key: cord-0917180-bvvnancq
authors: Joelsson, Adam; Green, Shannon; Siciliano, Nicholas; Dutta, Vikrant; Johnson, Ron
title: Validation Study for VERIPRO® SARS-CoV-2 Env Assay for the Detection of SARS-CoV-2 from Stainless Steel Environmental Surface Swabs: Emergency Response Validation-AOAC—AOAC Performance Tested Method(SM) 122001
date: 2021-04-19
journal: J AOAC Int
DOI: 10.1093/jaoacint/qsab048
sha: ce6f3e144e1a8f275cf64f86c1461491901abed9
doc_id: 917180
cord_uid: bvvnancq

BACKGROUND: The VERIPRO SARS-CoV-2 Env assay uses reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2, the causative agent of COVID-19, from stainless steel environmental sample swabs. OBJECTIVE: To validate the VERIPRO SARS-CoV-2 Env assay as part of the AOAC Research Institute’s Emergency Response Validation Performance Tested Method(SM) program. METHODS: The VERIPRO SARS-CoV-2 Env assay was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 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 specificity of the method in being able to detect target sequences and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the reference method. CONCLUSIONS: The VERIPRO SARS-CoV-2 Env assay is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on food contact surfaces. HIGHLIGHTS: The VERIPRO SARS-CoV-2 Env assay can be performed without the need for an optional RNA purification step to detect SARS-CoV-2 from environmental surfaces.

The VERIPRO SARS-CoV-2 Env assay is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on food contact surfaces.

Highlights: The VERIPRO SARS-CoV-2 Env assay can be performed without the need for an optional RNA purification step to detect SARS-CoV-2 from environmental surfaces.

In late 2019, Chinese authorities notified the World Health Organization (WHO) of a novel coronavirus (SARS-CoV-2) that caused an outbreak of pneumonia and has resulted in millions of confirmed human infections globally (1) . The detection and spread of the new respiratory viruses has led to uncertainty of its ability to survive on surfaces, including food contact surfaces (2) . Previous respiratory outbreaks have resulted in studies indicating the viability of organisms on surfaces for extended periods of time. Reducing the risk associated with these surfaces includes both effective cleaning, disinfection, and a robust environmental monitoring program, including rapid detection methods (3) . Having access to this information can lead toward infection prevention and improved control measures aimed at reducing surface-based transmission.

The VERIPRO SARS-CoV-2 Env test kit is a reverse transcriptase PCR (RT-PCR) assay for the molecular detection of SARS-CoV-2 from environmental surface swabs. The assay targets the highly conserved N1 and N2 regions of the nucleocapsid gene of SARS-CoV-2. It utilizes a multiplex detection method that detects the targets on the Cy5 channel and an RNA- The assay is designed to be compatible with purified RNA via approved RNA extraction kits and synthetic environmental sampling swabs. The Limit of Detection (LOD) of the assay is 5-10 genomic copies (1000-2000 genomic copies of sample per mL from either approved collection buffers or extracted samples). 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. (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. 

Note: For samples that will not be processed in <1 hour from collection, store swab/buffer at 2-6° (for up to 24 h).

Note: Alternatively, 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.

(a) After collection swabs are processed via a quick RNA extraction protocol or following purification:

(1) The Invisible Sentinel's quick RNA extraction method (steps b-i).

(2) The Qiagen QIAamp Viral RNA mini kit (Cat. No. 52904) following manufacturer's directions. After RNA purification, samples are processed as outlined in step (g).

(b) Transfer 100 μL of the OR-100 buffer from the environmental swab collection tube to a 1.5 mL microcentrifuge tube.

(c) Heat for 75°C for 10 min in water bath or heat block. (g) Transfer 5 μL from sample to a thawed VERIPRO® SARS-CoV-2 Env PCR tube.

Note: IMPORTANT: Open the VERIPRO® SARS-CoV-2 Env PCR tube only when adding sample and promptly close after addition to avoid cross contamination between tubes.

(h) Load 5 μL of RNA positive control in a separate reaction tube. RNA control is provided at 1000 genomic copies per 5 μL.

(i) Load 5 μL of RNA free water into a separate reaction tube. This will serve as a negative control.

(j) Place PCR Tube into GENE-UP PCR instrument, select "SARS-CoV-2 Env PCR" (f) Select "Calculate" to obtain the Ct/Cp values. Inspect the traces for the characteristic exponential amplification curve shape.

(g) The sample Ct/Cp results table from each channel can be exported as a text file by right clicking on the sample results table. These files can be opened in Microsoft Excel to record, organize, and evaluate the data. Reports can be generated by saving the analysis and navigating to the "Report" module.

Step (g)-(h) for each channel by toggling to each individual filter using the "Channel" Button.

(a) Amplification curves have a characteristic shape consisting of an initial lag phase, exponential amplification phase and a final plateau phase. The final plateau phase, which represents a decrease in reaction efficiency as reagents are consumed, may not be reached in reactions containing low levels of target RNA. Amplification curves that deviate from the ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com

characteristic shape should be interpreted with caution. For each VERIPRO® reaction, the cycle at which fluorescence signal rises above background fluorescence is determined and is called the "threshold cycle" (Ct) or "crossing point" (Cp), depending on the instrument. The Ct/Cp will occur at an earlier cycle for samples containing high levels of target organisms and will be delayed for reactions containing low levels of target organisms. The software algorithm utilizes a 2nd derivative max option to enhance the sensitivity of the method. The CY5 channel is designed to detect the N1 and N2 regions of the SARS-CoV-2 Nucleocapsid gene (as defined by CDC). The HEX channel serves as an internal amplification control (IAC) to indicate a successful PCR reaction and should be detected at a Ct/Cp value between ~26-30 cycles.

The study was conducted according to the procedures outlined in the AOAC Research

September 2020). The in silico analysis was performed by bioMérieux. Test portions for the matrix studies were prepared by the independent laboratory, MRIGlobal, and shipped blindcoded to bioMérieux for analysis. Additional AOAC RI PTM parameters, robustness and product consistency and stability will be submitted for full PTM certification by March 31, 2021 (4).

Robustness.-Data to be collected and submitted for final PTM certification. 

Results.-Of the target genomes analyzed, 15 759 were perfect matches for detection on the Cy5 target channel of our assay (99.97% inclusivity). For the variants that were not perfect matches, it is highly likely that these variants are rare, have ambiguous positions or possible sequence errors. See Table 1 for a summary of the inclusivity results.

All exclusivity organisms (near neighbors and background organisms) produced no matching sequences for the VERIPRO SARS-CoV-2 ENV primers and probes. See Table 2 for a summary of the results for the closest mismatches for the exclusivity testing. While the N2 region shows a stable UUCG hairpin, the N2 probe utilized in the assay does not bind at this site and should not pose an issue to method performance. Table 3 

information was provided by WRCEVA. GC/mL was determined by MRIGlobal as described below using one of the frozen viral stock aliquots.

Viral genomic copies per mL (GC/mL) was determined by quantitative RT-PCR using a Bio- Table 7 .

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/ L. 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 8 .

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. 

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 (CPE) was apparent by microscopic examination of the Vero cells.

Dilutions of SARS-CoV-2 virus stock were prepared in VTM from a frozen viral stock aliquot as shown in 

Swabs were processed according to the methodologies listed in the Sample Preparation section. Each test portion was evaluated using the rapid extraction protocol and using the QIAamp RNA purification assay.

Results.-As per criteria outlined in Appendix J of the Official Methods of Analysis Manual, fractional positive results were obtained for the CDC reference method. The probability of detection (POD) was calculated as the number of positive outcomes divided by the total number of trials (6). POD was 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 VERIPRO SARS-CoV-2 Env assay and the reference method indicated that there was a significant difference between the two methods, with the candidate method detecting more positive samples than the reference method, which is acceptable according to AOAC Appendix J policies. A summary of POD analyses are presented in Table   14 . Individual results are provided in Table 15 .

The VERIPRO SARS-CoV-2 Env assay successfully detected the target analyte from stainless steel environmental surface samples following two extraction procedures (Invisible Sentinel's quick RNA extraction procedure which requires no RNA purification step or the Qiagen QIAamp viral RNA purification kit). Once the virus begins to lyse on surfaces, RNA will begin to degrade, and some genes may be more labile than others. As the RNA fragments become smaller in size over time, methods that require only a single target may be more likely to detect these fragments. This increases the chances of the candidate method being positive at the limit of detection of the assay as it can likely detect both intact virus and viral RNA fragments from surfaces.

The VERIPRO SARS-CoV-2 Env method allows for the rapid screening of surfaces for the target strain. With no RNA purification protocol requirement, samples can be quickly swabbed and analyzed. Product instructions are well written and simple enough that a technician at any level of training would be able to follow it and achieve accurate results. The assay utilizes the GENE-UP instrument which allows the integration of this test into their routine environmental sampling workflow.

The data from this study supports the product claim that the VERIPRO SARS-CoV-2 -inoculated control level  1  -----2  -----3  -----4  -----5 -----Total 0/5 0/5 0/5 0/5 0/5 a Positive result indicates either the presence of the N1 gene, the N2 gene or both genes. 

CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel

Surface sampling of coronavirus disease (COVID-19): A practical "how to" protocol for health care and public health professionals

Evaluating SARS-CoV-2 Cleanup and Disinfection Practices

for the Washington State 2019-nCoV Case Investigation Team

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