Validation of the Thermo Scientific^™ SARS-CoV-2 RT-PCR Detection Workflow for the Detection of SARS-CoV-2 from Stainless-Steel Environmental Surface Swabs: AOAC Performance Tested Method^SM 012103

Abstract

Background

The Thermo Scientific^™ SARS-CoV-2 reverse transcription–polymerase chain reaction (RT-PCR) Detection Workflow, packaged with Applied Biosystems™ TaqMan™ 2019-nCoV Assay Kit v1 targets three different SARS-CoV-2 genomic regions in a single RT-PCR reaction.

Objective

To validate the Thermo Scientific SARS-CoV-2 RT-PCR Workflow, for the detection of SARS-CoV-2 virus on stainless-steel surfaces as part of the AOAC Performance Tested Method^SM Emergency Response Validation program.

Method

The Applied Biosystems TaqMan 2019-nCoV Assay Kit v1, as part of the Thermo Scientific SARS-CoV-2 RT-PCR Workflow, was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms. The Thermo Scientific SARS-CoV-2 RT-PCR Workflow was evaluated in an unpaired study for one environmental surface (stainless steel) and compared to the U.S. Centers for Disease Control and Prevention 2019-Novel Coronavirus RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020).

Results

In silico analysis showed that, of the 15 756 target SARS-CoV-2 genomes analyzed, 99% of the strains/isolates are perfectly matched to at least two of the three assays, and more than 90% have 100% homology to all three assays (ORF1ab, N-gene, S-gene) in the SARS-CoV-2 Kit. None of the 65 non-target strain genomes analyzed showed matching sequences. In the matrix study, the Thermo Scientific SARS-CoV-2 workflow showed comparable detection to the centers of disease control and prevention (CDC) method.

Conclusions

The Thermo Scientific SARS-CoV-2 RT-PCR Workflow is an effective procedure for detection of RNA from SARS-CoV-2 virus from stainless steel.

Highlights

The workflow provides equivalent performance results with the two tested RNA extraction platforms and the two tested RT-PCR instruments.

General Information

SARS-CoV-2 is a public health issue, and not a food-borne virus. However, it is obvious that some food production segments have faced contamination in the working environment. Coronaviruses have been shown to survive (remain viable) on different surfaces, for up to 28 days, under various conditions including temperature, relative humidity, and light (1). SARS-CoV-2 can be transmitted when a healthy person touches contaminated food or environmental contact surfaces (including packaging materials) (2).

Proper cleaning, surveillance, and the prevention of cross-contamination are critical in the control of foodborne illnesses. The application of sound principles of environmental sanitation, personal hygiene, and established food safety practices reduces the likelihood that harmful pathogens, including SARS-CoV-2, may threaten the safety of the food supply, regardless of how it is produced. In July 2020, Thermo Fisher Scientific applied to the SARS-CoV-2 Emergency Response Validation (ERV) program of the AOAC Research Institute’s (RI) Performance Tested Method(s)^SM (PTM) program for the detection of SARS-CoV-2 on environmental surfaces.

Principle

The Applied Biosystems™ TaqMan™ 2019-nCoV Assay Kit v1 contains a set of TaqMan reverse transcription–polymerase chain reaction (RT-PCR) assays for the detection and characterization of SARS-CoV-2. The kit includes three assays that target SARS-CoV-2 genes and one positive control assay that targets the human RNase P RPPH1 gene. Features of the TaqMan 2019-nCoV Assay Kit include: 2019-nCoV assays targeting three different viral genomic regions that are run together in a single PCR reaction and detected on the same channel, highly specific target sequences generated by robust bioinformatic selection that are unique to SARS-CoV-2 and show no cross-reactivity with one another, and an RNase P internal positive control run in duplex with the 2019-nCoV assays.

The TaqMan 2019-nCoV Assay Kit is used with the TaqMan 2019-nCoV Control Kit v1 to monitor assay-specific amplification. Additionally, the TaqMan 2019-nCoV Control Kit v1 is a synthetic positive control; the TaqMan 2019-nCoV Control Kit v1 contains target sequences for each of the assays included in the TaqMan 2019-nCoV Assay Kit v1. The control includes synthetic DNA target sequences for three SARS-CoV-2 genes (ORF1ab, S protein, and N protein) and the human RNase P RPPH1 gene. During RT-PCR, 1 μL of the 2019-nCoV Control v1 in 9.75 μL PCR-grade water is used as the sample for the positive-control reaction.

The positive control is intended to demonstrate correct setup of PCR reagents and the correct extraction protocol has been conducted, as well as presence of PCR inhibitors which may negatively impact the other targets. As long as a positive reaction is observed in each well for the positive control, PCR is proven to be a success. The control can be used in SARS-CoV-2 detection to verify assay performance and to help with troubleshooting.

Both the TaqMan 2019-nCoV Assay Kit and the TaqMan 2019-nCoV Control Kit v1 use Applied Biosystems TaqMan^® Minor Groove Binder (MGB) probes .

Scope of Method

• Analyte.—SARS-CoV-2 virus.

• Matrixes.—Stainless-steel environmental surface (e.g., such as those in production or laboratory settings).

• Summary of validated performance claims.—The Thermo Scientific SARS-CoV-2 Real Time PCR Workflow demonstrated comparable performance to the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time Diagnostic Panel Instructions for Use (Revision 4, Effective 6/12/2020; 2) for the detection of SARS-CoV-2 on stainless steel.

Definitions

• 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. 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).

• Difference of probabilities of detection (dPOD).—Difference of probabilities of detection is the difference between any two POD values. If the confidence interval (CI) of a dPOD does not contain zero, then the difference is statistically significant at the 5% level (3).

• In silico.—The use of computer simulation to evaluate target and non-target sequences for molecular methods.

Materials and Methods

Test Kit Information

• Kit names and catalog numbers.—The Thermo Scientific SARS-CoV-2 Real Time PCR Workflow using PrepSEQ™ Nucleic Acid Extraction Kit for Food and Environmental Testing with KingFisher™ Flex-96 Deep-Well Magnetic Particle Processor or MagMAX™ Express-96 Deep well Magnetic Particle Processor, TaqMan 2019-nCoV Assay Kit v1, TaqMan 2019-nCoV Control Kit v1 (Cat. No. A47533), RNA UltraSense™ One-Step qPCR Master Mix on Applied Biosystems QuantStudio™ Design and Analysis Software v1.5.1 or later or Applied Biosystems 7500 Software SDS v.1.4.2 or later.

• Ordering information.—All materials are available through the Thermo Fisher Microbiology ordering process or https.//thermofisher.com.

Test Kit Components

RNA extraction kit (one of the following):

• PreSEQ Nucleic Acid Extraction Kit (cat. No. 4480466; 100 total NA isolations).

  1. Lysis buffer.—2 × 50 mL.

  2. Magnetic particles.—2 × 1.5 mL.

  3. Binding solution (isopropanol).—One empty bottle provided.

  4. Wash buffer concentrate.—2 ×26 mL.

  5. Elution buffer.—1 × 25 mL.

  6. Proteinase K buffer.—1 × 1.25 mL.

• PrepSEQ Nucleic Acid Exctraction Kit (cat. No. 4428176; 300 total NA isolations).

  1. Lysis buffer.—6 × 50 mL.

  2. Magnetic particles.—6 × 1.5 mL.

  3. Binding solution (isopropanol).—Three empty bottles (user provides).

  4. Wash buffer concentrate.—6 × 26 mL.

  5. Elution buffer.—3 × 25 mL.

  6. Proteinase K buffer.—3 × 1.25 mL.

Automated Nucleic Acid Extraction System and Materials

Extraction instrument (one of the following):

• KingFisher Flex 96 Deepwell System.

  1. KingFisher Flex Magnetic Particle Processor with 96 Deepwell Head (Cat. No. A32681).

  2. KingFisher Flex Deepwell 96 Plate (Cat. No. 95040450).

  3. KingFisher 96 tip comb for Deepwell

     magnets (Cat. No. 97002534).

• MagMAX Express-96 Deepwell Magnetic Particle Processor.

  1. MagMAX Express-96 Deepwell Particle Processor with 96 Deepwell Head.

  2. KingFisher Flex Deepwell 96 Plate.

  3. KingFisher 96 tip comb for DW magnets.

Assay Kits

• TaqMan 2019nCoV Assay Kit v1 (Cat. No. A47532).

  1. 2019-nCoV (ORF1ab) Assay, FAM dye, 75 µL, 20X.

  2. 2019-nCoV (S protein) Assay, FAM dye, 75 µL, 20X.

  3. 2019-nCoV (N protein) Assay, FAM dye, 75 µL, 20X.

  4. RNAse P Assay, VIC dye, 250 µL, 20X.

• TaqMan 2019nCoV Control Kit v1 (Cat. No. A47533).—One tube containing 50 µL (1 x 10⁴ copies/µL).

Master Mix

• RNA UltraSense One-Step Quantitative RT-PCR System (Cat. No. 11732927).

  1. 250 µL RNA UltraSense Enzyme Mix.

  2. 1 mL RNA UltraSense 5X Reaction Mix.

  3. 300 µL 20X Bovine Serum Albumin (BSA).

  4. 1 mL 50-mM magnesium sulfate (MgSO₄).

  5. 100 µL ROX Reference Dye.

• Total RNA Control (Human, Cat. No. 4307281 or equivalent).

Real-Time PCR Instrument

Use one of the following:

• Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System (Cat. No. A36328 OR A36320).—96-well, 0.1 mL block, laptop, QuantStudio Design Analysis Software v1.5.1 or later (included).

• Applied Biosystems 7500 Fast Food Safety Real-Time PCR System (Cat. No. A30299 OR A30304).—96-well, 0.1 mL block, laptop, 7500 Software SDS Software v1.4 or later (included).

Apparatus

• Laboratory freezers, –30 to –10°C and ≤–70°C.

• Centrifuge, with a rotor that accommodates deepwell microplates.

• Microcentrifuge.

• Laboratory mixer, vortex, or equivalent.

• Single and multichannel adjustable pipettors.—1.00 to 1000 µL.

• Cold block.—96 well or 384 well or ice.

• MicroAmp™ Optical 96-Well Reaction Plate, 0.1 mL (Cat. No. 4346907 or equivalent)or MicroAmp Optical 8-Cap Strip (Cat. No. 4323032).

• MicroAmp Optical Adhesive Film (Cat. No. 4313663 or equivlalent) or MicroAmp Optical Fast 8-Tube Strip (0.1 mL; Cat. No. 4358293).

• Disposable gloves.

• Micropipet tips, aerosol resistant.

• Non-cellulose swabs with synthetic tip.

Reagents

• Thermo Scientific Oxoid™ Phosphate Buffered Saline Tablets (Cat. No. BR0014G).

• Ethanol, 95%.

• Isoproranol, 100%.

• Nuclease-free water.

Safety Precautions

• Resources.—The Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow should be performed by qualified and trained staff to avoid the risk of erroneous results. Samples should always be treated as if infectious and/or biohazardous in accordance with safe laboratory procedures.

  Follow necessary precautions when handling specimens. Use personal protective equipment (PPE) consistent with current guidelines for the handling of potentially infectious samples. Laboratories shall implement the method and ensure data reporting, in compliance with local regulations.

• Surface sampling and sample transport.—Follow good laboratory practices and all precautions and guidelines in these user guides to avoid cross-contamination between samples.

  Samples must be collected, transported, and stored using appropriate procedures and conditions. Improper collection, transport, or storage of specimens may hinder the ability of the assay to detect the target sequences.

  Refer to the ISO 15216–1: 2017 standard (4) and the “Surface sampling of coronavirus disease (COVID-19): A practical ‘how to’ protocol for health care and public health professionals”, published by the World Health Organization (WHO) (5).

• Real-time PCR.—This workflow uses purified RNA as a sample for analysis which is processed through an RT-PCR. The quality of the RNA recovered from environmental samples is essential for the quality of the results generated with the method. Following the completion or partial completion of a real-time PCR run, it is important that PCR tubes are not opened, because amplified DNA (amplicon) in the tubes could easily contaminate the equipment and surrounding laboratory environment, potentially leading to false-positive results in future analyses. The reagents should not pose a safety concern when used as directed in the method instructions. Dispose of waste lysate, eluted RNA, and PCR tubes according to local guidelines.

  Refer to the “Real-Time PCR Instrument Manual” for guidelines on cleaning equipment and handling possible amplicon contamination. Positive and negative test controls must be included to accurately interpret test results.

• Consumables and reagents.—For disposal of surface sampling or any of the reagents and materials included in the Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow and associated tests, refer to the manufacturer’s material safety data sheets and apply appropriate local guidelines.

Sample Collection and Storage

• Pre-moisten swab with phosphate buffered saline (PBS) or viral transport medium (VTM; the workflow utilized PBS in this study).

• When sampling, apply pressure with the wet swab onto the surface, move in at least two different directions while rotating the swab stick. Avoid letting the swab dry completely. Swab an area up to 2 × 2 inches, ensuring that the full area is covered in the sampling.

• Place the swab back into the transport tube containing 2 mL PBS.

• Place swabs at refrigeration temperature (2–8°C) no more than 15 min post sampling and maintain refrigeration until analysis.

• Analyze the swabs, preferably within 24 h. If the swabs are not likely to be analyzed within 48 h maximum, store the swabs, preferably between −70 and −80°C, and ship on dry ice.

Sample Preparation and RNA Extraction

The KingFisher Flex Purification System with 96 Deepwell Head or the MagMAX Express-96 Deepwell Magnetic Particle Processor can be used to extract RNA with the PrepSEQ Nucleic Acid Extraction Kit for Food and Environmental Testing.

• Ensure that the KingFisher Flex Magnetic Particle Processor with 96 Deepwell Head is set up with the KingFisher Flex 96 Deepwell Heating Block.

• Ensure that the PSNA_Flex_300ul script has been downloaded from the product page and loaded onto the instrument using the BindIt™ software. Use PSNA_MagMAX_300ul if you are using the MagMAX Express-96 Deepwell Magnetic Particle Processor.

• Prepare the PrepSEQ Nucleic Acid Extraction Kit reagents:

  1. Add 74 mL 95% ethanol to the wash buffer concentrate bottle.

• Add 35 mL 100% isopropanol to an empty binding solution bottle.

• Incubate the tube of magnetic particles at 37 ± 1°C for approximately 10 min.

Prepare the extraction processing plates and label the side of the processing plates according to Table 1. Cover the plates with an adhesive film, then store at room temperature for up to 1 h while setting up the sample plate.

Table 1.

Prepare extraction processing plates

Plate ID	Plate type	Reagent	Volume per well, μL
Sample	KingFisher Deepwell 96 Plates	Binding bead mix	700
Wash plate 1		Wash solution	300
Wash plate 2		Wash solution	300
Elution plate		Elution buffer	100
Tip comb plate	Place a KingFisher 96 tip comb for deepwell magnets in a KingFisher 96 microplate

Prepare the required amount of lysis bead mix on each day of use:

• Vortex the total nucleic acid magnetic beads to ensure that the bead mixture is homogeneous.

• For the number of required reactions, prepare the binding bead mix according to Table 2.

• Invert the binding bead mix gently five times, to mix, then add 700 μL to each sample well and the negative extraction control well in the sample plate.

  Note: Remix the binding bead mix by inversion frequently during pipetting to ensure even distribution of beads to all samples or wells. The binding bead mix is viscous, so pipet slowly to ensure that the correct amount is added. Do not reuse pipet tips to add binding bead mix to the samples, as the high viscosity will cause variations in the volumes added.

• Add 1 μL total human RNA control to each sample well and the negative extraction control well in the sample plate.

• Vigorously mix each sample tube containing the swab by vortexing for 30 s.

• Add 300 μL sample to each sample well.

• Add 300 μL nuclease-free water (not deionized and diethylpyrocarbonate (DEPC) treated) to the negative extraction control well in the sample plate.

• Select the PSNA_Flex_300ul on the KingFisher Flex Magnetic Particle Processor with 96 Deepwell Head. The PSNA_MagMAX_300ul script should be used with the MagMAX Express 96 DW Magnetic Particle Processor.

• Start the run, then load the prepared plates into position when prompted by the instrument.

• After the run is complete (∼40 min after start), immediately remove the elution plate from the instrument, then cover the plate with an adhesive film.

• Place the rlution plate on ice for immediate use in RT-PCR.

Table 2.

Prepare lysis bead mix

Component	Volume per well, μLa
Proteinase K buffer	340
Binding solution	325
Total nucleic acid magnetic beads	25
Proteinase K	10
Total binding bead mix per well	700

^a Include 10% overage when making for multiple reactions.

Note: RNA can be stored at –70°C for long term storage (up to 1 year).

RT-PCR and Data Analysis

• Use purified, non-degraded total nucleic acid that is free of RNase activity and RT-PCR inhibitors.

• Protect the assays and master mix from light.

• For each research sample, include the primers and probes for all three 2019-nCoV targets (FAM™ assay) in multiplex with the RNase P target (VIC™ assay).

• Before beginning, determine the number of required reactions. In addition to the nucleic acid test samples, include the following reactions:

  1. One negative extraction control per plate.

  2. One 2019-nCoV Control reaction per plate.

  3. One no-template control (NTC) per plate.

• Perform PCR using 2019-nCoV assay kit and 2019-nCoV control kit combined with the RNA UltraSense Master Mix.

PCR Setup using RNA UltraSense Master Mix

• On ice or using a cold block, prepare an RT-PCR Master Mix for the number of reactions required plus 10% overage. See Table 3.

• For each reaction combine the following components in PCR reaction tubes or plates as in Table 4.

• Cap or seal the reaction vessels, and gently tap on the bench top to make sure that all components are at the bottom of the amplification tube. Centrifuge briefly.

  Note: It is essential that no bubbles remain in the PCR reaction tubes. If bubbles remain after centrifugation, gently tap the reaction plate/tubes on the bench top and then briefly centrifuge again at a higher speed until all bubbles are removed.

• Place the PCR reaction tubes in a thermal cycler programmed as described below. The following software should be used with each instrument (Table 5).

• Set up and run real-time PCR instrument (Table 6).

Table 3.

Prepare RT-PCR master mix

Component	Volume per reaction (μL)
RNA UltraSense Enzyme Mix	1.0
RNA UltraSense 5X Reaction Mix	4.0
2019-nCoV ORF1ab Assay (20X)	1.0
2019-nCoV N Protein Assay (20X)	1.0
2019-nCoV S Protein Assay (20X)	1.0
RNAse P assay (20X)	1.0
MgSO4 (50 mM)	0.25
Total volume per reaction	9.25

Table 4.

RT-PCR master mix

Component	Volume per reaction, μL
Reaction mix	9.25
Sample (elution from RNA extraction) or 1 μl 2019-nCoV Control v1 + 9.75 μl RT-PCR grade water or NTC (RT-PCR grade water)	10.75
Total reaction volume	20.0

Table 5.

Instruments and software

Instrument	Software
7500 Fast Food Safety Real-Time PCR System	7500 Fast SDS v1.4 or later
QuantStudio 5 Food Safety Real-Time PCR System	QuantStudio Design and Analysis Software v1.5.1 or later

Table 6.

Thermal cycler program

Step	Stage	Temperature °C	Time
Reverse transcription	Hold	50	15 min
Polymerase activation	Hold	95	2 min
Amplification	45 cycles	95	3 s
		60	30 s
		Collect data

Software and Analysis

For more information about using a software, see the software user guide or help.

• Open the data file (EDS) in the same software used to run PCR.

• Perform analysis using the following settings (Table 7).

• Analyze the run with no reference dye.

• For each plate, confirm that the control reactions perform as expected (Table 8).

• Review all of the results for the 2019-nCoV assay to ensure that all positive results are detected. Evaluate the overall shape of the amplification curves. An exponential and continuous increase in signal amplification indicates positive amplification.

• Export the results.

Table 7.

Settings to perform analysis

Target	Baseline	Threshold
2019-nCoV assay (FAM dye)	Auto	Auto
RNAse P assay (VIC dye and IPC)

Table 8.

Confirm control reactions

Reaction	2019-nCoV assay (FAM dye)	RNAse P assay (IPC) (VIC dye)	About expected result
NTC	Undetermined		If the NTC has an amplification curve that crosses the threshold (false positive), sample contamination may have occurred. Repeat the test with new reagents, following good RT-PCR practices.
Negative extraction control	Undetermined	Ct < 40	If a Ct value is detected in the 2019-nCoV assay then contamination may have occurred during extraction. Repeat the test with new reagents.
2019-nCoV Control v1	Ct ≤ 35	Ct < 40	If the Ct value for either assay is above the expected result, repeat the test with new reagents.

Review Results

• Positive signals.

  1. Amplification curves are considered positive if they have a sudden increase in fluorescence into an exponential signal.

  2. Shallow or linear amplification curves are acceptable as positive if there is a continuous increase in fluorescence with little or no flattening of the curve after the initial increase.

• Note: If the amplification signal is weak, the software can be manipulated to allow for easier interpretation of results. This can be achieved with the following steps:

  1. Set the baseline to remove any anomalous data, e.g., 12–25 cycles.

  2. Remove the threshold by setting it to 1.

  3. Change the graph view to linear.

  4. Change the y axis range to minimum –100 and maximum 1 000 000.

  5. Change the x axis range to 12–45 cycles.

• (b) Negative signals.

  1. Amplification curves are considered negative if they have no continuous increase in fluorescence.

  2. If an increase in fluorescence is present, the signal must be minor and reach a plateau rapidly or decrease to result in no overall continuous increase in fluorescence.

(c) Review the amplification plots for the RNase P assay (IPC) and classify according to Table 9.

Table 9.

Interpretation of results

2019-nCoV assay (FAM dye)	RNase P assay (IPC) (VIC dye)	2019-nCoV assay result
Clear positive signal	Any valuea	Positive
Weak positive signal	Ct < 40	Positive
	Ct = Undetermined or Ct ≥ 40	Invalid. Proceed to troubleshooting.
No positive signal	Ct < 40	Negative
	Ct = Undetermined or Ct ≥ 40	Invalid. Re-purify the nucleic acid from the sample, then repeat the test.

^a An RNase P positive result is expected in most reactions. However, when a strong positive signal is detected in the 2019-nCoV assay the RNase P assay can occasionally give a negative result. These samples should be treated as positive if the amplification curve for the 2019-nCoV assay appears normal.

(c) For each test sample, interpret the results using the table 10. It is recommended that each lab perform accuracy testing with appropriate samples to establish guidelines for interpreting results. SeeTable 10.

Table 10.

Guidelines for interpreting results

2019-nCoV assay result	Interpretation of results
Clearly positive	High risk positive. Employ full hygiene intervention strategy.
Weak positive	Lower risk positive. Use risk assessment to employ appropriate hygiene intervention strategy.
Negative	No need for hygiene intervention strategy.

(d) SeeTable 11 for troubleshooting guidance.

Table 11.

Troubleshooting guidance

Observation	Possible cause	Recommended action
Inhibition of downstream PCR, indicated by non-detection of IPC reaction in samples with no or late target-specific signal.	Magnetic particles were in the elution plate.	Avoid disturbing the magnetic particles during transfer of eluted RNA to the lyophilized assay. Avoid transfer of magnetic particles using one of the following methods (optional): Place the Elution Plate on the 96-well magnetic ring stand during transfer of eluted RNA sample to the assay. Spin the plate at maximum speed in a plate centrifuge for the equivalent of approximately 4000 × g for approximately 30 s, to pellet the magnetic particles to the bottom of the plate.
	Elution plate contains other inhibitory substances.	Dilute the eluted RNA 1:5 with nuclease-free water to dilute PCR inhibitors and repeat the assay. If PCR remains inhibited, repeat the sample preparation.
In positive control wells, no target-specific signal is detected.	Positive control was omitted (pipetting error).	Repeat the assay. Make sure to pipet the positive control into all positive control wells.
In positive control wells or unknown sample wells, no IPC signal is detected, but target-specific signal is detected.	A high copy number of target RNA exists in samples, resulting in preferential amplification of the target-specific RNA.	No action is required. The result is considered positive if the target-specific amplification curve appears normal.
In negative extraction control wells, target-specific signal is detected.	Carryover contamination occurred.	Repeat the assay using fresh aliquots of all reagents and clean pipetting equipment. If the negative extraction control continues to show contamination, repeat the assay using a new kit. If the negative extraction control continues to show contamination, contact Technical Support.
Amplification curves appear abnormal (differ from an exponential and continuous increase).	Auto baseline settings cannot account for large deviations in CT values between samples.	Manually adjust the baseline settings for the sample under investigation.
	Inhibitory substances carried over from RNA extraction.	Dilute the eluted RNA 1:5 with nuclease-free water to dilute PCR inhibitors and repeat the assay. If PCR remains inhibited, repeat the sample preparation.
	Primers and probes have degraded.	Repeat the PCR assay using fresh reagents.

Validation Study

This validation study was conducted under the AOAC RI PTM program according to the procedures outlined in the AOAC RI ERV PTM Study Outline: Validation Outline for Molecular Methods that Detect SARS-CoV-2 on Surfaces (V10, July 2020). The inclusivity/exclusivity in silico evaluation was conducted by Thermo Fisher Scientific (San Francisco, CA; Vantaa, Finland; and Basingstoke, United Kingdom). The matrix study consisted of swabs taken from 2 × 2” stainless-steel surface areas. The surfaces were prepared and sampled by the independent laboratory, MRI Global (Kansas City, MO). Swabs were shipped blind-coded to Food Safety Net Services (FSNS; San Antonio, TX) for analysis by the Thermo Scientific SARS-CoV-2 RT-PCR Workflow. MRI Global analyzed the corresponding unpaired swabs by the CDC 2019-nCoV RT-PCR method. Additional PTM parameters, robustness, and product consistency and stability will be submitted for full PTM certification by March 31, 2021.

Inclusivity/Exclusivity Study

The in silico analysis of the primer and probe sequences was conducted according to the Validation Outline provided by AOAC RI using Appendix 1 with the minimal set of global initiative on sharing all influenza data (GISAID) accession numbers for the inclusivity testing, and Appendix 2 with the minimal set environmental genomes for the exclusivity testing.

The selection and design of the primer and probe sequences were developed according to similar quality measures, i.e., unimolecular folding, bimolecular hybridization.

In silico analysis was performed to determine inclusivity (reactivity) and exclusivity of the primer and probe sequences according to the Wuhan in silico Reactivity-Inclusivity Study Protocol. BLASTN 2.6.0+ version was used. Blast was performed using ncbi_blastn -query assays.fas -word_size 7-db <organism genome database>. 15, 829 sequences were assessed: 15, 764 SARS-CoV-2 genomes from GISAID and GenBank Viral national center for biotechnology information (NCBI databases) were assessed, eight near neighbor genomes, 26 environmental virus genomes, 23 environmental bacteria and fungi genomes, and eight eukaryotes environmental genomes. Homology was calculated using: %homology= identity_hsp/len_comp. identity_hsp: Identical nucleotide in the blast hsp (high scoring segment pair). Len_comp: length of the primer or probe. Individual components (forward and reverse primers and probe) of each were realigned against the sequence database. Alignments are reported for which of the three components (forward and reverse primers and probes) resulting in an alignment signal with the blast parameters listed above.

Inclusivity Study Results

A total of 15 756 accessions were tested from 15 764 accessions listed. The remaining eight sequences were withdrawn from the GISAID repository by the submitter.

Of the 15 756 strains/isolates analyzed, more than 90% have 100% homology to all three assays (ORF1ab, N-gene, S-gene) in the SARS-CoV-2 Kit and 99% have 100% homology to at least two of the three assays. The strains/isolates without 100% homology to at least two assays are shown in Table 12. The 15 756 tested genomes are predicted to be detected by at least one of the three 2019-nCoV assays. Continuous monitoring of the inclusivity/homology shall be conducted as new genomes are added to the databases.

Table 12.

Inclusivity results

Strain name	GenBank accession	Assay	Forward primer homology, %	Reverse primer homology, %	Probe homology, %
hCoV-19/Australia/VIC505/2020|EPI_ISL_426776|2020–03-22	EPI_ISL_426776	N gene	96	100	100
hCoV-19/Australia/VIC644/2020|EPI_ISL_426940|2020–03-28	EPI_ISL_426940	ORF1ab	96	100	100
hCoV-19/Australia/VIC644/2020|EPI_ISL_426940|2020–03-28	EPI_ISL_426940	S gene	100	96	100
hCoV-19/Australia/VIC648/2020|EPI_ISL_426944|2020–03-28	EPI_ISL_426944	ORF1ab	88	100	100
hCoV-19/Australia/VIC951/2020|EPI_ISL_430646|2020–03-26	EPI_ISL_430646	ORF1ab	96	100	100
hCoV-19/Australia/VIC951/2020|EPI_ISL_430646|2020–03-26	EPI_ISL_430646	S gene	100	96	100
hCoV-19/Brazil/CV17/2020|EPI_ISL_429677|2020–03-11	EPI_ISL_429677	ORF1ab	88	100	100
hCoV-19/Brazil/CV41/2020|EPI_ISL_429694|2020–03-18	EPI_ISL_429694	ORF1ab	88	100	100
hCoV-19/Brazil/CV46/2020|EPI_ISL_429699|2020–03-20	EPI_ISL_429699	N gene	78	78	100
hCoV-19/Canada/ON_PHL3917/2020|EPI_ISL_418322|2020–03-08	EPI_ISL_418322	ORF1ab	88	100	100
hCoV-19/Canada/ON_PHL3917/2020|EPI_ISL_418322|2020–03-08	EPI_ISL_418322	S gene	96	100	100
hCoV-19/Canada/ON_PHL3919/2020|EPI_ISL_418323|2020–03-14	EPI_ISL_418323	ORF1ab	88	100	100
hCoV-19/Canada/ON_PHL3919/2020|EPI_ISL_418323|2020–03-14	EPI_ISL_418323	S gene	96	100	100
hCoV-19/England/20104035803/2020|EPI_ISL_417238|2020–03-03	EPI_ISL_417238	ORF1ab	88	100	100
19/England/201361007/2020|EPI_ISL_421784|2020–03-26	EPI_ISL_421784	ORF1ab	100	96	100
hCoV-19/England/201380276/2020|EPI_ISL_421826|2020–03-23	EPI_ISL_421826	ORF1ab	100	96	100
hCoV-19/England/201380277/2020|EPI_ISL_421827|2020–03-24	EPI_ISL_421827	ORF1ab	100	96	100
hCoV-19/England/20139021104/2020|EPI_ISL_421880|2020–03-26	EPI_ISL_421880	S gene	96	100	100
hCoV-19/England/20146017604/2020|EPI_ISL_423580|2020–04-02	EPI_ISL_423580	ORF1ab	100	96	100
hCoV-19/France/OCC-4/2020|EPI_ISL_434619|2020–03	EPI_ISL_434619	ORF1ab	100	100	95
hCoV-19/France/OCC-4/2020|EPI_ISL_434619|2020–03	EPI_ISL_434619	S gene	96	100	100
hCoV-19/Iceland/143/2020|EPI_ISL_417794|2020–03-12	EPI_ISL_417794	N gene	96	100	100
hCoV-19/Iceland/194/2020|EPI_ISL_417821|2020–03-16	EPI_ISL_417821	ORF1ab	88	100	100
hCoV-19/Iceland/28/2020|EPI_ISL_417767|2020–03-03	EPI_ISL_417767	N gene	96	100	100
hCoV-19/Iceland/545/2020|EPI_ISL_424565|2020–03-26	EPI_ISL_424565	ORF1ab	100	96	100
hCoV-19/Iceland/86/2020|EPI_ISL_417870|2020–03-10	EPI_ISL_417870	N gene	96	100	100
hCoV-19/India/nimh-0834/2020|EPI_ISL_428485|2020–04-12	EPI_ISL_428485	N gene	100	100	0
hCoV-19/Mexico/Chiapas-InDRE_02/2020|EPI_ ISL_424666|2020–02-29	EPI_ISL_424666	S gene	100	68	100
hCoV-19/Netherlands/Gelderland_8/2020|EPI_ISL_ 422644|2020–03-13	EPI_ISL_422644	ORF1ab	88	100	100
hCoV-19/Netherlands/Gelderland_8/2020|EPI_ISL_ 422644|2020–03-13	EPI_ISL_422644	N gene	100	100	0
hCoV-19/USA/CA-CDPH-UC25/2020|EPI_ISL_417329|2020–03-04	EPI_ISL_417329	N gene	100	100	0
hCoV-19/USA/CA-CZB027/2020|EPI_ISL_429068|2020–03-25	EPI_ISL_429068	S gene	100	76	100
hCoV-19/USA/CA-CZB04/2020|EPI_ISL_429069|2020–03-25	EPI_ISL_429069	S gene	100	76	100
hCoV-19/Wales/PHWC-2433A/2020|EPI_ISL_419398|2020–03-18	EPI_ISL_419398	N gene	96	100	100
hCoV-19/Wales/PHWC-243FE/2020|EPI_ISL_419408|2020–03-17	EPI_ISL_419408	ORF1ab	100	96	100
hCoV-19/Wales/PHWC-243FE/2020|EPI_ISL_419408|2020–03-17	EPI_ISL_419408	S gene	96	100	100
hCoV-19/Wales/PHWC-24C11/2020|EPI_ISL_419500|2020–03-20	EPI_ISL_419500	S gene	100	100	0
hCoV-19/Wales/PHWC-254CD/2020|EPI_ISL_420979|2020–03-22	EPI_ISL_420979	ORF1ab	100	96	100
hCoV-19/Wales/PHWC-254CD/2020|EPI_ISL_420979|2020–03-22	EPI_ISL_420979	S gene	72	100	100
hCoV-19/Wales/PHWC-2704F/2020|EPI_ISL_422376|2020–03-28	EPI_ISL_422376	S gene	72	100	100
SARS-CoV-2/human/IRN/HGRC-01-IPI-8206/2020	MT281530.2	ORF1ab	88	100	100
SARS-CoV-2/human/USA/CT-UW-5036/2020	MT375469.1	ORF1ab	92	100	100
SARS-CoV-2/human/USA/WA-UW-1963/2020	MT326080.1	ORF1ab	100	64	100
SARS-CoV-2/human/USA/WA-UW-2225/2020	MT345837.1	S gene	96	100	100

Exclusivity Study Results

Analysis was conducted for the assays for potential off-target hybridization against the exclusivity listed in the Appendix 2 of the Validation Outline. Complete genomes were used for all. The background list was evaluated, and complete genomes were used when available. There were no primer/probe combinations where a homology was observed for more than one target sequence. The worst-case scenario is shown in Table 13.

Table 13.

Exclusivity worst case scenario

Assay_name	Blast_subject_id	forward primer homology, %	prob, homology, %	reverse primer homology, %
66792_S	Aedes albopictus NW_021837454.1	0	100%	0

Of the 65 non-target genomes analyzed against the primers and probes of the TaqMan 2019-nCoV Assay Kit v1, none showed matching sequences.

Quality Assessment of the Unimolecular Folding and Bimolecular Hybridization

The selection and design of the PCR primers and probes were done according to Thermo Fisher Scientific TaqMan assay development, good practices, and quality management procedures to avoid substantial unfolding of the target. This was done using an internal mapping tool for TaqMan Assays, that are proprietary to Thermo Fisher Scientific. Additionally, in regard to in silico analysis requirements, all analyses were performed using the RNAStructure program (6). Default salt concentration of the program was used throughout. Reverse primer (RT) analysis was performed as described, with a temperature of 50°C using the RNA mode of the folding program. Forward primer and probe analyses were performed using the DNA mode and probe orientation was taken into account by choosing the strand that the probe binds to. No folding was reported at annealing/extension temperature of 60°C. The DNA folding was set up at 37°C by default and the energy for folding was indicated much less than reported, i.e., 37°C. Note there are no energy values available in the RNAStructure program for Applied Biosystems TaqMan MGB probes.

The bimolecular folding was performed as instructed. The requested pictures of the primers and probes were presented respectively for 6673_nsp2_fwd primer, 6673_nsp2_probe, 6673_nsp2_reverse primer, 66792_S_fwd primer, 66792_S_probe, 66792_S_reverse primer, 66794_N_fwd primer, 66794_N_probe, and 66794_N_reverse primer.

The reverse primer (RP) binding region, forward primer (FP) binding region, the probe binding region, the primer and probe unimolecular folding, and the hybridization protocol have been accurately assessed. No significant predicted off target hybridization was observed.

Independent Laboratory Studies

Matrix study

Viral stock preparation.— The stainless-steel surface samples were prepared for this matrix study by MRI Global under Biosafety Level 3 (BSL-3) conditions. SARS-CoV-2 strain USA_WA1/2020, isolated from the first documented US case of a traveler from Wuhan, China (7) was sourced from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) for use in this study. The virus stock contained 3.6 × 10⁶ plaque forming units/mL (PFU/mL) pre-lyophilization. Upon receipt, the lyophilized virus stock was resuspended by MRI Global in 2 mL PBS, and single use aliquots of 50 µL were frozen and stored at –70°C.

Viral genomic copies per mL (GC/mL) were determined by quantitative RT-PCR using a Bio-Rad CFX96 Real-Time Detection System. The standard curve was prepared from synthetic SARS-CoV-2 RNA (ATCC No. VR-3276SD). The qPCR procedure used N1 primer and probe sequences published by the CDC (8). Primers and probes were purchased from Integrated DNA Technologies (IDT No. 10006713). TaqPath 1-step RT-qPCR Master Mix, CG was sourced from Thermo Fisher Scientific. Thermal cycling conditions followed those published in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel Instructions for Use (8).

The synthetic RNA standard curve consisted of the following concentrations: 1 × 10¹, 1 × 10², 1 × 10³, 1 × 10⁴, and 1 × 10⁵ 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. 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 × 10⁹ GC/mL.

The presence of infectious SARS-CoV-2 in the WRCEVA virus stock was verified using standard cell culture techniques. Briefly, 3 × 10⁶ Vero E6 cells were plated into a T75 flask with 15 mL infection media (Dulbecco's Modified Eagle's medium supplemented with 5% fetal bovine serum and nonessential amino acids) and incubated in a humidified incubator with 5% CO2. The following day the Vero cells were re-fed with infection media and inoculated with virus stock. Cells were incubated for 5 days at which point widespread cytopathic effect was apparent by microscopic examination of the Vero cells.

Test plate inoculation.— Dilutions of SARS-CoV-2 virus stock were prepared in VTM, prepared by MRI per CDC instructions (9) from a frozen viral stock aliquot and spread onto test areas as shown in Table 14. The same concentrations of virus were used for inoculating test areas for both the CDC 2019-nCoV RT-PCR and Thermo Scientific SARS-CoV-2 Workflow methods.

Table 14.

SARS-CoV-2 dilutions in VTM

Target contamination level	Method	Test area size	No. of test areas	GC/mLa	µL/test area	GC/test areab
High (1 POD)c	Reference	2 × 2”	5	1.3 × 105	135	1.8 × 104
Low (0.5 POD)	Reference	2 × 2”	20	1.3 × 104	135	1.8 × 103
VTM control (0 POD)	Reference	2 × 2”	5	0	135	0
High (1 POD)	Candidate	2 × 2”	5	1.3 × 105	135	1.8 × 104
Low (0.5 POD)	Candidate	2 × 2”	20	1.3 × 104	135	1.8 × 103
VTM control (0 POD)	Candidate	2 × 2”	5	0	135	0

a

 GC/mL = Number of gene copies of SARS-CoV-2 viral RNA per mL viral stock solution.

b

 GC/test area = Number of gene copies of SARS-CoV-2 viral RNA applied per 2 × 2” surface area based on range-finding studies using the CDC 2019-nCoV RT-PCR method.

c

 POD = 1 targets 5/5 positive responses, POD = 0.5 targets 10/20 positive responses, POD = 0 targets 0/5 responses.

Square 14 × 14” grade 304 stainless-steel plates were used for the studies to mimic food preparation surfaces. All test plates were cleaned, disinfected, and autoclaved prior to use. Test grids of 2 × 2” test areas were created on the test plates using laboratory tape. To inoculate the test plates, 135 µL (see Tables 15 and 16) was pipetted onto the appropriate test area and spread evenly over the entire test area with a sterile 10 µL inoculating loop. Inoculated plates were left until visibly dry (up to 1 h) in a biosafety cabinet (BSC) then transferred to a sealed plastic container and stored overnight at room temperature (21 h). Temperature and humidity ranged from 18.4–21.1°C and 30–35% relative humidity (RH) during the plate inoculation and drying process.

Table 16.

Statistical comparison of the Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow method results using KingFisher Flex 96 Deepwell System with Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System

Matrix	Inoculation strain	GC per test area	na	Thermo SARS-CoV-2 KF/QS5b			CDC 2019-nCoV RT-PCR				dPODCf	95% CIg
				xc	PODCd	95% CI	n	x	PODRe	95% CI
Stainless steel	SARS-CoV-2 USA-WA1/2020	0	5	0	0.00	0.00, 0.43	5	0	0.00	0.00, 0.43	0.00	–0.43, 0.43
		1.8 × 103	20	14	0.70	0.48, 0.86	20	11	0.55	0.34, 0.74	0.15	–0.14, 0.41
		1.8 × 104	5	5	1.00	0.57, 1.00	5	5	1.00	0.57, 1.00	0.00	–0.43, 0.43

a

 n = Number of test areas.

b

 KF/QS5 = Samples extracted using the Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow, KingFisher Flex 96 Deepwell System, RT-PCR by the Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System.

c

 x = Number of positive test areas.

d

 POD_C = Candidate method (Thermo SARS-CoV-2 Workflow) positive outcomes divided by the total number of trials.

e

 POD_R = Reference method (CDC 2019-nCoV RT-PCR) positive outcomes divided by the total number of trials.

f

 dPOD_C= Difference in POD values between the candidate method confirmed and reference method confirmed results.

g

 95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

CDC 2019-nCoV RT-PCR Method Plate Sampling

After drying overnight, test areas on the CDC 2019-nCoV RT-PCR Method test plates were sampled using Puritan foam tipped swabs (Cat. No. 25–1607 1PFSC). A swab was pre-moistened by dipping into a 15 mL conical tube containing 2.0 mL VTM. The pre-moistened 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. 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.

Thermo Scientific SARS-CoV-2 Workflow Method Plate Sampling

Test areas on the Thermo Scientific SARS-CoV-2 Workflow test plates were sampled using the same Puritan foam tipped swabs that were used for the CDC 2019-nCoV RT-PCR Method. A swab was pre-moistened by dipping into a 15 mL conical tube containing 2.0 mL PBS (provided by Thermo Fisher Scientific). The pre-moistened 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. After sampling the test area, the swab was snapped at the break point and placed back into the PBS tube. Each tube was capped and then wrapped with Parafilm to avoid moisture loss during shipping. A unique random ID number was assigned to each sample, and a key correlating test area sample to random ID number was created and sent to AOAC. Swab samples were shipped overnight to FSNS with ice packs on the day of sampling.

CDC 2019-nCoV RT-PCR Testing

Samples from the CDC 2019-nCoV RT-PCR method test plate were transferred to an operator, who was not aware of the blinded sample identities, for testing using the CDC 2019-nCoV RT-PCR Diagnostic Panel test kit. RNA was extracted from 140 µL of sample using the Qiagen QIAamp Viral RNA Mini Kit per the manufacturer’s instructions. Extracted RNA was tested on the CDC Panel on an Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument following published instructions. Fractional positive results were seen with the 0.5 POD sample set. Reference method test results were sent to AOAC for comparison with the Thermo Scientific SARS-CoV-2 Workflow.

Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow

The Thermo Scientific SARS-CoV-2 Workflow swab samples arrived at FSNS laboratories by 10:00 AM on the day following the sampling. The analyst receiving the samples noted that the ice packs used for shipping had melted, and the temperature of the test samples were 16°C (2–8°C for shipping was preferred). From each sample tube, four analyses were conducted; (1) KingFisher Flex 96 Deepwell System/Applied Biosystems 7500 Fast Food Safety Real-Time PCR System, (2) KingFisher Flex 96 Deepwell System/Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System, (3) MagMAX Express-96 Deepwell Magnetic Particle Processor/Applied Biosystems 7500 Fast Food Safety Real-Time PCR System, and (4) MagMAX Express-96 Deepwell Magnetic Particle Processor/Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System. Results from each analysis were recorded and provided to AOAC for blind sample decoding and POD analysis.

Results

As per criteria outlined in Appendix J of the Official Methods of Analysis Manual (3), fractional positive results were obtained for the CDC 2019-nCoV RT-PCR (reference method for this purpose) at the low level of contamination. POD was calculated for the Thermo Scientific SARS-CoV-2 Workflow (the candidate method) 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 calculations for each sample extraction/PCR analysis are presented in Tables 15–19. For all analyses, there were no positive responses at the 0/5 contamination level (POD = 0), and there were 5/5 positive responses at the high contamination level (POD = 1). At the low contamination level, there were 11/20 positive responses for the CDC 2019-nCoV RT-PCR (positive for N1 and N2), 14/20 for the KingFisher Flex Purification System with both the 7500 Fast and QuantStudio 5 Real-Time PCR systems, 16/20 for the MagMax Magnetic Processor with the 7500 Fast Real-Time PCR System, and 15/20 for the MagMax Magnetic Processor with the QuantStudio 5 Real-Time PCR System. There were no statistically significant differences seen between the methods, based on POD analysis, with the exception of the comparison between the MagMax Magnetic Particle Processor with PrepSEQ Nucleic Acid Extraction kit with the 7500 Fast Real-Time PCR analysis (dPOD = 0.25, 95% CI = −0.04, −0.49). If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

Table 15.

Statistical comparison of Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow method results using KingFisher Flex 96 Deepwell System with Applied Biosystems 7500 Fast Food Safety Real-Time PCR System

Matrix	Inoculation strain	GCper test area	na	Thermo SARS-CoV-2 KF/7500 Fastb			CDC 2019-nCoV RT-PCR				dPODCf	95% CIg
				xc	PODCd	95% CI	n	x	PODRe	95% CI
Stainless steel	SARS-CoV-2 USA-WA1/2020	0	5	0	0.00	0.00, 0.43	5	0	0.00	0.00, 0.43	0.00	–0.43, 0.43
		1.8 × 103	20	14	0.70	0.48, 0.86	20	11	0.55	0.34, 0.74	0.150	–0.14, 0.41
		1.8 × 104	5	5	1.00	0.57, 1.00	5	5	1.00	0.57, 1.00	0.00	–0.43, 0.43

a

 n = Number of test areas.

b

 KF/7500 Fast = Samples extracted using KingFisher Flex 96 Deepwell System, RT-PCR by the Applied Biosystems 7500 Fast Food Safety Real-Time PCR System.

c

 x = Number of positive test areas.

d

 POD_C = Candidate method (Thermo SARS-CoV-2 Workflow) positive outcomes divided by the total number of trials.

e

 POD_R = Reference method (CDC 2019-nCoV RT-PCR) positive outcomes divided by the total number of trials.

f

 dPOD_C = Difference in POD values between the candidate method confirmed and reference method confirmed results.

g

 95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

Table 17.

Statistical comparison of the Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow method results using MagMAX Express-96 Deepwell Magnetic Particle Processor and Applied Biosystems 7500 Fast Food Safety Real-Time PCR System

Matrix	Inoculation strain	GC per test area	na	Thermo SARS-CoV-2 MM/7500 Fastb			CDC 2019-nCoV RT-PCR				dPODCf	95% CIg
				xc	PODCd	95% CI	n	x	PODRe	95% CI
Stainless steel	SARS-CoV-2 USA-WA1/2020	0	5	0	0.00	0.00, 0.43	5	0	0.00	0.00, 0.43	0.00	–0.43, 0.43
		1.8 × 103	20	16	0.80	0.58, 0.92	20	11	0.55	0.34, 0.74	0.25	–0.04, –0.49
		1.8 × 104	5	5	1.00	0.57, 1.00	5	5	1.00	0.57, 1.00	0.00	–0.43, 0.43

a

 n = Number of test areas.

b

 MM/7500 Fast = Samples extracted using MagMAX Express-96 Deepwell Magnetic Particle Processor, RT-PCR by the Applied Biosystems 7500 Fast Food Safety Real-Time PCR System.

c

 x = Number of positive test areas.

d

 POD_C = Candidate method (Thermo SARS-CoV-2 Workflow) positive outcomes divided by the total number of trials.

e

 POD_R = Reference method (CDC 2019-nCoV RT-PCR) confirmed positive outcomes divided by the total number of trials.

f

 dPODC= Difference in POD values between the candidate method confirmed and reference method confirmed results.

g

 95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

Table 18.

Statistical comparison of the Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow method results using MagMAX Express-96 Deepwell Magnetic Particle Processor and Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System

Matrix	Inoculation strain	GC per test area	na	Thermo SARS-CoV-2 MM/QS5b			CDC 2019-nCoV RT-PCR				dPODCf	95% CIg
				xc	PODCd	95% CI	n	x	PODRe	95% CI
Stainless steel	SARS-CoV-2 USA-WA1/2020	0	5	0	0.00	0.00, 0.43	5	0	0.00	0.00, 0.43	0.00	−0.43, 0.43
		1.8 × 103	20	15	0.75	0.53, 0.89	20	11	0.55	0.34, 0.74	0.20	−0.09, 0.45
		1.8 × 104	5	5	1.00	0.57, 1.00	5	5	1.00	0.57, 1.00	0.00	−0.43, 0.43

a

 n = Number of test areas.

b

 MM/QS5 = Samples extracted using MagMAX Express-96 Deepwell Magnetic Particle Processor, RT-PCR by the Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System.

c

 x = Number of positive test areas.

d

 POD_C = Candidate method (Thermo SARS-CoV-2 Workflow) positive outcomes divided by the total number of trials.

e

 POD_R = Reference method (CDC 2019-nCoV RT-PCR) confirmed positive outcomes divided by the total number of trials.

f

 dPOD_C = Difference in POD values between the candidate method confirmed and reference method confirmed results.

g

 95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

Table 19.

The Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow and CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel test kit method summary results

Matrix	Inoculation strain	GC per test area	na	Thermo SARS-CoV-2 workflow				CDC 2019-nCoV RT-PCRb
				KF/7500 Fastc	KF/QS5d	MM/7500 Faste	MM/QS5f	N1 positive	N2 positive	Final result
Stainless steel	SARS-CoV-2 USA-WA1/2020	0	5	0	0	0	0	0	0	0
		1.8 x 103	20	14	14	16	15	11	18	11
		1.8 x 104	5	5	5	5	5	5	5	5

a

 n = Number of test areas.

b

 Both the N1 and N2 targets must give positive responses to call a sample positive. For the Thermo SARS-CoV-2 workflow, at least one must be detected to call a sample positive.

c

 KF/7500 Fast = Samples extracted using KingFisher Flex 96 Deepwell System, RT-PCR by the Applied Biosystems 7500 Fast Food Safety Real-Time PCR System.

c

 KF/QS5 = Samples extracted using KingFisher Flex 96 Deepwell System, PCR by the Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System.

e

 MM/7500 Fast = Samples extracted using MagMAX Express-96 Deepwell Magnetic Particle Processor, RT-PCR by the Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System.

f

dMM/QS5 = Samples extracted using MagMAX Express-96 Deepwell Magnetic Particle Processor, RT-PCR by the Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System.

Discussion

Inclusivity

Of the 15 756 target SARS-CoV-2 genomes analyzed, 99% of the strains/isolates are perfectly matched to at least two of the three assays, more than 90% have 100% homology to all three assays (ORF1ab, N-gene, S-gene) in the SARS-CoV-2 Kit.

Of the tested genomes, 15 756 out of 15 756 are predicted to be detected by at least one of the three SARS-CoV-2 assays: 100% of the strains/isolates matched with at least one of the three assays (ORF1ab, N-gene, S-gene) in the SARS-CoV-2 Kit.

Exclusivity

Of the 65 non-target genomes analyzed against the primers and probes of the TaqMan 2019-nCoV Assay Kit v1, none showed matching sequences.

Matrix Study

Data from the matrix study demonstrated that all four options for the Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow, KingFisher Flex Purification System with 96 Deepwell Head used to extract RNA with the PrepSEQ Nucleic Acid Extraction Kit procedure with the Applied Biosystems 7500 Fast or the Applied Biosystem QuantStudio 5 Real-Time PCR Systems and the MagMAX Express-96 Deepwell Magnetic Particle Processor used to extract RNA with the PrepSEQ Nucleic Acid Extraction Kit extraction procedure with the Applied Biosystems 7500 Fast or the Applied Biosystems QuantStudio 5 Real-Time PCR Systems were able to detect low levels of SARS-CoV-2 viral RNA on stainless steel. Each option gave statistically comparable or slightly better results than the CDC 2019-nCoV RT-PCR method, based on POD analysis. It should be noted that for the CDC to call a sample positive, both the N1 and N2 targets must give positive responses. For the Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow, at least one of the targets must give a positive response to call a sample positive. In this study, the N2 target for the CDC method gave significantly more positive responses than the N1 (18 versus 11, dPOD = –0.35, CIs = –0.57, –0.07). The reason for this difference has not been determined but could depend on degradation of virus during the application and drying onto the surface for this study. However, experiments to examine this have not been conducted as part of this evaluation. When comparing the Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow results to the CDC N2 results only, no statistically significant differences were seen (data not shown), even with the largest differences between the methods (14 positive results versus 18 positive results, dPOD = –0.20, CIs = −0.43, 0.05). It should also be noted that the test samples shipped to FSNS arrived at an elevated temperature (16°C versus 2–8°C), and although there do not appear to be any adverse effects on the results, there was a potential for the candidate method samples to be compromised. As presented in this study, the data show that all evaluated options for the Thermo Scientific SARS-CoV-2 Workflow are a comparable alternative to the CDC 2019-nCoV Real-Time RT-PCR method for detecting SARS-CoV-2 on stainless-steel surfaces.

Conclusions

The Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow is targeting three different SARS-CoV-2 genomic regions in one single RT-PCR reaction. A flexibility feature is offered with the use of multiple extraction and real-time PCR instruments The RNA extraction can be run either with the KingFisher Flex 96 Deepwell System or the MagMAX Express-96 Deepwell Magnetic Particle Processor; the Real-Time PCR can be conducted either with the Applied Biosystems QuantStudio 5 Food Safety Real-Time PCR System or the Applied Biosystems 7500 Fast Food Safety Real-Time PCR System. Data interpretation supports risk management and decision-making to develop relevant hygiene control measures.

During the ERV AOAC PTM program, the Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow delivered outstanding robustness, specificity, and sensitivity. All 15 756 SARS-CoV-2 genomes analyzed in the in silico analysis are predicted to be detected with Thermo Scientific SARS-CoV-2 Real-Time PCR Monitoring Workflow. None of the 65 near neighbor genomes and other environmental microorganisms’ genomes showed matching sequences with the primers and probes of the TaqMan 2019-nCoV Assay Kit. The Thermo Scientific SARS-CoV-2 Real-Time PCR workflow showed comparable detection to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 from stainless-steel surface swabs, whatever the RNA extraction procedures and RT-PCR instruments.

The Thermo Scientific SARS-CoV-2 Real-Time PCR Workflow is an effective procedure for SARS-CoV-2 detection on stainless-steel surfaces (such as those found in a food production environment) in evaluating the efficiency of control measures and develop proper hygiene control measures .

Submitting Company

Thermo Fisher Scientific

Wade Road

Basingstoke, Hampshire

RG24 8PW, UK

Participating Laboratories

Thermo Fisher Scientific

Wade Road

Basingstoke

Hampshire

RG24 8PW, UK

Thermo Fisher Scientific

Ratastie 2

01620 Vantaa, Finland

Food Safety Net Services (FSNS)

199 W. Rhapsody

San Antonio, TX 78216, USA

Independent Laboratory

MRI Global

Kansas City, MO 64110, USA

Reviewers

John SantaLucia

Wayne State University

42 W Warren Ave

Detroit, MI 48202, USA

William Burkhardt

FDA GCSL

1 Iberville Drive

Dauphin Island, AL 36528, USA

Sanjiv Shah

U.S. Environmental Protection Agency’s Office of Research and Development

1200 Pennsylvania Ave NW

Washington, DC 20004, USA