Diagnostic Power of One-step and Two-step RT-qPCR Methods to SARS‑CoV‑2 Detection

Overview

Journal BMC Infect Dis

Publisher Biomed Central

Specialty Infectious Diseases

Date 2022 Jun 1

PMID 35641907

Authors

Asra Malekshahi

Sayyad Khanizadeh

Shirzad Fallahi

Gholamreza Talei

Mehdi Birjandi

Faezeh Hajizadeh

Affiliations

Soon will be listed here.

Abstract

Background: Coronavirus-2019 (COVID-2019) is a novel coronavirus known as Acute Respiratory Syndrome (SARS-CoV-2). The premier standard test for SARS-CoV-2 diagnosis is a one-step RT-qPCR method, which requires specific probes and reagents. Therefore, detection on a large scale is expensive and cannot be very accurate.

Methods: A cost-effective technique based on SYBR green was evaluated in the current study. The specific primers for S and N genes were designed, then performed the cross-reactivity test with other coronavirus and respiratory viruses positive samples. Moreover, the analytical sensitivity test was carried out with 8 dilutions (1:10). Lastly, the SARS-CoV-2 clinical samples (n = 210) were tested by these two methods, and receiver operating characteristic (ROC) analysis was performed to investigate the incremental diagnostic value of each gene in the study methods.

Results: The two-step method detected up to 6th dilutions of the SARS-CoV-2 samples and did not show any amplification of the positive samples of other respiratory viruses. ROC analysis revealed a diagnostic ability of the two-step method for SARS-CoV-2 with an area under the ROC curve of ≥ 0.7 (P ˂ 0.05) and relatively high sensitivity and specificity. The combination of N and S genes increased the sensitivity up to 88%, specificity up to 86%, and area under the ROC curve up to 0.85 (95% confidence interval (95% CI) 0.72 to 0.93, P = 0.0461).

Conclusion: Our findings indicated that the two-step method has comparable sensitivity and specificity to the one-step method. Therefore, this method can be considered a potential diagnostic method for diagnosing and monitoring COVID-19 patients. It suggests that when the one-step RT-qPCR method is not available, the two-step RT-qPCR can be used to identify SARS-CoV-2.

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References

Freire-Paspuel B, Vega-Marino P, Velez A, Cruz M, Garcia-Bereguiain M . "Sample pooling of RNA extracts to speed up SARS-CoV-2 diagnosis using CDC FDA EUA RT-qPCR kit". Virus Res. 2020; 290:198173. PMC: 7513759. DOI: 10.1016/j.virusres.2020.198173. View

Tajadini M, Panjehpour M, Javanmard S . Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Adv Biomed Res. 2014; 3:85. PMC: 3988599. DOI: 10.4103/2277-9175.127998. View

. Early Epidemiological and Clinical Characteristics of 28 Cases of Coronavirus Disease in South Korea. Osong Public Health Res Perspect. 2020; 11(1):8-14. PMC: 7045878. DOI: 10.24171/j.phrp.2020.11.1.03. View

Liang Y, Wang M, Chien C, Yarmishyn A, Yang Y, Lai W . Highlight of Immune Pathogenic Response and Hematopathologic Effect in SARS-CoV, MERS-CoV, and SARS-Cov-2 Infection. Front Immunol. 2020; 11:1022. PMC: 7236801. DOI: 10.3389/fimmu.2020.01022. View

Jiang M, Pan W, Arasthfer A, Fang W, Ling L, Fang H . Development and Validation of a Rapid, Single-Step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) System Potentially to Be Used for Reliable and High-Throughput Screening of COVID-19. Front Cell Infect Microbiol. 2020; 10:331. PMC: 7313420. DOI: 10.3389/fcimb.2020.00331. View

Wightman F, Godoy Herz M, Munoz J, Stigliano J, Bragado L, Nieto Moreno N . A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2. RNA Biol. 2021; 18(12):2218-2225. PMC: 8174584. DOI: 10.1080/15476286.2021.1926648. View

Zhou X, Zhang T, Song D, Huang T, Peng Q, Chen Y . Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus. Mol Cell Probes. 2017; 33:36-41. DOI: 10.1016/j.mcp.2017.02.002. View

Figueroa S, Freire-Paspuel B, Vega-Marino P, Velez A, Cruz M, Cardenas W . High sensitivity-low cost detection of SARS-CoV-2 by two steps end point RT-PCR with agarose gel electrophoresis visualization. Sci Rep. 2021; 11(1):21658. PMC: 8568942. DOI: 10.1038/s41598-021-00900-8. View

Dorlass E, Monteiro C, Viana A, Soares C, Machado R, Matsumiya Thomazelli L . Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR. Braz J Microbiol. 2020; 51(3):1117-1123. PMC: 7411266. DOI: 10.1007/s42770-020-00347-5. View

10.

Chang F, Chen H, Chen P, Ho M, Hsieh S, Lin J . Immunologic aspects of characteristics, diagnosis, and treatment of coronavirus disease 2019 (COVID-19). J Biomed Sci. 2020; 27(1):72. PMC: 7270518. DOI: 10.1186/s12929-020-00663-w. View

11.

Watzinger F, Ebner K, Lion T . Detection and monitoring of virus infections by real-time PCR. Mol Aspects Med. 2006; 27(2-3):254-98. PMC: 7112306. DOI: 10.1016/j.mam.2005.12.001. View

12.

Kenyeres B, Anosi N, Banyai K, Matyus M, Orosz L, Kiss A . Comparison of four PCR and two point of care assays used in the laboratory detection of SARS-CoV-2. J Virol Methods. 2021; 293:114165. PMC: 8051013. DOI: 10.1016/j.jviromet.2021.114165. View

13.

Gomes-Ruiz A, Nascimento R, de Paula S, da Fonseca B . SYBR green and TaqMan real-time PCR assays are equivalent for the diagnosis of dengue virus type 3 infections. J Med Virol. 2006; 78(6):760-3. DOI: 10.1002/jmv.20620. View

14.

Marinowic D, Zanirati G, Rodrigues F, Grahl M, Alcara A, Machado D . A new SYBR Green real-time PCR to detect SARS-CoV-2. Sci Rep. 2021; 11(1):2224. PMC: 7838253. DOI: 10.1038/s41598-021-81245-0. View

15.

Espy M, Uhl J, Sloan L, Buckwalter S, Jones M, Vetter E . Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev. 2006; 19(1):165-256. PMC: 1360278. DOI: 10.1128/CMR.19.1.165-256.2006. View

16.

Pereira-Gomez M, Fajardo A, Echeverria N, Lopez-Tort F, Perbolianachis P, Costabile A . Evaluation of SYBR Green real time PCR for detecting SARS-CoV-2 from clinical samples. J Virol Methods. 2020; 289:114035. PMC: 7831559. DOI: 10.1016/j.jviromet.2020.114035. View

17.

Kudo E, Israelow B, Vogels C, Lu P, Wyllie A, Tokuyama M . Detection of SARS-CoV-2 RNA by multiplex RT-qPCR. PLoS Biol. 2020; 18(10):e3000867. PMC: 7571696. DOI: 10.1371/journal.pbio.3000867. View

18.

Zhu N, Zhang D, Wang W, Li X, Yang B, Song J . A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; 382(8):727-733. PMC: 7092803. DOI: 10.1056/NEJMoa2001017. View

19.

Candel F, Barreiro P, San Roman J, Abanades J, Barba R, Barberan J . Recommendations for use of antigenic tests in the diagnosis of acute SARS-CoV-2 infection in the second pandemic wave: attitude in different clinical settings. Rev Esp Quimioter. 2020; 33(6):466-484. PMC: 7712344. DOI: 10.37201/req/120.2020. View

20.

Suo T, Liu X, Feng J, Guo M, Hu W, Guo D . ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens. Emerg Microbes Infect. 2020; 9(1):1259-1268. PMC: 7448897. DOI: 10.1080/22221751.2020.1772678. View