Can Multiplex SYBR Green Real-Time PCR Assay Serve as a Detection and Quantification Method Comparable to the TaqMan Method for SARS-CoV-2 Diagnosis?

Authors

  • Helyatul Rasmah Mahali
  • Nur Athirah Yusof

DOI:

https://doi.org/10.51200/bijb.v3i.4526

Keywords:

RT-qPCR, SARS-CoV-2, SYBR Green, multiplex, COVID-19, Gold standard diagnosis

Abstract

The reopening of schools, business, and social sectors during the COVID-19 pandemic has caused a current increase in the number of COVID-19 cases and clusters all over the globe. While the COVID-19 pandemic is far from over, the reopening and resumption of all economic sectors are essential to recovering the world economy. Health experts all over the world have determined that the real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) method is the gold standard for diagnosing COVID-19 infections due to the test’s high sensitivity and specificity. During the past 3 years when WHO declared the COVID-19 pandemic, the cost of laboratory diagnosis of COVID-19 using a robust RT-qPCR assay is still considerably expensive, especially for low and middle-income countries. Therefore, numerous studies have reported optimized SYBR green methods which are more economical than the qPCR probe assay. Continuous diagnostic testing is vital to mitigate the spread of COVID-19. However, there is a question as to whether SYBR Green may serve as an excellent detection and quantification method for molecular diagnosis to perform SARS-CoV-2 screening. This review summarizes the numerous studies using SYBR Green RT-PCR to detect SARS-CoV-2. The reliability of SYBR Green qPCR assays for determining gene expression based on their performance is justified and the quality is comparable to the TaqMan method.

Author Biographies

Helyatul Rasmah Mahali

Biotechnology Research Institute,
Jalan UMS, Universiti Malaysia Sabah,
88400 Kota Kinabalu, Sabah, Malaysia

Nur Athirah Yusof

Biotechnology Research Institute,
Jalan UMS, Universiti Malaysia Sabah,
88400 Kota Kinabalu, Sabah, Malaysia

References

Aleem, A., Akbar Samad, A. B., & Vaqar, S. (2023). Emerging Variants of SARS-CoV-2 and Novel Therapeutics Against Coronavirus (COVID-19). In: StatPearls Publishing.

Alimohamadi, Y., Sepandi, M., Taghdir, M., & Hosamirudsari, H. (2020). Determine the most common clinical symptoms in COVID-19 patients: a systematic review and meta-analysis. Journal of preventive medicine and hygiene, 61(3), E304–E312. https://doi.org/10.15167/2421-4248/jpmh2020.61.3.1530

Alvarez, E., Bielska, I. A., Hopkins, S., Belal, A. A., Goldstein, D. M., Slick, J., Pavalagantharajah, S., Wynfield, A., Dakey, S., Gedeon, M.-C., Alam, E., & Bouzanis, K. (2023). Limitations of COVID-19 testing and case data for evidence-informed Health Policy and practice. Health Research Policy and Systems, 21(1). https://doi.org/10.1186/s12961-023-00963-1

American Society for Microbiology (2020). Laboratory Supply Shortages Are Impacting

COVID-19 and Non-COVID Diagnostic Testing. Accessed on 21 July 2023. https://asm.org/Articles/2020/September/Laboratory-Supply-Shortages-Are-Impacting-COVID-19

Bernama (2021). RTK-Ag should be conducted by trained operator- Health DG. Assessed on 21

July 2023. https://www.thesundaily.my/local/rtk-ag-should-be-conducted-by-trained-operator-health-dg-XX6264145

Boro, E. & Stoll, B. (2022). Barriers to COVID-19 Health Products in Low-and Middle-Income Countries During the COVID-19 Pandemic: A Rapid Systematic Review and Evidence Synthesis. Frontiers in public health, 10, 928065. https://doi.org/10.3389/fpubh.2022.928065

Brihn, A., Chang, J., OYong, K., Balter, S., Terashita, D., Rubin, Z., Yeganeh, N. (2021). Diagnostic Performance of an Antigen Test with RT-PCR for the Detection of SARS-CoV-2 in a Hospital Setting - Los Angeles County, California, June-August 2020. MMWR Morb Mortal Wkly Rep, 70(19), 702-706. https://doi.org/10.15585/mmwr.mm7019a3.

Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 55(4), 611-22. https://doi.org/10.1373/clinchem.2008.112797

Butt, A.A., Dargham, S.R., Tang, P., Chemaitelly, H., Hasan, M.R., Coyle, P.V., Kaleeckal, A.H., Latif, A.N., Loka, S., Shaik, R.M., Zaqout, A., Almaslamani, M.A., Al Khal, A., Bertollini, R., Abou-Samra, A.B., & Abu-Raddad, L.J. (2022). COVID-19 disease severity in persons infected with the Omicron variant compared with the Delta variant in Qatar. Journal of global health, 12, 05032. https://doi.org/10.7189/jogh.12.05032

Chung, Y.S., Lee, N.J., Woo, S.H. et al. (2021). Validation of real-time RT-PCR for detection of SARS-CoV-2 in the early stages of the COVID-19 outbreak in the Republic of Korea. Sci Rep 11, 14817. https://doi.org/10.1038/s41598-021-94196-3

Corman, V. M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D. K., Bleicker, T., Brünink, S., Schneider, J., Schmidt, M. L., Mulders, D. G., Haagmans, B. L., van der Veer, B., van den Brink, S., Wijsman, L., Goderski, G., Romette, J. L., Ellis, J., Zambon, M., Peiris, M., … Drosten, C. (2020). Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, 25(3), 2000045. https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045

Dhawan, M., Sharma, A., Priyanka, Thakur, N., Rajkhowa, T. K., & Choudhary, O.P. (2022). Delta variant (B.1.617.2) of SARS-CoV-2: Mutations, impact, challenges and possible solutions. Human vaccines & immunotherapeutics, 18(5), 2068883. https://doi.org/10.1080/21645515.2022.2068883

Dorlass, E.G., Monteiro, C.O., Viana, A.O., Soares, C.P., Machado, R.R.G., Thomazelli, L.M., Araujo, D.B., Leal, F.B., Candido, E.D., Telezynski, B.L., Valério, C.A., Chalup, V.N., Mello, R., Almeida, F.J., Aguiar, A.S., Barrientos, A.C.M., Sucupira, C., De Paulis, M., Sáfadi, M.A.P., Silva, D.G.B.P. & Oliveira, D.B.L. (2020). Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology], 51(3), 1117–1123. https://doi.org/10.1007/s42770-020-00347-5

Dragan, A.I., Pavlovic, R., McGivney, J.B. et al. (2012). SYBR Green I: Fluorescence Properties and Interaction with DNA. J Fluoresc 22, 1189–1199. https://doi.org/10.1007/s10895-012-1059-8

Duffy, S. (2018) Why are RNA virus mutation rates so damn high?. PLoS biology, 16(8), e3000003. https://doi.org/10.1371/journal.pbio.3000003

Feng, Z., Zhang, Y., Pan, Y., Zhang, D., Zhang, L., & Wang, Q. (2022). Mass screening is a key component to fight against SARS-CoV-2 and return to normalcy. Medical review (Berlin, Germany), 2(2), 197–212. https://doi.org/10.1515/mr-2021-0024

Filip, R., Gheorghita Puscaselu, R., Anchidin-Norocel, L., Dimian, M., & Savage, W. K. (2022). Global Challenges to Public Health Care Systems during the COVID-19 Pandemic: A Review of Pandemic Measures and Problems. Journal of personalized medicine, 12(8), 1295. https://doi.org/10.3390/jpm12081295

Gili, R., & Burioni, R. (2023). SARS-CoV-2 before and after Omicron: two different viruses and two different diseases? Journal of translational medicine, 21(1), 251. https://doi.org/10.1186/s12967-023-04095-6

Groff, A., Kavanaugh, M., Ramgobin, D., McClafferty, B., Aggarwal, C. S., Golamari, R., & Jain, R. (2021). Gastrointestinal Manifestations of COVID-19: A Review of What We Know. The Ochsner journal, 21(2), 177–180. https://doi.org/10.31486/toj.20.0086

Gupta, S.K., Minocha, R., Thapa, P.J., Srivastava, M., & Dandekar, T. (2022). Role of the Pangolin in Origin of SARS-CoV-2: An Evolutionary Perspective. International journal of molecular sciences, 23(16), 9115. https://doi.org/10.3390/ijms23169115

Hardt, M., Föderl-Höbenreich, E., Freydl, S., Kouros, A., Loibner, M., & Zatloukal, K. (2022). Pre-analytical sample stabilization by different sampling devices for PCR-based COVID-19 Diagnostics. New Biotechnology, 70, 19–27. https://doi.org/https://doi.org/10.1016/j.nbt.2022.04.001

Hashim, J. H., Adman, M. A., Hashim, Z., Mohd Radi, M. F., & Kwan, S. C. (2021). Covid-19 epidemic in Malaysia: Epidemic progression, challenges, and response. Frontiers in Public Health, 9. https://doi.org/https://doi.org/10.3389/fpubh.2021.560592

Hu, B. et al. (2020) ‘Characteristics of SARS-COV-2 and COVID-19’, Nature Reviews Microbiology, 19(3), pp. 141–154. https://doi.org/10.1038/s41579-020-00459-7

Iacobucci G. (2021). Covid-19: Runny nose, headache, and fatigue are commonest symptoms of omicron, early data show. BMJ (Clinical research ed.), 375, n3103. https://doi.org/10.1136/bmj.n3103

Ibrahim N.K. (2020). Epidemiologic surveillance for controlling Covid-19 pandemic: types, challenges and implications. Journal of infection and public health, 13(11), 1630–1638. https://doi.org/10.1016/j.jiph.2020.07.019

Jacofsky, D., Jacofsky, E. M., & Jacofsky, M. (2020). Understanding Antibody Testing for COVID-19. The Journal of arthroplasty, 35(7S), S74–S81. https://doi.org/10.1016/j.arth.2020.04.055

Johansson, M. A., Quandelacy, T. M., Kada, S., Prasad, P. V., Steele, M., Brooks, J. T., Slayton, R. B., Biggerstaff, M., & Butler, J. C. (2021). SARS-COV-2 transmission from people without COVID-19 symptoms. JAMA Network Open, 4(1). https://doi.org/10.1001/jamanetworkopen.2020.35057

Kaye, A.D., Okeagu, C.N., Pham, A.D., Silva, R.A., Hurley, J.J., Arron, B.L., Sarfraz, N., Lee, H.N., Ghali, G.E., Gamble, J.W., Liu, H., Urman, R.D., & Cornett, E.M. (2021). Economic impact of COVID-19 pandemic on healthcare facilities and systems: International perspectives. Best practice & research. Clinical anaesthesiology, 35(3), 293–306. https://doi.org/10.1016/j.bpa.2020.11.009

Khandker, S.S., Nik Hashim, N.H.H., Deris, Z.Z., Shueb, R.H., Islam, M.A. (2022). Diagnostic Accuracy of Rapid Antigen Test Kits for Detecting SARS-CoV-2: A Systematic Review and Meta-Analysis of 17,171 Suspected COVID-19 Patients. J Clin Med, 10(16), 3493. https://doi.org/10.3390/jcm10163493

Koelle, K., Martin, M. A., Antia, R., Lopman, B., & Dean, N. E. (2022). The changing epidemiology of SARS-CoV-2. Science (New York, N.Y.), 375(6585), 1116–1121. https://doi.org/10.1126/science.abm4915

Lamy, E., Rubio, C. P., Carreira, L., Capela e Silva, F., Martinez-Subiela, S., Tecles, F., Lopez-Jornet, P., Ceron, J. J., & Tvarijonaviciute, A. (2022). Effect of thermal and chemical treatments used for SARS-COV-2 inactivation in the measurement of saliva analytes. Scientific Reports, 12(1). https://doi.org/https://doi.org/10.1038/s41598-022-13491-9

Malekshahi, A., Khanizadeh, S., Fallahi, S., Talei, G., Birjandi, M., & Hajizadeh, F. (2022). Diagnostic power of one-step and two-step RT-qPCR methods to SARS CoV 2 detection. BMC infectious diseases, 22(1), 505. https://doi.org/10.1186/s12879-022-07478-0

Marinowic, D.R., Zanirati, G., Rodrigues, F.V.F. et al. (2021) A new SYBR Green real-time

PCR to detect SARS-CoV-2. Sci Rep 11, 2224 (2021). https://doi.org/10.1038/s41598-021-81245-0

Medical Device Authority, Ministry of Health Malaysia. Conditional Approval for COVID-19

RTK (Self-Test). Accessed on 21 July 2023. https://portal.mda.gov.my/component/fsf?catid=6&view=faq

Neopane, P., Nypaver, J., Shrestha, R., Beqaj, S.S. (2021). SARS-CoV-2 Variants Detection

Using TaqMan SARS-CoV-2 Mutation Panel Molecular Genotyping Assays. Infect Drug Resist, 14, 4471-4479. https://doi.org/10.2147/IDR.S335583

Pavia, C.S., & Plummer, M. (2021). The evolution of rapid antigen detection systems and their application for COVID-19 and other serious respiratory infectious diseases. Journal of microbiology, immunology, and infection, 54(5), 776–786. https://doi.org/10.1016/j.jmii.2021.06.003

Peeling, R. W., Heymann, D. L., Teo, Y. Y., & Garcia, P. J. (2022). Diagnostics for COVID-19: moving from pandemic response to control. Lancet (London, England), 399(10326), 757–768. https://doi.org/10.1016/S0140-6736(21)02346-1Pereira-Gómez, M., Fajardo, Á., Echeverría, N., López-Tort, F., Perbolianachis, P., Costábile, A., Aldunate, F., Moreno, P., & Moratorio, G. (2021). Evaluation of SYBR Green real time PCR for detecting SARS-CoV-2 from clinical samples. Journal of virological methods, 289, 114035. https://doi.org/10.1016/j.jviromet.2020.114035

Radvak, P., Kwon, H.-J., Kosikova, M., Ortega-Rodriguez, U., Xiang, R., Phue, J.-N., Shen, R.-F., Rozzelle, J., Kapoor, N., Rabara, T., Fairman, J., & Xie, H. (2021). SARS-COV-2 B.1.1.7 (alpha) and B.1.351 (beta) variants induce pathogenic patterns in K18-hACE2 transgenic mice distinct from early strains. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-26803-w

Rahmasari, R., Raekiansyah, M., Azallea, S.N., Nethania, M., Bilqisthy, N., Rozaliyani, A., Bowolaksono, A., Sauriasari, R. (2022) Low-cost SYBR Green-based RT-qPCR assay for detecting SARS-CoV-2 in an Indonesian setting using WHO-recommended primers. Heliyon, 8(11), e11130. https://doi.org/10.1016/j.heliyon.2022.e11130

Ranzenigo, M., Bruzzesi, E., Galli, L., Castagna, A., & Ferrari, G. (2021). Symptoms and signs of conjunctivitis as predictors of disease course in COVID-19 syndrome. Journal of ophthalmic inflammation and infection, 11(1), 35. https://doi.org/10.1186/s12348-021-00264-0

Ren, S.Y., Wang, W.B., Gao, R.D., & Zhou, A.M. (2022). Omicron variant (B.1.1.529) of SARS-CoV-2: Mutation, infectivity, transmission, and vaccine resistance. World journal of clinical cases, 10(1), 1–11. https://doi.org/10.12998/wjcc.v10.i1.1

Rong, G., Zheng, Y., Chen, Y., Zhang, Y., Zhu, P., & Sawan, M. (2023). COVID-19 Diagnostic Methods and Detection Techniques. Encyclopedia of Sensors and Biosensors, 17–32. https://doi.org/10.1016/B978-0-12-822548-6.00080-7

Ritchie, H., Ortiz-Ospina, E., Beltekian, D., Mathieu, E., Hasell, J., & Macdonald, B. et al. (2021). Coronavirus Pandemic (COVID-19). Our World in Data. Retrieved 4 July 2023, from https://ourworldindata.org/coronavirus.

Saberiyan, M., Karimi, E., Khademi, Z., Movahhed, P., Safi, A., & Mehri-Ghahfarrokhi, A. (2022). SARS-CoV-2: phenotype, genotype, and characterization of different variants. Cellular & molecular biology letters, 27(1), 50. https://doi.org/10.1186/s11658-022-00352-6

Sanduzzi, A., & Zamparelli, S. S. (2020). Nasopharyngeal and Oropharyngeal Swabs, And/Or Serology for SARS COVID-19: What Are We Looking For?. International journal of environmental research and public health, 17(9), 3289. https://doi.org/10.3390/ijerph17093289

Sanjuán, R., & Domingo-Calap, P. (2016). Mechanisms of viral mutation. Cellular and molecular life sciences : CMLS, 73(23), 4433–4448. https://doi.org/10.1007/s00018-016-2299-6

Sarkar, S.L., Alam, A.S.M.R.U., Das, P.K. et al. (2022). Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis. Sci Rep 12, 6501. https://doi.org/10.1038/s41598-022-10413-7

Shafie, M. H., Antony Dass, M., Ahmad Shaberi, H. S., & Zafarina, Z. (2023). Screening and confirmation tests for SARS-CoV-2: benefits and drawbacks. Beni-Suef University journal of basic and applied sciences, 12(1), 6. https://doi.org/10.1186/s43088-023-00342-3

Shereen, M., Khan, S., Kazmi, A., Bashir, N., & Siddique, R. (2020). COVID-19 infection: Emergence, transmission, and characteristics of human coronaviruses. Journal Of Advanced Research, 24, 91-98. https://doi.org/10.1016/j.jare.2020.03.005

Song, C. et al. (2022) ‘SARS-COV-2: The monster causes covid-19’, Frontiers in Cellular and Infection Microbiology, 12. https://doi.org/10.3389/fcimb.2022.835750.

Struyf, T., Deeks, J. J., Dinnes, J., Takwoingi, Y., Davenport, C., Leeflang, M. M., Spijker, R., Hooft, L., Emperador, D., Domen, J., Tans, A., Janssens, S., Wickramasinghe, D., Lannoy, V., Horn, S. R. A., Van den Bruel, A., & Cochrane COVID-19 Diagnostic Test Accuracy Group (2022). Signs and symptoms to determine if a patient presenting in primary care or hospital outpatient settings has COVID-19. The Cochrane database of systematic reviews, 5(5), CD013665. https://doi.org/10.1002/14651858.CD013665.pub3

Tao, Y., Yue, Y., Qiu, G. et al. (2022). Comparison of analytical sensitivity and efficiency for SARS-CoV-2 primer sets by TaqMan-based and SYBR Green-based RT-qPCR. Applied Microbiology Biotechnology 106, 2207–2218. https://doi.org/10.1007/s00253-022-11822-4

Udugama, B., Kadhiresan, P., Kozlowski, H. N., Malekjahani, A., Osborne, M., Li, V.Y.C., Chen, H., Mubareka, S., Gubbay, J.B., & Chan, W.C.W. (2020). Diagnosing COVID-19: The Disease and Tools for Detection. ACS nano, 14(4), 3822–3835. https://doi.org/10.1021/acsnano.0c02624

Wan, Z., Zhao, Y., Lu, R., Dong, Y., Zhang, C. (2021) Rapid antigen detection alone may not be sufficient for early diagnosis and/or mass screening of COVID-19. J Med Virol, 93(12), 6462-6464. https://doi.org/10.1002/jmv.27236

Xie, J. W., He, Y., Zheng, Y. W., Wang, M., Lin, Y., & Lin, L. R. (2022). Diagnostic accuracy of rapid antigen test for SARS-CoV-2: A systematic review and meta-analysis of 166,943 suspected COVID-19 patients. Microbiological research, 265, 127185. https://doi.org/10.1016/j.micres.2022.127185

Published

2023-12-22

How to Cite

Mahali, H. R., & Nur Athirah Yusof. (2023). Can Multiplex SYBR Green Real-Time PCR Assay Serve as a Detection and Quantification Method Comparable to the TaqMan Method for SARS-CoV-2 Diagnosis?. Borneo International Journal of Biotechnology (BIJB), 3, 80–99. https://doi.org/10.51200/bijb.v3i.4526
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