Development and validation of a rapid five-minute nucleic acid extraction method for respiratory viruses.

Publisher: BioMed Central Country of Publication: England NLM ID: 101231645 Publication Model: Electronic Cited Medium: Internet ISSN: 1743-422X (Electronic) Linking ISSN: 1743422X NLM ISO Abbreviation: Virol J Subsets: MEDLINE

Original Publication: [London] : BioMed Central, 2004-

RNA, Viral*/isolation & purification ; RNA, Viral*/genetics ; RNA, Viral*/analysis ; Real-Time Polymerase Chain Reaction*/methods; Humans ; Influenza A virus/isolation & purification ; Influenza A virus/genetics ; Respiratory Tract Infections/virology ; Respiratory Tract Infections/diagnosis ; Specimen Handling/methods ; Time Factors ; Viruses/isolation & purification ; Viruses/genetics ; Influenza, Human/diagnosis ; Influenza, Human/virology ; Molecular Diagnostic Techniques/methods

Background: The rapid transmission and high pathogenicity of respiratory viruses significantly impact the health of both children and adults. Extracting and detecting their nucleic acid is crucial for disease prevention and treatment strategies. However, current extraction methods are laborious and time-consuming and show significant variations in nucleic acid content and purity among different kits, affecting detection sensitivity and efficiency. Our aim is to develop a novel method that reduces extraction time, simplifies operational steps, and ensures high-quality acquisition of respiratory viral nucleic acid.
Methods: We extracted respiratory syncytial virus (RSV) nucleic acid using reagents with different components and analyzed cycle threshold (Ct) values via quantitative real-time polymerase chain reaction (qRT-PCR) to optimize and validate the novel lysis and washing solution. The performance of this method was compared against magnetic bead, spin column, and precipitation methods for extracting nucleic acid from various respiratory viruses. The clinical utility of this method was confirmed by comparing it to the standard magnetic bead method for extracting clinical specimens of influenza A virus (IAV).
Results: The solution, composed of equal parts glycerin and ethanol (50% each), offers an innovative washing approach that achieved comparable efficacy to conventional methods in a single abbreviated cycle. When combined with our A Plus lysis solution, our novel five-minute nucleic acid extraction (FME) method for respiratory viruses yielded superior RNA concentrations and purity compared to traditional methods. FME, when used with a universal automatic nucleic acid extractor, demonstrated similar efficiency as various conventional methods in analyzing diverse concentrations of respiratory viruses. In detecting respiratory specimens from 525 patients suspected of IAV infection, the FME method showed an equivalent detection rate to the standard magnetic bead method, with a total coincidence rate of 95.43% and a kappa statistic of 0.901 (P < 0.001).
Conclusions: The FME developed in this study enables the rapid and efficient extraction of nucleic acid from respiratory samples, laying a crucial foundation for the implementation of expedited molecular diagnosis.
(© 2024. The Author(s).)

J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Aug 5;822(1-2):54-60. (PMID: 15982935)
Expert Rev Anti Infect Ther. 2011 Aug;9(8):615-26. (PMID: 21819328)
Glob Health Res Policy. 2023 Feb 1;8(1):2. (PMID: 36726143)
Front Cell Dev Biol. 2020 Jun 04;8:468. (PMID: 32582718)
Front Med (Lausanne). 2020 Sep 18;7:549860. (PMID: 33043036)
Int J Toxicol. 2019 Nov/Dec;38(3_suppl):6S-22S. (PMID: 31840548)
J Virol Methods. 2021 Dec;298:114281. (PMID: 34530011)
Emerg Infect Dis. 2020 Jul;26(7):1478-1488. (PMID: 32267220)
ACS Cent Sci. 2020 May 27;6(5):591-605. (PMID: 32382657)
Drug Discov Today. 2012 Nov;17(21-22):1199-207. (PMID: 22819926)
Nature. 2020 Mar;579(7798):270-273. (PMID: 32015507)
Clin Chem. 2015 Jan;61(1):89-99. (PMID: 25451869)
Analyst. 2019 Nov 18;144(23):7032-7040. (PMID: 31651914)
Microbiol Spectr. 2021 Dec 22;9(3):e0066821. (PMID: 34756088)
J Biomed Biotechnol. 2009;2009:574398. (PMID: 20011662)
Nat Commun. 2020 Sep 23;11(1):4812. (PMID: 32968075)
Clin Microbiol Rev. 2021 May 12;34(3):. (PMID: 33980687)
PLoS Biol. 2020 Oct 2;18(10):e3000896. (PMID: 33006983)
Biotechnol Adv. 2020 Jul - Aug;41:107537. (PMID: 32199980)
Anal Chem. 2016 Oct 4;88(19):9403-9411. (PMID: 27264110)
Emerg Infect Dis. 2006 Jan;12(1):15-22. (PMID: 16494711)
Appl Health Econ Health Policy. 2022 May;20(3):395-404. (PMID: 35001292)
J Biotechnol. 2002 Apr 11;94(3):217-24. (PMID: 11861080)
Expert Rev Anti Infect Ther. 2010 Nov;8(11):1273-92. (PMID: 21073292)
J Sep Sci. 2022 Jan;45(1):172-184. (PMID: 34453482)
J Innate Immun. 2020;12(1):4-20. (PMID: 31610541)
Appl Microbiol Biotechnol. 2006 Dec;73(3):495-504. (PMID: 17063328)
J Med Virol. 2020 Sep;92(9):1518-1524. (PMID: 32104917)
J Clin Microbiol. 1990 Mar;28(3):495-503. (PMID: 1691208)
Fam Med. 2005 May;37(5):360-3. (PMID: 15883903)
PLoS One. 2019 Apr 18;14(4):e0215753. (PMID: 30998749)
J Med Virol. 2020 Oct;92(10):2096-2104. (PMID: 32383254)
Clin Chim Acta. 2021 May;516:1-7. (PMID: 33485902)
Nucleic Acids Res. 1994 Oct 25;22(21):4543-4. (PMID: 7971285)
Biotechniques. 2021 Oct;71(4):510-515. (PMID: 34528832)
BMC Biotechnol. 2014 Nov 14;14:94. (PMID: 25394494)
J Clin Virol. 2020 Sep;130:104579. (PMID: 32795959)
PLoS Biol. 2017 Nov 21;15(11):e2003916. (PMID: 29161268)
Biomed Res Int. 2017;2017:9306564. (PMID: 28785592)
Nucleic Acids Res. 2007;35(10):e74. (PMID: 17478499)
Biotechniques. 2020 Mar;68(3):159-162. (PMID: 31870171)
ACS Nano. 2020 Apr 28;14(4):3822-3835. (PMID: 32223179)
J Virol Methods. 2021 Jan;287:114008. (PMID: 33160015)
Diagnostics (Basel). 2020 Jun 11;10(6):. (PMID: 32545412)
J Clin Virol. 2020 Jul;128:104433. (PMID: 32405254)

2022B1111020003 Research and Development Project in the Key Areas of Guangdong Province; 2022B1111020003 Research and Development Project in the Key Areas of Guangdong Province; EKPG21-13 Emergency Key Program of Guangzhou Laboratory

Keywords: Five-minute nucleic acid extraction; Glycerin and ethanol; Respiratory viruses

0 (RNA, Viral)

Date Created: 20240818 Date Completed: 20240818 Latest Revision: 20240821

20240821

PMC11331601

10.1186/s12985-024-02381-3

39155366