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Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay


Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices.

Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis

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  1. World Health Organization. Global tuberculosis report 2015 ( Last accessed 15 February 2016.

  2. Dheda K, Ruhwald M, Theron G, Peter J, Yam WC. Respirology. 2013;18:217–32.

    Article  Google Scholar 

  3. Wilson ML. Clin Infect Dis. 2011;52:1350–5.

    Article  Google Scholar 

  4. Piersimoni C, Scarparo C. J Clin Microbiol. 2003;41:5355–65.

    Article  CAS  Google Scholar 

  5. Ioannidis P, Papaventsis D, Karabela S, Nikolaou S, Panagi M, Raftopoulou E, et al. J Clin Microbiol. 2011;49:3068–70.

    Article  Google Scholar 

  6. Asiello PJ, Baeumner AJ. Lab Chip. 2011;11:1420–30.

    Article  CAS  Google Scholar 

  7. Niemz A, Ferguson TM, Boyle DS. Trends Biotechnol. 2011;29:240–50.

    Article  CAS  Google Scholar 

  8. Li J, Macdonald J. Biosens Bioelectron. 2015;64:196–211.

    Article  CAS  Google Scholar 

  9. Zhao Y, Chen F, Li Q, Wang L, Fan C. Chem Rev. 2015;115:12491–545.

    Article  CAS  Google Scholar 

  10. Aryan E, Makvandi M, Farajzadeh A, Huygen K, Bifani P, Mousavi S-L, et al. Microbiol Res. 2010;165:211–20.

    Article  CAS  Google Scholar 

  11. Manage DP, Chui L, Pilarski L. Microfluid Nanofluid. 2013;14:731–41.

    Article  CAS  Google Scholar 

  12. Roskos K, Hickerson AI, Lu H-W, Ferguson TM, Shinde DN, Klaue Y, et al. PLoS One. 2013;8, e69355.

    Article  CAS  Google Scholar 

  13. Boyle DS, McNerney R, Low HT, Leader BT, Pérez-Osorio AC, Meyer JC, et al. PLoS One. 2014;9:e103091.

    Article  Google Scholar 

  14. Ng BYC, Xiao W, West NP, Wee EJH, Wang Y, Trau M. Anal Chem. 2015;87:10613–8.

    Article  CAS  Google Scholar 

  15. Ng BYC, Wee EJH, West NP, Trau M. Sci Rep. 2015;5:15027.

    Article  CAS  Google Scholar 

  16. Shin Y, Perera AP, Tang WY, Fu DL, Liu Q, Sheng JK, et al. Biosens Bioelectron. 2015;68:30–396.

    Google Scholar 

  17. Ng BYC, Wee EJH, West NP, Trau M (2015) ACS sens DOI: 10.1021/acssensors.5b00171

  18. Motré A, Kong R, Li Y. J Microbiol Meth. 2011;84:343–5.

    Article  Google Scholar 

  19. Torres-Chavolla E, Alocilja E. Biosens Bioelectron. 2011;26:4614–8.

    Article  CAS  Google Scholar 

  20. Ao W, Aldous S, Woodruff E, Hicke B, Rea L, Kreiswirth B, et al. J Clin Microbiol. 2012;50:2433–40.

    Article  CAS  Google Scholar 

  21. Barreda-García S, González-Álvarez MJ, de-los-Santos-Álvarez N, Palacios-Gutiérrez JJ, Miranda-Ordieres AJ, Lobo-Castañón MJ. Biosens Bioelectron. 2015;68:122–8.

    Article  Google Scholar 

  22. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Nucleic Acids Res. 2000;28:e63.

    Article  CAS  Google Scholar 

  23. Vincent M, Xu Y, Kong H. EMBO Rep. 2004;5:795–800.

    Article  CAS  Google Scholar 

  24. Poepenburg O, Williams C, Stemple DL, Armes NA. PLoS Biol. 2006;4:1115–21.

    Google Scholar 

  25. Patterson AS, Hsieh K, Soh HT, Plaxco KW. Trends Biotech. 2013;31:704–12.

    Article  CAS  Google Scholar 

  26. Moura-Melo S, Miranda-Castro R, de-los-Santos Álvarez N, Miranda-Ordieres AJ, Ribeiro J, Fonseca R, et al. Anal Chem. 2015;87:8547–54.

    Article  CAS  Google Scholar 

  27. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Nucleic Acid Res. 2012;40:e115.

    Article  CAS  Google Scholar 

  28. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T. BMC Bioinformatics. 2012;13:134.

    Article  CAS  Google Scholar 

  29. Zuker M. Nucleic Acid Res. 2003;31:3406–15.

    Article  CAS  Google Scholar 

  30. Jezewska MJ, Lucius AL, Bujalowski W. Biochem. 2005;44:3877–90.

    Article  CAS  Google Scholar 

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This research has been supported by the Spanish Ministerio de Economía y Competitividad (project CTQ2012-31157), the European Regional Development Fund, and Principado de Asturias government (FC-15-GRUPIN14-025). RMC thanks Principado de Asturias government and FICYT for a Clarín post-doctoral contract. We thank Dr. Juan José Palacios-Gutiérrez from Hospital Universitario de Asturias for providing the clinical samples.

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Correspondence to M. Jesús Lobo-Castañón.

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Published in the topical collection Isothermal Nucleic Acid Amplification in Bioanalysis with guest editor Maria Jesus Lobo Castañón.

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Barreda-García, S., Miranda-Castro, R., de-los-Santos-Álvarez, N. et al. Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay. Anal Bioanal Chem 408, 8603–8610 (2016).

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  • Genomagnetic assay
  • Helicase
  • Isothermal amplification
  • PCR
  • Mycobacterium tuberculosis