Comparison of Automated and Manual Viral Nucleic Acid Extraction Kits for Covid-19 Detection Using qRT-PCR
Main Article Content
Abstract
Introduction: The emergence of a novel Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted
in a pandemic. Rapid and accurate diagnosis method is crucial to reduce the disease burden and to improve early
diagnosis approaches to control of the disease. Real time Reverse transcriptase PCR (qRT-PCR) has been identified by
the World Health Organization as the most sensitive and specific method of detection. However, the success of this
assay relies on the quantity and quality of the extracted viral RNA. Methods: Various methods have been developed
for nucleic acid extraction however, the methods have not been assessed. RNA extraction was performed from 24
nasopharyngeal swab samples using a manual extraction kit (GF-1) and an automated extraction kit (Genolution).
The concentration and purity of the extracted RNA samples were measured, and its performance were tested using
qRT-PCR. Results: The average concentration and purity of the RNA samples extracted using GF-1 kit was higher
compared to Genolution. Similarly, the qRT-PCR assay using the RNA samples extracted using manual extraction
was better compared to automated kit. Conclusion: Both the manual and automated extraction kits have its advantages and disadvantages in terms of yield and purity. However, with proper optimization, both methods may be used
for routine molecular diagnostic of COVID-19 in laboratories.
Downloads
Article Details
References
Gorbalenya AE, Baker SC, Baric RS, de Groot RJ,
Drosten C, Gulyaeva AA, et al. The species Severe
acute respiratory syndrome-related coronavirus:
classifying 2019-nCoV and naming it SARS-CoV-2.
Nat Microbiol. 2020;5(4):536–44.
World Health Organization. (2020). Novel
Coronavirus (2019-nCoV): situation report, 10.
World Health Organization. https://apps.who.int/
iris/handle/10665/330775
Tahamtan A, Ardebili A. Real-time RT-PCR in
COVID-19 detection: issues affecting the results.
Expert Rev Mol Diagn. 2020;20(5):453–4.
World Health Organization. (2020). Laboratory
testing for coronavirus disease 2019 (COVID-19)
in suspected human cases: interim guidance, 2
March 2020. World Health Organization. https://
apps.who.int/iris/handle/10665/331329.
Carter LJ, Garner LV, Smoot JW, Li Y, Zhou Q,
Saveson CJ, Sasso JM, Gregg AC, Soares DJ, Beskid
TR, Jervey SR, Liu C. Assay Techniques and Test
Development for COVID-19 Diagnosis. ACS Cent
Sci. 2020; 27;6(5):591-605.
Ali N, Rampazzo RDCP, Costa ADiT, Krieger MA.
Current Nucleic Acid Extraction Methods and Their
Implications to Point-of-Care Diagnostics. Biomed
Res Int. 2017;2017:9306564.
de Paula VS, Villar LM, Coimbra Gaspar AM.
Comparison of four extraction methods to detect
hepatitis A virus RNA in serum and stool samples.
Braz J Infect Dis. 2003;7(2):135–41.
Yang G, Erdman DE, Kodani M, Kools J, Bowen
MD, Fields BS. Comparison of commercial
systems for extraction of nucleic acids from DNA/
RNA respiratory pathogens. J Virol Methods.
;171(1):195-199.
Borlido L, Azevedo AM, Roque ACA, Aires-Barros
MR. Magnetic separations in biotechnology.
Biotechnol Adv. 2013;31(8):1374–85.
McGaughey KD, Yilmaz-Swenson T, Elsayed
NM, Cruz DA, Rodriguez RM, Kritzer MD,
et al. Correction: Comparative evaluation of
a new magnetic bead-based DNA extraction
method from fecal samples for downstream nextgeneration 16S rRNA gene sequencing. PLoS ONE.
;13:8 (e0202858). DOI: 10.1371/journal.
pone.0202858). PLoS One. 2019;14(2):1–16.
Kasteren PB Van, Veer B Van Der, Brink S Van
Den, Wijsman L, Jonge J De, Brandt A Van Den,
et al. Comparison of seven commercial RT-PCR
diagnostic kits for COVID-19. J Clin Virol [Internet].
;128:104412.
Oberacker P, Stepper P, Bond DM, et al. BioOn-Magnetic-Beads (BOMB): Open platform
for high-throughput nucleic acid extraction and
manipulation. PLoS Biol. 2019;17(1):e3000107.
doi:10.1371/journal.pbio.3000107
Mengelle C, Mansuy JM, Da Silva I, Davrinche C,
Izopet J. Comparison of 2 highly automated nucleic
acid extraction systems for quantitation of human
cytomegalovirus in whole blood. Diagn Microbiol
Infect Dis. 2011;69(2):161-166. doi:10.1016/j.
diagmicrobio.2010.08.011
Waller JV, Kaur P, Tucker A, Lin KK, Diaz
MJ, Henry TS, Hope M. Diagnostic Tools for
Coronavirus Disease (COVID-19): Comparing
CT and RT-PCR Viral Nucleic Acid Testing. AJR
Am J Roentgenol. 2020 Oct;215(4):834-838. doi:
2214/AJR.20.23418. Epub 2020 May 15. PMID:
Kalmár A, Péterfia B, Wichmann B, Patai ÁV,
Barták BK, Nagy ZB, Furi I, Tulassay Z, Molnár
B. Comparison of Automated and Manual
DNA Isolation Methods for DNA Methylation
Analysis of Biopsy, Fresh Frozen, and FormalinFixed, Paraffin-Embedded Colorectal Cancer
Samples. J Lab Autom. 2015;20(6):642-51. doi:
1177/2211068214565903. Epub 2015 Jan 9.
PMID: 25576093.
Zora, Piskata., Eliska, Pospisilova., Gabriela,
Borilova. (2017) Comparative study of DNA
extraction methods from fresh and processed
yellowfin tuna muscle tissue, International Journal
of Food Properties. 2017;20. https://doi.org/10.108
/10942912.2017.1297953.
Wilfinger, WW., Mackey, K., Chomczynski, P. Effect
of pH and ionic strength on the spectrophotometric
assessment of nucleic acid purity. Biotechniques.
;22(3):474-481. doi:10.2144/97223st01.
Jue, E., Witters, D. & Ismagilov, R.F. Two-phase
wash to solve the ubiquitous contaminantcarryover problem in commercial nucleic-acid
extraction kits. Sci Rep 10. 2020;1940. https://doi.
org/10.1038/s41598-020-58586-3.
Sathiamoorthy, S., Malott, R.J., Gisonni-Lex, L. et
al. Selection and evaluation of an efficient method
for the recovery of viral nucleic acids from complex
biologicals. npj Vaccines. 2018;3:31. https://doi.
org/10.1038/s41541-018-0067-3