briefly centrifuge the PCR tubes or PCR plate containing the Labeling PCR product. (j) Add 18 μL of Hybridization Buffer to each well of the Labeling PCR product within the 96-well PCR plate or tubes, pipette up and down to mix. It is important that no cross-contamination occurs during this step. The PCR product and the Hybridization Buffer mix constitute the Hybridization Cocktail. (k) Aspirate Pre-hybridization Buffer from the arrays. Caution: Do not allow the arrays to air dry. Avoid contact with the array surface. (l) Immediately add 68 μL (Total Volume of PCR Reaction + Hyb Buffer) of the Hybridization Cocktail to each array of the 96-well being careful not to touch the array surface with the pipette tip. Ensure that the sample ID and location are recorded. (m) Close the Hybridization Chamber lid. (n) Allow to hybridize for 30 min at room temperature in the Hybridization Chamber. Post hybridization PathogenDx slide processing.-(a) Prepare Wash Buffer according to the number of microarray wells to be used in the 96-well plate. (Table 7 ). Washing must be performed according to the protocol to ensure detectable signal and adequate washing to prevent elevated background signals. (e) Close the tray, select "Close Tray". (f) Select "Scan". (g) Select the number of wells that are being scanned in the 96-well plate (ex. 1, 2, 3, 4) Note: All other information on this screen is preprogrammed -do not alter. (h) Select the Blue Arrow to begin the scanning process. (i) While the plate is being scanned, select "Result Overview" to review the images of the wells. (j) When the plate is finished scanning and the screen displays the digital image of a plate with all ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com green wells, select the Red X to exit the scanning process. (k) Open the tray, select "Open Tray". (l) Remove the plate and store in the moisture barrier bag with the desiccant packets. (m) Close the tray, select "Close Tray". (n) Exit the Array Reader application, select "Exit". (o) On the Sensovation Scanner desktop, select the folder "Scan Results". (p) Locate the folder associated with your plate and rename the folder with the slide barcode number by scanning the barcode located either on the outside of the barrier bag or on the plate itself. (ex. rename: ScanJob-191108130334_1 to 7024001001) (q) Submit the whole barcode labeled folder to the "Image Folder" within Dropbox. (r) The folder will automatically begin uploading, the PathogenDx Augury © Software will analyze the data and directly deposit the reports into the "Reports" folder within Dropbox. (Table 8) . The study was conducted according to the procedures outlined in the AOAC Research Institute Additional PTM parameters, robustness and product consistency and stability will be submitted for full PTM certification by March 31, 2021. Primer which is obtained by dividing the data in Row 3 by Primer Length. The method of data analysis in Row 3 (prevalence summed over the entire primer length) is as suggested by GISAID. The analysis in Row 4 is length independent and defines the average mismatch probability per nucleotide in the probe sequence. ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com With the exception of one PCR Primer (N3, Locus, FP) which displayed an average mismatch frequency of 1.68%, the frequency of mismatches within the Primers was ≤1%, indicating that the prevalence of the mismatches was sporadic among the primers. It should be noted that the relatively higher values obtained for N3, Locus, FP was due to a mutation at the 25 th base from the 3' end of the primer, otherwise it would be equivalent to that observed for the N3 Primer (CDC N3 FP) in the published CDC assay (0.98%). The Middle Panel is an analysis of the last 5 bases at the 3' terminus of each of the PCR Primers used, relative to Primer mismatch frequency calculations for a published q-RT-PCR based test from the CDC (bold). The 3' terminus is known to be the region within a PCR Primer where mismatches will most readily affect PCR efficiency, due to its role as the initiation site for PCR elongation. Consequently, GISAID has suggested that this alternative PCR Primer Inclusivity analysis be used (GISAID, https://www.gisaid.org). Data are presented in two formats: Average Mismatch Frequency (%) in the 5-base-long 3' PCR primer sequence ( which is obtained by dividing the data in Row 3 by (5). With the exception of a single PCR Primer (N1, Locus, RP) which displayed an average mismatch frequency of 0.24% in its 3' region (identical to CDC's), the frequency of mismatches within all other PCR Primers was <0.1%, indicating that prevalence of the mismatches within the last 5 bases near the 3' terminus was sporadic among the Primer terminus region and occurred at a lower incidence than the overall incidence of mismatching throughout the full Primer region. The Bottom Panel is an analysis of the entire sequence of each Hybridization Probe used, relative to Probe mismatch frequency calculation for a published q-RT-PCR based test from the CDC (bold). Data are presented in two formats: ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com is as suggested by GISAID. That in Row 4 is length independent and defines the average probability per nucleotide in the probe. With the exception of 2 Probes (N1 RE. PCR primer inclusivity.-At the low incidence observed, the risk that the calculated PCR Primer mismatches would result in a significant loss in reactivity to cause a false negative result is extremely low due to the design of the PCR Primers of this assay, with melting temperatures of 65°C and with annealing temperature at 55°C that can thus tolerate up to 3 mismatches (i.e., 12% mismatching, so long as removed from the 3'terminus. Hybridization probe inclusivity.-Similarly, at the low incidence observed, the risk that the calculated Hybridization Probe mismatches would result in a significant loss in reactivity causing a false negative result is extremely low due to the design of the Hybridization Probes of this assay, with melting temperatures of 60°C and with hybridization temperature at 25°C that can thus tolerate up to 2 mismatches (8%) anywhere in the span of the probe. Results.- Figure 5 homology analysis to compare the sequence of the PCR primers used to support the 2-step tandem PCR reaction of the Enviro X -Rv test (i.e "Locus" and Labelling") for each of the 3 SARS-Cov-2 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com Page 20 of 32 domains analyzed (N1, N2, N3). The Figure also shows homology analysis for each of the microarray probes used in the Enviro X -Rv test to interrogate the PCR product of the tandem PCR assay. Analysis was performed on all available "High Quality Genomes", as defined by GISAID, resident in the GISAID database as of July 15, 2020 (@41 000 in total). Details of the analysis are described in more detail in the Appendix. In all cases, as suggested by GISAID in their own analysis of Inclusivity for the various q-RT-PCR assays, the PCR primer and q-RT-PCR probe sequences designed by the CDC have been used as a standard in these calculations. There is a single example where there is an "apparent" 40% loss of inclusivity relative to the CDC PCR Enviro X -Rv test is calculated to be comparable to Probe Inclusivity seen with the assay (CDC q-RT-PCR) chosen by GISAID as a reference standard. Results.-In silico analysis of the recommend organisms (Table 9 ) and the SARS-CoV-2 N1, N2 and N3 PCR primers was performed. No primer homology greater than 80% was detected among any of the microbial target sequences in the NCBI/Genbank non-redundant databases [GISAID databases only contain SARS-CoV-2/Influenza sequences]. The only partial homology detected was in the human genome for the N1-F Locus primer for Enviro X -Rv, which showed 90% complementarity to a single site within the Human genome. However, the homology included a 3' inhibitory mismatch, which would have been expected to disable N1-F primer function in PCR and there were no human sequences found suitable to serve as a complementary primer to N1-F within 20 kb of the above mentioned 90% homologous region. Further, the lack of cross-reactivity in the human genome has been confirmed among the numerous negative human matrix samples tested with no evidence of cross-reactivity. In addition, an in silico analysis demonstrated that the PCR amplicon derived from each of the 4 RT-PCR reactions (N1, N2, N3, RNaseP) produced an amplicon which would only hybridize to its cognate probes and not measurably to microarray probes specific for other SARS-CoV-2 regions or to any of the "species-specific" probes not engaged directly in SARS-CoV-2 analysis ( Table 1 ). The amplicon sequences for each SARS-CoV-2 target and RNaseP were compared using BLAST against each of the probe sequences in the array. Calculated cross-hybridization for 5 closely related SARS-CoV-2 probes, per GISAID were detected above the 80% cutoff: 86% PANG-1 and -2, 81% BAT2, 80% SARS-rel, 88% hCoV19 homology, none of these probes are utilized in the Enviro X -Rv assay and based on LoD and clinical experiments we did not observe a measurable cross-hybridization signal that impacted the correct SARS-CoV-2 call ( Figure 6 ). Results.-Cross reactivity analysis through wet-testing was also performed to determine the specificity of the Enviro X -Rv assay against closely related and recommended viruses and bacteria. The Exact Table 10 with no cross-reactivity detected. The remaining wet-testing samples will be conducted within 90 days of approval. We have been unable to obtain these samples rapidly due to delays with our vendors. Methodology.-The purpose of performing the thermodynamic folding simulations is to deduce if the PCR primers and probe are able to bind to their targets without substantial unfolding of the target. Primers that require substantial unfolding or the target are often "fragile" and can give false negatives if a mutation occurs at a primer binding site or if the salt concentrations vary slightly (e.g., due to a bad master mix lot or user intentionally diluting reagents). The steps below provide a recipe for determining the quality of the designs. The numbering referred to below is the numbering for the sense strand of the RNA virus. Use a program (e.g., MFOLD, RNAStructure, VisualOMP, etc.) to predict the secondary structure of the RNA and DNA target regions. An expert will evaluate the reported results to deduce if primers and probes are likely to have problems. We understand that providing primer and probe sequences are sensitive information and thus we request only the amplicon positions, though if the user does provide the primer and probe sequences, then we can provide more specific guidance can be given about the potential problems of the user's design. This is DG (total). Folding of the sequences (amplicons ± 150 nucleotides), primers, and probes were carried out with MFOLD program with the default conditions except the conditions specified in Table 2 and 4. In the folding of the target RNA, complementary DNA, and sense DNA sequences, the approximate locations and the start and the end of the amplicons are marked with red and blue arrows, respectively. The lowest energy structure for each molecule was presented in the file. We analyzed the RNA reverse primer binding region with additional 150 nts on either side of the amplicon, using MFOLD. In Table 11 , we display the resulting total DG of the reverse primer binding region at a temperature of 45°C, Mg2+ salt concentration at 1 mM, and default monovalent concentration. In Figures 7-9 , we have the MFOLD structures for each sequence. Make the reverse compliment of the target region and convert all U's to T's. This is the DNA complementary strand to which the Forward primer will bind (which we refer to as the cDNA target strand). Predict the secondary structure of the cDNA target region. Perform the folding using "DNA" as the strand type and use the annealing temperature for the PCR reaction (e.g., 60°C) and a salt concentration We analyzed the cDNA Forward Primer target region with additional 150 nts on either side of the amplicon, using MFOLD. In Table 12 , we display the resulting total DG of the reverse transcription primer binding region at a temperature of 55°C, Mg2+ salt concentration at 1 mM, and default monovalent concentration. In Figures 10-12 , we have the MFOLD structures for each sequence. If the probe binds to the antisense strand, then use the cDNA region described in step 3. If the probe binds to the sense strand, then use the region described in step 1, but convert all the U's to T's (call this "DNA sense strand"). Whichever is the appropriate strand, we will call this region the "probe-binding DNA target strand". Predict the secondary structure of the probe-binding DNA target strand. Perform the folding using "DNA" as the strand type and use the extension temperature for the PCR reaction (e.g., 72°C) and a salt We analyzed the cDNA of the Probe target region with additional 150 nts on either side of the amplicon, using MFOLD. In Table 13 , we display the resulting total DG of the PCR extension temperature at 25°C, Mg2+ salt concentration at 1 mM, and 600 mM Na+ concentration. In Figures 13-15 , we have the MFOLD structures for each sequence. For each primer and probe, predict their secondary structure. Perform the folding using "DNA" as the strand type and use the annealing temperature for the PCR reaction (e.g., 60°C) and a salt concentration We analyzed the DNA of the Primer and Probe target region with additional 150 nts on either side of the amplicon, using MFOLD. In Table 14 , we display the resulting total DG of the PCR extension temperature at 25°C, 45°C, or 55°C, Mg2+ salt concentration at 1 mM, and appropriate monovalent concentration (80 mM Primers and 600 mM Probes). Perform simulation of 2-state bimolecular hybridization for FP binding to the cDNA target (from step 3) under PCR salt conditions and using the annealing temperature. See Table 15 . Perform simulation of 2-state bimolecular hybridization for RP binding to the RNA target (from step 1) under the PCR salt conditions and using the reverse transcription temperature. See Table 16 . Perform simulation of 2-state bimolecular hybridization for RP binding to the DNA target (from step 1 but with U's converted to T's) under the PCR salt conditions and using the annealing temperature. See Table 17 . Perform simulation of 2-state bimolecular hybridization for Probe binding to the DNA target (from step 5) under the PCR salt conditions and using the extension temperature. See Table 18 . Methodology.-The SARS-CoV-2 isolate used for these studies, USA_WA1/2020, was isolated from the first documented US case of a traveler from Wuhan, China (5). This isolate was sourced from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). The virus stock was received from WRCEVA as a 1 mL lyophilizate. Upon receipt the lyophilizate was resuspended in 2 mL of PBS and singleuse aliquots frozen at -70°C. Table 19 summarizes the characteristics of the SARS-CoV-2 stock used for these studies. The PFU/mL quantitation information was provided by WRCEVA. GC/mL was determined by MRIGlobal as described below using one of the frozen viral stock aliquots. Table 20 . The synthetic RNA standard curve consisted of the following concentrations: 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 and 1×10 5 GC/ mL. SARS-CoV-2 virus stock was diluted in nuclease-free water for testing at the following dilutions: 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 . Master mix was prepared as noted in Table 21 . For the RT-PCR reaction, 15 µL of prepared master mix was added to each well followed by 5 µL of standard or sample, for a final total volume of 20 µL per reaction well. Both RNA standards and SARS-CoV-2 sample dilutions were run in triplicate wells. The GC/mL of the SARS-CoV-2 dilutions was determined using the slope and y-intercept of the synthetic RNA standard curve, as determined by linear regression analysis. The GC/mL of the virus stock was determined based on the average of the triplicate well results for all dilutions within the standard curve range. For the SARS-CoV-2 stock used for these studies, the concentration was calculated to be 1.6 × 10 9 GC/mL. Dilutions of SARS-CoV-2 virus stock were prepared in VTM from a frozen viral stock aliquot as shown in Table 23 . After drying overnight, test areas on the Reference Method test plates were sampled as follows: A swab was pre-moistened by dipping into a 15 mL conical tube containing 2.0 mL of VTM. The premoistened swab was used to sample the 2" × 2" test area by rubbing the swab in at least two different directions while applying pressure to the surface and rotating the swab head. It will take between 10-15 s to complete. After sampling the test area, the swab was snapped at the break point and placed back into the VTM tube. A random sample ID was assigned to each test area sample. Swab samples were placed in a refrigerator (2-8°C) within 15 min of test area sampling and stored overnight (22 h) before nucleic acid extraction. Components used in the Reference Method test plate sampling are listed in Table 24 . ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com Test areas on the Candidate Method test plates were sampled using the provided World Bioproducts swab samplers. Each swab sampler contains a swab fitted to a threaded cap inside a tube containing transport media. Briefly, the excess transport media was expressed from the wet swab by pressing on the inside of the collection tube above the level of the liquid. The pre-moistened swab was then used to sample the 2" × 2" test area by rubbing the swab back and forth in at least two different directions while applying pressure to the surface and rotating the swab head. After sampling the test area, the swab was screwed back into the collection tube containing transport media. Each sample tube was assigned a unique random ID number (a key correlating test area sample to random ID number was created and sent to AOAC). Swab samples were shipped overnight to PathogenDx with an ice pack the day of sampling. Components used in the Candidate Method test plate sampling are listed in Table 25 . Page 30 of 32 calculated for the candidate presumptive results, POD C and the reference method, POD R as well as the difference in the candidate and reference methods, dPOD C . The POD analysis between the Enviro X-Rv assay and the reference method indicated that there was a statistically significant difference when compared to the results of the CDC method utilized in this study, with the candidate method resulting in more positive replicates as it only requires 1 target to be present for a positive sample. A summary of POD analyses is presented in Table 27 . Individual results are provided in Table 28 . In the matrix study, the Enviro X-Rv assay successfully detected the target analyte from stainless steel environmental surface samples. The Enviro X-Rv method demonstrated a high level of specificity in detecting the target analyte in over 40 000 accession numbers from the GISAD database and showing low affinity for non-target organisms. The POD statistical analysis in Table 28 (PODc 0.90 vs PODr 0.55), indicated that the candidate method performance was statistically different than the reference method (95% CI 0.12, 0.61) with the candidate method detecting more positive samples. Based on the principle for detection of the candidate and CDC method, it is not surprising that a difference in the number of positive results was obtained. The CDC method result interpretation requires more than one signature being positive (N1 and N2), increasing the probability of a positive result being due to the presence of intact virus. This makes practical sense as the CDC method is designed for clinical use and has been adapted for surface detection with the WHO sampling method in this validation study. The Enviro X -Rv assay requires only a single target to be present (N1 and/or N2 and/or N3 conserved target regions), making it more likely to call a sample positive due to these RNA fragments than the CDC method which requires both targets to be present to be called positive. ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com The data from this study supports the product claim that the Enviro X-Rv assay can detect SARS-CoV-2 from stainless steel environmental surface samples (LOD of 2000 genomic copies per 2" × 2" test area). Data from the in silico analysis indicates the method is highly specific and can detect a wide range of target sequences and discriminate them from background organisms and near neighbors. The results obtained by the POD analysis of the method comparison study demonstrated that the candidate methods performance was not statistically different than that of the reference method. -36 .9 N/A 75.0 N/A a cDNA = Complementary DNA reverse transcribed from viral RNA. b Probe = Oligonucleotide probes for detection of viral target sequence in complementary DNA strand. c N/A = The probe is tethered to the surface, rather than in solution, and we are unable to accurately predict any secondary structure formation. d N3_Probe(T) = Degenerate nucleotide Y converted to T in the analysis. e N3_Probe(C) = Degenerate nucleotide Y converted to C in the analysis. -------Total 0/5 0/5 0/5 0/5 0/5 0/5 0/5 a N3 only positive targets may be positive for SARS-CoV-1 or SARS-CoV-2. Samples are considered positive. Refer to Table 8 CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel ScholarOne Support Recommended List of organisms to be analyzed in silico Human genome (taxid:9606) Streptococcus pneumoniae taxid:694009) Human parainfluenza virus 2 (taxid:11214) Human coronavirus 229E (taxid:11137) Respiratory syncytial virus (taxid:11250) Human coronavirus OC43 Human coronavirus HKU1 (taxid:290028) Parainfluenza virus 4b (taxid:11226) Human coronavirus NL63 (taxid:277944) Haemophilus influenza MERS-coronavirus (taxid:1335626) Legionella pneumophila Human Metapneumovirus (taxid:162145) Mycobacterium tuberculosis (taxid:1773) Adenovirus (taxid:1643649) Streptococcus pyogenes (taxid:1314) Parainfluenza virus 1 (taxid:11210) Bordetella pertussis (taxid:520) Parainfluenza virus 3 (taxid:11216) Mycoplasma pneumonia (taxid:2104) Parainfluenza virus 4a (taxid:11224) Pneumocystis jirovecii (taxid:42068) Influenza A (taxid:11320) Candida albicans (taxid:5476) Influenza B (taxid:11520 Pseudomonas aeruginosa Enterovirus (taxid:12059) Staphylococcus epidermis (taxid:1282) Parainfluenza virus 4a (taxid:1124) Streptococcus salivarius (taxid:1304) Parainfluenza virus 4b (taxid:1126) Chlamydia pneumonia Homology was obtained using NCBI BLAST using Accession nos. NC_045512.2 for COVID-19 and NM_006413.5 for RNAse P. *ND -Less than 25% calculated homology with PCR Primer generated targets Homology <80% is not expected to contribute to hybridization signals as discussed by FDA SARS specificity control -Probes with SARS sequence within test loci.