key: cord-0813068-cljqoo82
authors: Poindexter, M. R.; Xu, T.; Swift, C. M.; Proctor, C. M.; Kara-Murdoch, F.; Morehouse, Z. P.; Ryan, G. L.; Löffler, F. E.; Nash, R. J.
title: Comparison of mechanical homogenization versus enzymatic digestion sample preparation methodologies for SARS-CoV-2 detection in saliva for surveillance of variants of concern on the University of Tennessee campus in early 2021.
date: 2022-05-16
journal: nan
DOI: 10.1101/2022.05.11.22274949
sha: 272d331e7fbe2ee6b92fef538eba54cb3e186d9f
doc_id: 813068
cord_uid: cljqoo82

The SARS-CoV-2 pandemic has profoundly impacted communities across the globe, requiring accurate and accessible diagnostic technologies in support of public health mitigation efforts. As testing has evolved throughout the course of the pandemic, varying sample preparation methodologies have been employed. Herein we perform a comparison of three commercial sample preparation methods: two mechanical homogenization workflows and one enzymatic digestion approach for the detection of SARS-CoV-2 from biomarker genes in 20 human saliva pools. SARS-CoV-2 variants of concern were also identified on the University of Tennessee, Knoxville campus during the spring semester of 2021 utilizing the commercial PerkinElmer PKamp VariantDetect SARS-CoV-2 RT-PCR Assay kit. Two hundred and ten (210) human saliva pools were selected and analyzed for the presence of SARS-CoV-2 variants of concern providing insight into the utility of these various commercial workflows for integration into current public health SARS-CoV-2 surveillance measures.

In this study, we compare three qPCR based methodologies for SARS-CoV-2 diagnostics using 72 human saliva samples collected on the University of Tennessee, Knoxville campus. Two of these 73 methodologies, the SalivaDirect with Proteinase K and the KingFisher RNA Extraction method, 74 are commercially available and were compared to the Omni Direct-to-PCR (dPCR) method [9-75 11]. This comparative evaluation provides insight into the ability of different workflows to 76 accurately detect viral RNA within saliva samples. While less invasive for the patient to provide, 77 these samples often have increased PCR inhibitors present, making saliva a more difficult sample 78 to process and achieve sensitive detection of SARS-CoV-2 biomarker genes. Thus, comparative 79 studies assessing the impact of sample preparation workflows are crucial for establishing 80 confidence in diagnostic testing with this sample medium. 81 Additionally, the human saliva samples collected for this study were screened for variants of 82 concern utilizing the commercial PerkinElmer PKamp VariantDetect SARS-CoV-2 RT-PCR 83 Assay kit. These samples were collected throughout the 2021 spring semester, and demonstrated 84 the ability of this kit to accurately detect and differentiate between variants of SARS-CoV-2 as 85 they spread throughout the campus community. The Omni dPCR method utilizes samples homogenized by the Bead Ruptor Elite (Omni, Cat. 93 No. 19-042E) and then added directly to the qPCR reactions. The KingFisher extraction protocol 94 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 relies upon viral particle lysis and mechanical disruption of the virus. The SalivaDirect protocol 95 combines both Proteinase K and heat treatment to denature human saliva samples.

The samples utilized in this study were obtained from the University of Tennessee, Knoxville 97 campus in the Spring 2021 semester. All participants were consented via written and oral consent 98 prior to providing saliva samples. Saliva samples provided for the research in this study were 99 deidentified prior to being handed over to the lab for further use. All research was approved 100 under protocol IBC-20-547-2 approved on July 6 2020 by the UTK institutional review board.

In early summer 2020, the University of Tennessee, Knoxville campus developed and validated 102 protocols to detect SARS-CoV-2 for public health surveillance using RT-qPCR assays that target The de-identified human saliva samples used in this study were chosen based upon previous 114 testing using the protocols established by the University of Tennessee SARS-CoV-2 Surveillance 115 Testing Laboratory to include known SARS-CoV-2 positive and known SARS-CoV-2 negative 116 human saliva samples. Table 1 below shows the published primer and probe sets specifically 117 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 targeting the E, RdRP, and RNase P genes that were used in this study [3, 4, 6] . Although the 118 viral N gene was originally included in the multiplexed qPCR assays during previous testing, it is 119 excluded in this study as both the E and RdRP targets are sufficient for detecting the presence of 120 the viral genome in the saliva samples. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 CoV-2 mutations which include the Alpha variant (B1.1.7 -United Kingdom, September 2020), April 2021) (Table 3) . is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274949 doi: medRxiv preprint 1) Omni Direct-to-PCR method: Each human saliva pool (500 µL) was transferred to a 2-165 mL Omni bead tube (Omni, . The bead tubes were placed inside the CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. CoV-2 RT-PCR Assay kit with Table 4 showing the results of the variant tests for the pools used is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 imbalance between primers, template and non-template RNA concentrations [14] . The negative 265 control for the Omni dPCR method showed no amplification, which leads us to attribute the 266 amplification of the E gene in the SARS-CoV-2 negative human saliva samples to the Omni 267 dPCR method itself. The test was repeated 4 separate times with new reagents each time to 268 assure contamination was not the issue. Each repetition produced similar results. Contamination 269 of the kit was ruled out as a contributing factor from the manufacturer due to the quality control Table 4 , we are able to gain some valuable information. The increased rates of mutation present in our sampling community. By monitoring the rate of these 287 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 mutations in the population, we can monitor the rate of transmission of this particular lineage and 288 potentially detect upcoming variants of interest.

It is interesting to note that there were no confirmed cases of the Delta variant on the University CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 the presence and proportionality of multiple variants of concern across the university's campus 311 during the spring 2021 semester. When evaluating variants, an observed shift from wild type to 312 alpha variant was seen surrounding spring break. This shift corresponds with a national trend 313 seen at that time as the alpha variant transitioned to becoming the dominant variant across the 314 US. An unidentified variant was seen as the semester progressed, but it is likely that that is the 315 emergence of a recombination seen following spring break with large portions of the student 316 body traveling and was an event that did not correspond with a variant of concern identified in 317 the utilized assay. Further evaluation of the unidentified variant could be considered for future 318 studies on the genomic diversity of SARS-CoV-2 across university populations. Overall, this 319 manuscript successfully demonstrates the ability of multiple commercially available assays to 320 successfully utilize pooled saliva samples for highly sensitive SARS-CoV-2 detection.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) All data pertaining to this study is presented and available in the manuscript without alterations or 342 restrictions.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 

Systematic review with meta-analysis of the accuracy of diagnostic tests

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274949 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274949 doi: medRxiv preprint