key: cord-0829156-dfmhkce8
authors: Brzoska, Pius; Crabtree, David; Evans, Katharine; Leonte, Ana-Maria; Manolis, Amanda; Salavirta, Heikki; Stephenson, Patrick; Sohier, Daniele
title: 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
date: 2021-04-05
journal: J AOAC Int
DOI: 10.1093/jaoacint/qsab050
sha: 29fd896306d61aa86bddf0b1307b6786226fa06f
doc_id: 829156
cord_uid: dfmhkce8

BACKGROUND: The Thermo Scientific™ SARS-CoV-2 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. METHODS: 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 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.

Test Kit Information (1) Lysis Buffer.-2 x 50 mL.

(2) Magnetic Particles.-2 x 1.5 mL. (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 (MgSO 4 

(a) Pre-moisten swab with PBS or viral transport medium (the workflow utilized PBS in this study).

(b) 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 x 2 inches, ensuring that the full area is covered in the sampling.

(c) Place the swab back into the transport tube containing 2 mL PBS.

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

(e) Analyze the swabs preferably in the coming 24 h. If the swabs are not likely to be analyzed within 48 h maximum, store the swabs preferably between -70°C and -80°C, and ship on dry ice.

The (i) Start the run, then load the prepared plates into position when prompted by the instrument.

(j) After the run is complete (~40 minutes after start), immediately remove the Elution Plate from the instrument, then cover the plate with an adhesive film.

(k) Place the Elution Plate on ice for immediate use in RT-PCR.

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

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

(b) Protect the assays and master mix from light.

(c) 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).

(d) Before beginning, determine the number of required reactions. In addition to the nucleic acid test samples, include the following reactions: Table 3 .

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

(c) 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.

(d) Place reactions in a thermal cycler programmed as described below. The following software should be used with each instrument (Table 5) .

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

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

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

ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com

Page 14 of 27 (b) Perform analysis using the following settings (Table 7) .

(c) Analyze the run with no reference dye.

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

(e) 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.

(f) Export the results.

(a) 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.

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:

Set the baseline to remove any anomalous data. Example: 12 -25 cycles.

Remove the threshold by setting it to 1.

Change the graph view to linear.

Change the y axis range to minimum -100 and maximum 1,000,000.

Change the x axis range to 12-45 cycles.

(b) Negative Signals:

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(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 .

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

See Table 10 .

(e) See Table 11 for Troubleshooting guidance. 

U N C O R R E C T E D P R O O F Page 16 of 27

The in silico analysis of the primer and probe sequences was conducted according to the Validation Outline provided by AOAC Research Institute using Appendix 1 with the minimal set of 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 mentioned in the Appendix 6, i.e. unimolecular folding, bimolecular hybridization.

In silico analysis was performed to determine inclusivity (reactivity) and exclusivity the primer and 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.

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

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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.

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 .

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

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 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 (7). Default salt concentration of the program was used throughout. Reverse primer (RT) analysis was performed as described, with a ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com

Page 18 of 27 temperature of 50°C using the RNA mode of the folding program. Forward primer and probe analysis 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 is 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. recorded and provided to AOAC for blind sample decoding and POD analysis. dPOD does not contain zero, then the difference is statistically significant at the 5% level.

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 

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

Data from the matrix study demonstrated that all four options for the Thermo Scientific SARS-CoV-2 

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 If the C t value for either assay is above the expected result, repeat the test with new reagents. 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.

ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com 

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. 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 targetspecific 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 targetspecific RNA.

No action is required. The result is considered positive if the target-specific amplification curve appears normal.

In negative extraction control wells, targetspecific signal is detected.

Carryover contamination occurred. 

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