Use of The Heat Treatment Method as An Alternative to Commercial Kit-Based Nucleic Acid Extraction Method for Detecting SARS-CoV-2 Using RT-qPCR Technique under Reagent Limited Conditions
Abstract
The increase in the number of COVID-19 cases could cause disruptions to the global supply chain of commercial kits for the extraction of SARS-CoV-2 nucleic acid so that the COVID-19 screening process has the potential to be hampered. Another alternative method that can be used is the heat-treatment (HT) method. This study aims to assess the quality of the results of the RT-qPCR examination for the detection of SARS-CoV-2 using the HT nucleic acid extraction method. The samples were nasopharyngeal and oropharyngeal swabs and then underwent nucleic acid extraction using the magnetic beads extraction (MBE) / commercial kit and the HT method on the same day (less than 6 hours). The nucleic acid extraction results from both methods were then amplified using the RT-qPCR technique, then compared and analyzed the results. The results showed that qualitatively there was a very significant difference in the interpretation of the results between the two nucleic acid extraction methods (McNemar test, p = 0; p < 0.01). Qualitative comparative analysis also showed that there were very significant differences in ct (cycle threshold) values in the SARS-CoV-2 genes detected between the two nucleic acid extraction methods (McNemar test, p = 0; p < 0.01). While the value of ct in human gene between the two methods of nucleic acid extraction qualitatively there was no significant difference (McNemar test, p = 1; p > 0.05). The use of HT method for extraction will be more effective on cells or microorganisms that have genetic material in the form of DNA than RNA. The HT method is not recommended for use in the diagnosis of COVID-19. The use of this method as a screening in moderate to severe symptomatic populations under conditions of limited extraction reagents may be considered.
Keywords: SARS-CoV-2, COVID-19, Heat-Treatment Method, Nucleic Acid Extraction, RT-qPCR
Full Text:
PDFReferences
Shereen MA, Khan S, Kazmi A, Bashir N, Siddique R. COVID-19 Infection: Origin, Transmission, and Characteristics of Human Coronaviruses. J Adv Res. 2020 Mar 16; 24: 91 – 98. https://doi.org/10.1016/j.jare.2020.03.005.
Rothan HA and Byrareddy SN. The Epidemiology and Pathogenesis of Coronavirus Disease (COVID-19) Outbreak. J Autoimmun. 2020 May; 109: 102433. https://doi.org/10.1016/j.jaut.2020.102433.
World Health Organization (WHO). COVID-19 Weekly Epidemiological Update. 22 June 2022. Available at: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports.
World Health Organization (WHO). A Timeline of WHO’s Responseto COVID-19 in The WHO European Region: A Living Document (Update to Version 2.0 from 31 December 2019 to 31 July 2021). Availbale at: https://www.who.int/europe/publications/i/item/WHO-EURO-2021-1772-41523-60739.
Cheng MP, Papenburg J, Desjardins M, Kanjilal S, Quach C, Libman M, et al. Diagnostic Testing for Severe Acute Respiratory Syndrome–Related Coronavirus-2. Ann Intern Med. 2020 Apr 13: M20 – 1301. https://doi.org/10.7326%2FM20-1301.
Oliveira BA, de Oliveira LC, Sabino EC, Okay TS. SARS-CoV-2 and The COVID-19 Disease: A Mini Review on Diagnostic Methods. Rev Inst Med Trop Sao Paulo. 2020; 62: e44. https://doi.org/10.1590/s1678-9946202062044.
Gupta N. DNA Extraction and Polymerase Chain Reaction. J Cytol. 2019 Apr-Jun; 36(2): 116-117. https://doi.org/10.4103%2FJOC.JOC_110_18.
Nolan T, Hands RE, Bustin SA. Quantification of mRNA Using Real-Time RT-PCR
Pearson JD, Trcka D, Lu S, Hyduk SJ, Jen M, Aynaud M-M, et al. Comparison of SARS-CoV-2 Indirect and Direct RT-qPCR Detection Methods. Virol J. 2021; 18: 99. https://doi.org/10.1186/s12985-021-01574-4.
Tan SC, Yiap BC. DNA, RNA, and Protein Extraction: The Past and The Present. J Biomed Biotechnol. 2009; 2009: 574398. https://doi.org/10.1155%2F2009%2F574398.
Wozniak A, Cerda A, Henríquez C-I, Sebastian V, Armijo G, Lamig L, et al. A Simple RNA Preparation Method for SARS-CoV-2 Detection by RT-qPCR. Sci Rep. 2020. 10: 16608. https://doi.org/10.1038/s41598-020-73616-w.
Klein S, Müller TG, Khalid D, Sonntag-Buck V, Heuser A-M, Glass B, et al. SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP. Viruses. 2020 Aug; 12(8): 863. https://doi.org/10.3390%2Fv12080863.
Abdallah NMA, Zaki AM, Abdel-Salam SA. Stability of MERS-CoV RNA on Spin Columns of RNA Extraction Kit at Room Temperature. Diagn Microbiol Infect Dis. 2020 Dec; 98(4): 115182. https://doi.org/10.1016%2Fj.diagmicrobio.2020.115182.
CDC. 2019-Novel Coronavirus (2019-nCoV) Real Time RT-PCR Diagnostic Panel. Centers for Disease Control and Prevention. Division of Viral Diseases. pp. 49-52.
Dashti AA, Jadaon MM, Abdulsamad AM, Dashti HM. Heat Treatment of Bacteria: A Simple Method of DNA Extraction for Molecular Techniques. Kuwait Medical Journal. 2009; 41 (2): 117-122. Available at: http://applications.emro.who.int/imemrf/kmj_2009_41_2_117.pdf.
Song F, Kuehl JV, Chandran A, Arkin AP. A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis. mSystems 6: e00224-21. https://doi.org/10.1128/mSystems.00224-21.
Dimitrakopoulou M-E, Stavrou V, Kotsalou C, Vantarakis A. Boiling Extraction Method VS Commercial Kits for Bacterial DNA Isolation from Food Samples. J Food Sci Nutr Res. 2020; 3(4): 311-319. https://www.doi.org/10.26502/jfsnr.2642-11000057.
Fenner F, Bachmann PA, Gibbs EPJ, Murphy FA, Studdert MJ, White DO. Structure and Composition of Viruses. Veterinary Virology. 1987: 3-19. https://doi.org/10.1016%2FB978-0-12-253055-5.50005-0.
Abraham JP, Plourde BD, Cheng L. Using Heat to Kill SARS‐CoV‐2. Rev Med Virol. 2020 Sep; 30(5): e2115. https://doi.org/10.1002%2Frmv.2115.
Batéjat C, Grassin Q, Manuguerra J-C, Leclercq I. Heat Inactivation of The Severe Acute Respiratory Syndrome Coronavirus 2. Journal of Biosafety and Biosecurity. 2021 June; 3(1): 1-3. https://doi.org/10.1016/j.jobb.2020.12.001.
Astuti I, Ysrafil. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An Overview of Viral Structure and Host Response. Diabetes Metab Syndr. 2020 July-August; 14(4): 407-412. https://doi.org/10.1016%2Fj.dsx.2020.04.020.
Wang M-Y, Zhao R, Gao L-J, Gao X-F, Wang D-P, Cao J-M. SARS-CoV-2: Structure, Biology, and Structure-Based Therapeutics Development. Front Cell Infect Microbiol. 2020; 10: 587269. https://doi.org/10.3389%2Ffcimb.2020.587269.
Fordyce SL, Kampmann M-L, van Doorn NL, Gilbert MTP. Review: Long-Term RNA Persistence in Postmortem Contexts. Investig Genet. 2013 Apr 23; 4(1): 7. https://doi.org/10.1186/2041-2223-4-7.
Zhang K, Hodge J, Chatterjee A, Moon TS, Parker KM. Duplex Structure of Double-Stranded RNA Provides Stability against Hydrolysis Relative to Single-Stranded RNA. Environ. Sci. Technol. 2021; 55: 8045-8053. https://doi.org/10.1021/acs.est.1c01255.
Houseley J, Tollervey D. The Many Pathways of RNA Degradation. 2009 February 20; 136(4): 763-776. https://doi.org/10.1016/j.cell.2009.01.019.
Chu L-Y, Hsieh T-J, Golzarroshan B, Chen Y-P, Agrawal S, Yuan HS. Structural Insights into RNA Unwinding and Degradation by RNase R. Nucleic Acids Res. 2017 Nov 16; 45(20): 12015-12024. https://doi.org/10.1093%2Fnar%2Fgkx880.
Vincent HA, Deutscher MP. Insights Into How RNase R Degrades Structured RNA: Analysis of the Nuclease Domain. J Mol Biol. 2009 Apr 3; 387(3): 570-583. https://doi.org/10.1016%2Fj.jmb.2009.01.068.
Tan SC, Yiap BC. DNA, RNA, and Protein Extraction: The Past and The Present. J Biomed Biotechnol. 2009; 2009: 574398. https://doi.org/10.1155%2F2009%2F574398.
Shin JH. Nucleic Acid Extraction Techniques. Advanced Techniques in Diagnostic Microbiology. 2012 Apr 5: 209-225. https://doi.org/10.1007%2F978-1-4614-3970-7_11.
Weng CL, Yazid H, Appalasamy S, Geng BJ, Nasir WMNWM, Muhammad NMN, et al. Optimization of Binding, Washing and Elution Buffer for Development of DNA Isolation Kit. IOP Conf. Ser.: Earth Environ. Sci. 2020; 596: 012008.
Li Y, Chen S, Liu N, Ma L, Wang T, Veedu RN, et al. A Systematic Investigation of Key Factors of Nucleic Acid Precipitation Toward Optimized DNA/RNA Isolation. 2020; 68(4): 191-199. https://doi.org/10.2144/btn-2019-0109.
Jue E, Witters D, Ismagilov RF. Two-Phase Wash to Solve the Ubiquitous Contaminant-Carryover Problem in Commercial Nucleic-Acid Extraction Kits. Sci Rep. 2020; 10. https://doi.org/10.1038/s41598-020-58586-3.
Singanayagam A, Patel M, Charlett A, Bernal JL, Saliba V, Ellis J, et al. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Euro Surveill. 2020 Aug 13; 25(32): 2001483. https://doi.org/10.2807%2F1560-7917.ES.2020.25.32.2001483.
Rabaan AA, Tirupathi R, Sule AA, Aldali J, Al Mutair A, Alhumaid S, et al. Viral Dynamics and Real-Time RT-PCR Ct Values Correlation with Disease Severity in COVID-19. Diagnostics (Basel). 2021 Jun 15; 11(6): 1091. https://doi.org/10.3390/diagnostics11061091.
AlBahrani S, Alghamdi M, Zakary N, Jebakumar AZ, AlZahrani SJ, ElGezery MH, et al. Initial Viral Cycle Threshold Values in Patients with COVID-19 and their Clinical Significance. Eur J Med Res. 2022: 27(101): 1-9. https://doi.org/10.1186/s40001-022-00729-5.
DOI: http://dx.doi.org/10.30872/j.kes.pasmi.kal.v7i2.15797
Refbacks
- There are currently no refbacks.
Indexing by :
