A Rapid RT-LAMP Assay for SARS-CoV-2 with Colorimetric Detection Assisted by a Mobile Application
Abstract
:1. Introduction
2. Materials and Methods
2.1. Origin of Samples
2.2. Real-Time RT-PCR
2.3. Primers and Initial Conditions for LAMP
2.4. Mobile-Assisted Visualization
2.5. Optimization to Avoid RNA Purification
2.6. Rapid LAMP Strategy
2.7. Detection Limit of the Rapid Assay
2.8. Cross-Reactivity
2.9. Performance of the Rapid LAMP Assay
3. Results
3.1. Setting Up the Conditions
3.2. Detection Limit of a Selected Strategy
3.3. Cross-Reactivity
3.4. Performance of the Visual Approach
3.5. Performance of Assay with Mobile Application
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
- Cucinotta, D.; Vanelli, M. WHO Declares COVID-19 a Pandemic. Acta Biomed. 2020, 91, 157–160. [Google Scholar] [CrossRef] [PubMed]
- Thompson, D.; Lei, Y. 2020. Mini Review: Recent Progress in RT-LAMP Enabled COVID-19 Detection. Sens. Actuators Rep. 2020, 2, 100017. [Google Scholar] [CrossRef] [PubMed]
- Yang, Q.; Meyerson, N.R.; Clark, S.K.; Paige, C.L.; Fattor, W.T.; Gilchrist, A.R.; Barbachano-Guerrero, A.; Healy, B.G.; Worden-Sapper, E.R.; Wu, S.S.; et al. Saliva TwoStep for Rapid Detection of Asymptomatic SARS-CoV-2 Carriers. Elife 2021, 10, e65113. [Google Scholar] [CrossRef]
- Mautner, L.; Baillie, C.K.; Herold, H.M.; Volkwein, W.; Guertler, P.; Eberle, U.; Ackermann, N.; Sing, A.; Pavlovic, M.; Goerlich, O.; et al. Rapid Point-of-Care Detection of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP). Virol. J. 2020, 17, 160. [Google Scholar] [CrossRef]
- Ali, Z.; Aman, R.; Mahas, A.; Rao, G.S.; Tehseen, M.; Marsic, T.; Mahfouz, M.M. iSCAN: An RT-LAMP-Coupled CRISPR-Cas12 Module for Rapid, Sensitive Detection of SARS-CoV-2. Virus Res. 2020, 288. [Google Scholar] [CrossRef]
- Schermer, B.; Fabretti, F.; Damagnez, M.; Di Cristanziano, V.; Heger, E.; Arjune, S.; Tanner, N.A.; Imhof, T.; Koch, M.; Ladha, A.; et al. Rapid SARS-CoV-2 Testing in Primary Material Based on a Novel Multiplex RT-LAMP Assay. PLoS ONE 2020, 15, e0238612. [Google Scholar] [CrossRef]
- Zhu, X.; Wang, X.; Han, L.; Chen, T.; Wang, L.; Li, H.; Li, S.; He, L.; Fu, X.; Chen, S.; et al. Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Combined with Nanoparticle-Based Lateral Flow Biosensor for the Diagnosis of COVID-19. Biosens. Bioelectron. 2020, 166, 112437. [Google Scholar] [CrossRef]
- Huang, W.E.; Lim, B.; Hsu, C.C.; ** Countries. Int. J. Infect. Dis. 2021, 104, 303–305. [Google Scholar] [CrossRef]
- Wilson-Davies, E.S.W.; Mahanama, A.I.K.; Samaraweera, B.; Ahmed, N.; Friar, S.; Pelosi, E. Concerns Regarding the Sensitivity of the OptiGene Direct SARS-CoV-2 LAMP Assay and Its Suitability for Use in at-Risk Groups and Hospital Staff. J. Infect. 2021, 82, 282–327. [Google Scholar] [CrossRef] [PubMed]
- Centers for Disease Control and Prevention. Research Use Only Real-Time RT-PCR Protocol for Identification of 2019-nCoV. 2020. Available online: https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html (accessed on 29 March 2021).
- El-Tholoth, M.; Bau, H.H.; Song, Y. A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry. ChemRxiv 2020. [Google Scholar] [CrossRef]
- Sicasys Software GmbH; Spotxel® Reader : Germersheim, Germany, 2017.
- Medeiros da Silva, R.C.; Nogueira Marinho, L.C.; de Araújo Silva, D.N.; Costa de Lima, K.; Pirih, F.Q.; Luz de Aquino Martins, A.R. Saliva as a Possible Tool for the SARS-CoV-2 Detection: A Review. Travel Med. Infect. Dis. 2020, 38, 101920. [Google Scholar] [CrossRef] [PubMed]
- Rabe, B.A.; Cepko, C. SARS-CoV-2 Detection Using Isothermal Amplification and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification. Proc. Nat. Acad. Sci. USA 2020, 117, 24450–24458. [Google Scholar] [CrossRef]
- Meyerson, N.R.; Yang, Q.; Clark, S.K.; Paige, C.L.; Fattor, W.T.; Gilchrist, A.R.; Barbachano-Guerrero, A.; Sawyer, S.L. A Community-Deployable SARS-CoV-2 Screening Test Using Raw Saliva with 45 Minutes Sample-to-Results Turnaround. Medrxiv 2020. [Google Scholar] [CrossRef]
- Dinnes, J.; Deeks, J.J.; Adriano, A.; Berhane, S.; Davenport, C.; Dittrich, S.; Emperador, D.; Takwoingi, Y.; Cunningham, J.; Beese, S.; et al. Cochrane COVID-19 Diagnostic Test Accuracy Group. Rapid, Point-of-Care Antigen and Molecular-Based Tests for Diagnosis of SARS-CoV-2 Infection. Cochrane Database Syst. Rev. 2020, 8, CD013705. [Google Scholar] [CrossRef] [PubMed]
- Jones, T.C.; Biele, G.; Mühlemann, B.; Veith, T.; Schneider, J.; Beheim-Schwarzbach, J.; Bleicker, T.; Tesch, J.; Schmidt, M.L.; Sander, L.E.; et al. Estimating Infectiousness throughout SARS-CoV-2 Infection Course. Science 2021, 373, eabi5273. [Google Scholar] [CrossRef]
- van Kampen, J.J.A.; van de Vijver, D.A.M.C.; Fraaij, P.L.A.; Haagmans, B.L.; Lamers, M.M.; Okba, N.; van den Akker, J.P.C.; Endeman, H.; Gommers, D.A.M.P.J.; Cornelissen, J.J.; et al. Duration and Key Determinants of Infectious Virus Shedding in Hospitalized Patients with Coronavirus Disease-2019 (COVID-19). Nat. Commun. 2021, 12, 267. [Google Scholar] [CrossRef]
- Kawasuji, H.; Takegoshi, Y.; Kaneda, M.; Ueno, A.; Miyajima, Y.; Kawago, K.; Fukui, Y.; Yoshida, Y.; Kimura, M.; Yamada, H.; et al. Transmissibility of COVID-19 Depends on the Viral Load around Onset in Adult and Symptomatic Patients. PLoS ONE 2020, 15, e0243597. [Google Scholar] [CrossRef]
- Juscamayta-López, E.; Valdivia, F.; Horna, H.; Tarazona, D.; Linares, L.; Rojas, N.; Huaringa, M. A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2. Front. Cell Infect. Microbiol. 2021, 11, 653616. [Google Scholar] [CrossRef]
- Escalante-Maldonado, O.; Vidal-Anzardo, M.; Donaires, F.; Solis-Sanchez, G.; Gallesi, I.; Pampa-Espinoza, L.; Huaringa, M.; Rojas-Serrano, N.; García, C.; Angles-Yanqui, E.; et al. Standardization and Validation of an in House RT-LAMP Molecular Test for the Diagnosis of SARS-CoV-2. Rev. Peru Med. Exp. Salud. Publica 2021, 38, 7–16. [Google Scholar] [CrossRef] [PubMed]
NPS | NPA | SAL | ||||||
---|---|---|---|---|---|---|---|---|
Estimated No Copies | Positives Visual | Positives SpotXel | Estimated No Copies | Positives Visual | Positives SpotXel | Estimated No Copies | Positives Visual | Positives SpotXel |
564,849 | 4/4 | 4/4 | 558,801 | 4/4 | 4/4 | 582,730 | 4/4 | 4/4 |
48,810 | 4/4 | 4/4 | 112,814 | 4/4 | 4/4 | 88,682 | 3/4 | 4/4 |
22,594 | 4/4 | 4/4 | 22,563 | 2/4 | 3/4 | 23,309 | 0/4 | 2/4 |
7001 | 1/4 | 2/4 | 10,827 | 2/4 | 2/4 | 7906 | 1/4 | 2/4 |
621 | 2/4 | 2/4 | 1381 | 3/4 | 3/4 | 933 | 0/4 | 1/4 |
181 | 1/4 | 1/4 | 179 | 3/4 | 2/4 | 186 | 1/4 | 2/4 |
36 | 0/4 | 2/4 | 36 | 1/4 | 3/4 | 37 | 0/4 | 0/4 |
0.0 | 0/4 | 0/0 | 0.0 | 0/4 | 0/4 | 0.0 | 0/4 | 0/4 |
Virus | No. Samples Tested * | Positives, Visual | Positives, SpotXel |
---|---|---|---|
AdV | 4 | 0/4 | 0/4 |
InfA | 3 | 0/3 | 0/3 |
InfB | 2 | 0/2 | 0/2 |
hMPNV | 3 | 0/3 | 0/3 |
Parainf (1–3) | 6 | 0/6 | 0/6 |
RSV | 4 | 0/4 | 0/4 |
HCoV-OC43 | 1 | 0/1 | 0/1 |
RT-LAMP Visual | |||||
---|---|---|---|---|---|
Positives | Faint | Negatives | Total | ||
qRT-PCR CDC | Positives | 62 | 35 | 34 | 131 |
Faint | 0 | 0 | 0 | 0 | |
Negatives | 4 | 14 | 59 | 77 | |
Total | 66 | 49 | 93 | 208 | |
Accuracy * | 67.8 (61.03–74.1) | ||||
Kappa index * | 0.34 (0.26–0.42) |
Global qPCR (+) = 131 qPCR (−) = 77 | NPS qPCR (+) = 61 qPCR (−) = 25 | NPA qPCR (+) = 37 qPCR (−) = 25 | Saliva qPCR (+) = 33 qPCR (−) = 27 | |
---|---|---|---|---|
Accuracy | 80.6 (74.6–85.8) | 54.8 (43.7–65.6) | 69.8 (56.8–80.8) | 79.5 (67.1–88.8) |
Sensitivity | 74.8 (66.5–82.0) | 42.62 (30.0–55.9) | 46.0 (29.5–63.1) | 57.6 (39.2–74.5) |
Specificity | 83.1 (72.9–90.7) | 60.0 (38.7–78.9) | 80.0 (59.3–93.2) | 88.9( 70.8–97.7) |
Positive predictive value | 65.51 (53.4–75.9) | 31.4 (20.7–44.5) | 49.6 (29.4–69.9) | 68.9 (42.4–87.0) |
Negative predictive value | 88.5 (84.9–91.3) | 70.9 (62.4–78.2) | 77.5 (70.8–83.1) | 83.0 (76.3–88.1) |
Fold change (average ± sd) | tp = 1.20 ± 0.16 | tp = 1.17 ± 0.14 | tp = 1.22 ± 0.17 | tp = 1.25 ± 0.15 |
tn = 0.89 ± 0.07 | tn = 0.91 ± 0.04 | tn = 0.91 ± 0.05 | tn = 0.85 ± 0.09 | |
fp = 1.13 ± 0.07 | fp = 1.10 ± 0.07 | fp = 1.01 ± 0.01 | fp = 1.16 ± 0.05 | |
fn = 0.92 ± 0.06 | fn = 0.94 ± 0.04 | fn = 0.90 ± 0.04 | fn = 0.90 ± 0.08 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Londono-Avendano, M.A.; Libreros, G.; Osorio, L.; Parra, B. A Rapid RT-LAMP Assay for SARS-CoV-2 with Colorimetric Detection Assisted by a Mobile Application. Diagnostics 2022, 12, 848. https://doi.org/10.3390/diagnostics12040848
Londono-Avendano MA, Libreros G, Osorio L, Parra B. A Rapid RT-LAMP Assay for SARS-CoV-2 with Colorimetric Detection Assisted by a Mobile Application. Diagnostics. 2022; 12(4):848. https://doi.org/10.3390/diagnostics12040848
Chicago/Turabian StyleLondono-Avendano, María Aurora, Gerardo Libreros, Lyda Osorio, and Beatriz Parra. 2022. "A Rapid RT-LAMP Assay for SARS-CoV-2 with Colorimetric Detection Assisted by a Mobile Application" Diagnostics 12, no. 4: 848. https://doi.org/10.3390/diagnostics12040848