Evaluating and optimizing Acid-pH and Direct Lysis RNA extraction for SARS-CoV-2 RNA detection in whole saliva.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
25 Mar 2024
25 Mar 2024
Historique:
received:
21
09
2023
accepted:
09
02
2024
medline:
26
3
2024
pubmed:
26
3
2024
entrez:
26
3
2024
Statut:
epublish
Résumé
COVID-19 has been a global public health and economic challenge. Screening for the SARS-CoV-2 virus has been a key part of disease mitigation while the world continues to move forward, and lessons learned will benefit disease detection beyond COVID-19. Saliva specimen collection offers a less invasive, time- and cost-effective alternative to standard nasopharyngeal swabs. We optimized two different methods of saliva sample processing for RT-qPCR testing. Two methods were optimized to provide two cost-efficient ways to do testing for a minimum of four samples by pooling in a 2.0 mL tube and decrease the need for more highly trained personnel. Acid-pH-based RNA extraction method can be done without the need for expensive kits. Direct Lysis is a quick one-step reaction that can be applied quickly. Our optimized Acid-pH and Direct Lysis protocols are reliable and reproducible, detecting the beta-2 microglobulin (B2M) mRNA in saliva as an internal control from 97 to 96.7% of samples, respectively. The cycle threshold (Ct) values for B2M were significantly higher in the Direct Lysis protocol than in the Acid-pH protocol. The limit of detection for N1 gene was higher in Direct Lysis at ≤ 5 copies/μL than Acid-pH. Saliva samples collected over the course of several days from two COVID-positive individuals demonstrated Ct values for N1 that were consistently higher from Direct Lysis compared to Acid-pH. Collectively, this work supports that each of these techniques can be used to screen for SARS-CoV-2 in saliva for a cost-effective screening platform.
Identifiants
pubmed: 38527999
doi: 10.1038/s41598-024-54183-w
pii: 10.1038/s41598-024-54183-w
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
7017Subventions
Organisme : CIHR
ID : 177776
Pays : Canada
Informations de copyright
© 2024. Crown.
Références
Riemersma, K. K. et al. Shedding of infectious SARS-CoV-2 despite vaccination. https://doi.org/10.1101/2021.07.31.21261387 (2021).
Puhach, O. et al. Infectious viral load in unvaccinated and vaccinated individuals infected with ancestral, Delta or Omicron SARS-CoV-2. Nat. Med. 1–1. https://doi.org/10.1038/s41591-022-01816-0 (2022).
Bradley, E. H., An, M.-W. & Fox, E. Reopening colleges during the coronavirus disease 2019 (COVID-19) pandemic—one size does not fit all. JAMA Netw. Open 3, e2017838–e2017838 (2020).
pubmed: 32735333
doi: 10.1001/jamanetworkopen.2020.17838
Vogels, C. B. F. et al. SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity. Medicine https://doi.org/10.1016/j.medj.2020.12.010 (2020).
doi: 10.1016/j.medj.2020.12.010
Brook, C. E., Northrup, G. R., Ehrenberg, A. J., Doudna, J. A. & Boots, M. Optimizing COVID-19 control with asymptomatic surveillance testing in a university environment. Epidemics 37, 100527 (2021).
pubmed: 34814094
pmcid: 8591900
doi: 10.1016/j.epidem.2021.100527
Corchis-Scott, R. et al. Averting an outbreak of SARS-CoV-2 in a university residence hall through wastewater surveillance. Microbiol. Spect. 9, e00792-e821 (2021).
Scott, L. C. et al. Targeted wastewater surveillance of SARS-CoV-2 on a university campus for COVID-19 outbreak detection and mitigation. Environ. Res. 200, 111374 (2021).
pubmed: 34058182
pmcid: 8163699
doi: 10.1016/j.envres.2021.111374
Levy, J. I., Andersen, K. G., Knight, R. & Karthikeyan, S. Wastewater surveillance for public health. Science 379, 26–27 (2023).
pubmed: 36603089
pmcid: 10065025
doi: 10.1126/science.ade2503
Barrera-Avalos, C. et al. The rapid antigen detection test for SARS-CoV-2 underestimates the identification of COVID-19 positive cases and compromises the diagnosis of the SARS-CoV-2 (K417N/T, E484K, and N501Y) variants. Front. Public Health 9, 780801 (2021).
pubmed: 35047474
doi: 10.3389/fpubh.2021.780801
Osterman, A. et al. Impaired detection of omicron by SARS-CoV-2 rapid antigen tests. Med. Microbiol. Immunol. https://doi.org/10.1007/s00430-022-00730-z (2022).
doi: 10.1007/s00430-022-00730-z
pubmed: 36370197
pmcid: 9660148
Drain, P. K. Rapid diagnostic testing for SARS-CoV-2. N. Engl. J. Med. 386, 264–272 (2022).
pubmed: 34995029
doi: 10.1056/NEJMcp2117115
Senok, A. et al. Saliva as an alternative specimen for molecular COVID-19 testing in community settings and population-based screening. IDR 13, 3393–3399 (2020).
doi: 10.2147/IDR.S275152
Tan, S. H., Allicock, O., Armstrong-Hough, M. & Wyllie, A. L. Saliva as a gold-standard sample for SARS-CoV-2 detection. Lancet Respir. Med. 9, 562–564 (2021).
pubmed: 33887248
pmcid: 8055204
doi: 10.1016/S2213-2600(21)00178-8
Balamane, M. et al. Detection of HIV-1 in saliva: Implications for case-identification, clinical monitoring and surveillance for drug resistance. Open Virol. J. 4, 88–93 (2010).
pubmed: 21673840
pmcid: 3111737
doi: 10.2174/1874357901004010088
Chen, K. M. et al. Human papilloma virus prevalence in a multiethnic screening population. Otolaryngol. Head Neck Surg. 148, 436–442 (2013).
pubmed: 23300223
pmcid: 3707492
doi: 10.1177/0194599812471938
Kwok, H., Chan, K. W., Chan, K. H. & Chiang, A. K. S. Distribution, persistence and interchange of Epstein–Barr Virus strains among PBMC, plasma and saliva of primary infection subjects. PLOS ONE 10, e0120710 (2015).
pubmed: 25807555
pmcid: 4373854
doi: 10.1371/journal.pone.0120710
Pinninti, S. G. et al. Comparison of saliva PCR assay versus rapid culture for detection of congenital cytomegalovirus infection. Pediatr. Infect. Dis. J. 34, 536–537 (2015).
pubmed: 25876092
pmcid: 4400866
doi: 10.1097/INF.0000000000000609
Bonaldo, M. C. et al. Isolation of infective Zika virus from urine and saliva of patients in Brazil. PLOS Neglect. Trop. Dis. 10, e0004816 (2016).
doi: 10.1371/journal.pntd.0004816
Podzimek, S., Vondrackova, L., Duskova, J., Janatova, T. & Broukal, Z. Salivary markers for periodontal and general diseases. Dis. Mark. 2016, 9179632 (2016).
Kaczor-Urbanowicz, K. E. et al. Saliva diagnostics—Current views and directions. Exp. Biol. Med. (Maywood) 242, 459–472 (2017).
pubmed: 27903834
doi: 10.1177/1535370216681550
Matuck, B. F. et al. Salivary glands are a target for SARS-CoV-2: a source for saliva contamination. J. Pathol. 254, 239–243 (2021).
pubmed: 33834497
pmcid: 8250228
doi: 10.1002/path.5679
Azzi, L. et al. Saliva is a reliable tool to detect SARS-CoV-2. J. Infect. 81, e45–e50 (2020).
pubmed: 32298676
pmcid: 7194805
doi: 10.1016/j.jinf.2020.04.005
To, K.K.-W. et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: An observational cohort study. Lancet Infect. Dis. 20, 565–574 (2020).
pubmed: 32213337
pmcid: 7158907
doi: 10.1016/S1473-3099(20)30196-1
De Santi, C. et al. Concordance between PCR-based extraction-free saliva and nasopharyngeal swabs for SARS-CoV-2 testing. HRB Open Res. 4, 85 (2021).
pubmed: 34522839
pmcid: 8408542
doi: 10.12688/hrbopenres.13353.2
Johnson, A. J. et al. Saliva testing is accurate for early-stage and presymptomatic COVID-19. Microbiol. Spectr. 9, e0008621 (2021).
pubmed: 34259552
doi: 10.1128/Spectrum.00086-21
Marais, G. et al. Improved oral detection is a characteristic of Omicron infection and has implications for clinical sampling and tissue tropism. J. Clin. Virol. 152, 105170 (2022).
pubmed: 35525108
doi: 10.1016/j.jcv.2022.105170
Salmona, M. et al. Detection of SARS-CoV-2 in saliva and nasopharyngeal swabs according to viral variants. Microbiol. Spect. 10, e02133-e2222 (2022).
Barat, B. et al. Pooled saliva specimens for SARS-CoV-2 testing. J. Clin. Microbiol. 59, e02486-e2520 (2021).
pubmed: 33262219
pmcid: 8106731
doi: 10.1128/JCM.02486-20
Vander Schaaf, N. A. et al. Routine, cost-effective SARS-CoV-2 surveillance testing using pooled saliva limits viral spread on a residential college campus. Microbiol. Spect. 9, e01089–21 (2021).
Fábryová, H. & Celec, P. On the origin and diagnostic use of salivary RNA. Oral. Dis. 20, 146–152 (2014).
pubmed: 23517132
doi: 10.1111/odi.12098
Abdalhamid, B. et al. Assessment of specimen pooling to conserve SARS CoV-2 testing resources. Am. J. Clin. Pathol. 153, 715–718 (2020).
pubmed: 32304208
pmcid: 7188150
doi: 10.1093/ajcp/aqaa064
Harris, P. A. et al. Research electronic data capture (REDCap)—A metadata-driven methodology and workflow process for providing translational research informatics support. J. Biomed. Inf. 42, 377–381 (2009).
doi: 10.1016/j.jbi.2008.08.010
Harris, P. A. et al. The REDCap consortium: Building an international community of software platform partners. J. Biomed. Inf. 95, 103208 (2019).
doi: 10.1016/j.jbi.2019.103208
Wozniak, A. et al. A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR. Sci. Rep. 10, 16608 (2020).
pubmed: 33024174
pmcid: 7538882
doi: 10.1038/s41598-020-73616-w
Ostheim, P. et al. Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes. Sci. Rep. 12, 2312 (2022).
pubmed: 35145126
pmcid: 8831573
doi: 10.1038/s41598-022-05670-5
Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46, W537–W544 (2018).
pubmed: 29790989
pmcid: 6030816
doi: 10.1093/nar/gky379
Avendano, C. et al. SARS-CoV-2 variant tracking and mitigation during in-person learning at a Midwestern University in the 2020–2021 school year. JAMA Netw. Open 5, e2146805 (2022).
pubmed: 35113163
pmcid: 8814910
doi: 10.1001/jamanetworkopen.2021.46805
Ehrenberg, A. J. et al. Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus. PLOS ONE 16, e0251296 (2021).
pubmed: 34038425
pmcid: 8153421
doi: 10.1371/journal.pone.0251296
Hamilton, J. R. et al. Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples. PLOS ONE 16, e0255690 (2021).
pubmed: 34351984
pmcid: 8341588
doi: 10.1371/journal.pone.0255690
Jarvinen, P., Oivanen, M. & Lonnberg, H. Interconversion and phosphoester hydrolysis of 2’,5’- and 3’,5’-dinucleoside monophosphates: kinetics and mechanisms. J. Org. Chem. 56, 5396–5401 (1991).
doi: 10.1021/jo00018a037
Khan, J. M. et al. Protonation favors aggregation of lysozyme with SDS. Soft Matter. 10, 2591–2599 (2014).
pubmed: 24647567
doi: 10.1039/c3sm52435c
Jafari, M. & Mehrnejad, F. Molecular insight into human lysozyme and its ability to form amyloid fibrils in high concentrations of sodium dodecyl sulfate: A view from molecular dynamics simulations. PLOS ONE 11, e0165213 (2016).
pubmed: 27768744
pmcid: 5074503
doi: 10.1371/journal.pone.0165213
Jafari, M., Mehrnejad, F., Rahimi, F. & Asghari, S. M. The molecular basis of the sodium dodecyl sulfate effect on human ubiquitin structure: A molecular dynamics simulation study. Sci. Rep. 8, 2150 (2018).
pubmed: 29391595
pmcid: 5794983
doi: 10.1038/s41598-018-20669-7
Ochert, A. S., Boulter, A. W., Birnbaum, W., Johnson, N. W. & Teo, C. G. Inhibitory effect of salivary fluids on PCR: Potency and removal. Genome Res. 3, 365–368 (1994).
doi: 10.1101/gr.3.6.365
Ståhlberg, A., Zoric, N., Aman, P. & Kubista, M. Quantitative real-time PCR for cancer detection: The lymphoma case. Expert Rev. Mol. Diagn. 5, 221–230 (2005).
pubmed: 15833051
doi: 10.1586/14737159.5.2.221
Nolan, T., Hands, R. E. & Bustin, S. A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1, 1559–1582 (2006).
pubmed: 17406449
doi: 10.1038/nprot.2006.236
Ranoa, D. R. E. et al. Saliva-based molecular testing for SARS-CoV-2 that bypasses RNA extraction. bioRxiv 2020.06.18.159434. https://doi.org/10.1101/2020.06.18.159434 (2020).
Smyrlaki, I. et al. Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR. Nat. Commun. 11, 4812 (2020).
pubmed: 32968075
pmcid: 7511968
doi: 10.1038/s41467-020-18611-5
Lai, J. et al. Comparison of saliva and midturbinate swabs for detection of SARS-CoV-2. Microbiol. Spect. 10, e00128-e222 (2022).
Qian, Y. et al. Safety management of nasopharyngeal specimen collection from suspected cases of coronavirus disease 2019. Int. J. Nurs. Sci. 7, 153–156 (2020).
pubmed: 32292635
pmcid: 7129371
Uddin, M. K. M. et al. Diagnostic performance of self-collected saliva versus nasopharyngeal swab for the molecular detection of SARS-CoV-2 in the clinical setting. Microbiol. Spect. 9, e00468-e521 (2021).
Lee, R. A., Herigon, J. C., Benedetti, A., Pollock, N. R. & Denkinger, C. M. Performance of saliva, oropharyngeal swabs, and nasal swabs for SARS-CoV-2 molecular detection: A systematic review and meta-analysis. J. Clin. Microbiol. 59, e02881-e2920 (2021).
pubmed: 33504593
pmcid: 8091856
doi: 10.1128/JCM.02881-20
Ostheim, P. et al. Overcoming challenges in human saliva gene expression measurements. Sci. Rep. 10, 11147 (2020).
pubmed: 32636420
pmcid: 7341869
doi: 10.1038/s41598-020-67825-6
Chen, Y., Liu, F. & Lee, L. P. Quantitative and ultrasensitive in situ immunoassay technology for SARS-CoV-2 detection in saliva. Sci. Adv. 8, eabn3481 (2022).
Farsaeivahid, N., Grenier, C., Nazarian, S. & Wang, M. L. A rapid label-Free disposable electrochemical salivary point-of-care sensor for SARS-CoV-2 detection and quantification. Sensors 23, 433 (2023).
doi: 10.3390/s23010433
Jaewjaroenwattana, J., Phoolcharoen, W., Pasomsub, E., Teengam, P. & Chailapakul, O. Electrochemical paper-based antigen sensing platform using plant-derived monoclonal antibody for detecting SARS-CoV-2. Talanta 251, 123783 (2023).
pubmed: 35977451
doi: 10.1016/j.talanta.2022.123783