Skip to main content
SpringerLink
Log in

Automated on-chip analysis of tuberculosis drug-resistance mutation with integrated DNA ligation and amplification

  • Communication
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Detection of a single base mutation in Mycobacterium tuberculosis DNA can provide fast and highly specific diagnosis of antibiotic-resistant tuberculosis. Mutation-specific ligation of padlock probes (PLPs) on the target followed by rolling circle amplification (RCA) is highly specific, but challenging to integrate in a simple microfluidic device due to the low temperature stability of the phi29 polymerase and the interference of phi29 with the PLP annealing and ligation. Here, we utilized the higher operation temperature and temperature stability of Equiphi29 polymerase to simplify the integration of the PLP ligation and RCA steps of an RCA assay in two different strategies performed at uniform temperature. In strategy I, PLP annealing took place off-chip and the PLP ligation and RCA were performed in one pot and the two reactions were clocked by a change of the temperature. For a total assay time of about 1.5 h, we obtained a limit of detection of 2 pM. In strategy II, the DNA ligation mixture and the RCA mixture were separated into two chambers on a microfluidic disc. After on-disc PLP annealing and ligation, the disc was spun to mix reagents and initiate RCA. For a total assay time of about 2 h, we obtained a limit of detection of 5 pM.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

References

  1. World Health Organization. Global tuberculosis report 2018 - Geneva. WHO Rep; 2018

  2. Chen CY, Weng JY, Huang HH, Yen WC, Tsai YH, Cheng TC, et al. A new oligonucleotide array for the detection of multidrug and extensively drug-resistance tuberculosis. Sci Rep. 2019;9:4425.

    Article  CAS  Google Scholar 

  3. Nguyen TNA, Le Berre VA, Bañuls AL, Nguyen TVA. Molecular diagnosis of drug-resistant tuberculosis; a literature review. Front Microbiol. 2019;10:794.

    Article  Google Scholar 

  4. Brossier F, Guindo D, Pham A, Reibel F, Sougakoff W, Veziris N, et al. Performance of the new version (v2.0) of the GenoType MTBDRsl test for detection of resistance to second-line drugs in multidrug-resistant mycobacterium tuberculosis complex strains. J Clin Microbiol. 2016;54:1573–80.

    Article  CAS  Google Scholar 

  5. Barreda-García S, Miranda-Castro R, De-los-Santos-Álvarez N, Miranda-Ordieres AJ, Lobo-Castañón MJ. Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay. Anal Bioanal Chem. 2016;408:8603–10.

    Article  CAS  Google Scholar 

  6. Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary BP, Landegren U. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science. 1994;265:2085–8.

    Article  CAS  Google Scholar 

  7. Banér J, Nilsson M, Mendel-Hartvig M, Landegren U. Signal amplification of padlock probes by rolling circle replication. Nucleic Acids Res. 1998;26:5073–8.

    Article  Google Scholar 

  8. Pavankumar AR, Engström A, Liu J, Herthnek D, Nilsson M. Proficient detection of multi-drug-resistant Mycobacterium tuberculosis by padlock probes and lateral flow nucleic acid biosensors. Anal Chem. 2016;88:4277–84.

    Article  CAS  Google Scholar 

  9. Hu B, Guo J, Xu Y, Wei H, Zhao G, Guan Y. A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology. Anal Bioanal Chem. 2017;409:4819–25.

    Article  CAS  Google Scholar 

  10. Mezger A, Fock J, Antunes P, Østerberg FW, Boisen A, Nilsson M, et al. Scalable DNA-based magnetic nanoparticle agglutination assay for bacterial detection in patient samples. ACS Nano. 2015;9:7374–82.

    Article  CAS  Google Scholar 

  11. Tian B, Fock J, Minero GAS, Garbarino F, Hansen MF. Ultrasensitive real-time rolling circle amplification detection enhanced by nicking-induced tandem-acting polymerases. Anal Chem. 2019;91:10102–9.

    Article  CAS  Google Scholar 

  12. Lin C, Zhang Y, Zhou X, Yao B, Fang Q. Naked-eye detection of nucleic acids through rolling circle amplification and magnetic particle mediated aggregation. Biosens Bioelectron. 2013;47:515–9.

    Article  CAS  Google Scholar 

  13. Zhang S, Wu Z, Shen G, Yu R. A label-free strategy for SNP detection with high fidelity and sensitivity based on ligation-rolling circle amplification and intercalating of methylene blue. Biosens Bioelectron. 2009;24:3201–7.

    Article  CAS  Google Scholar 

  14. Pashchenko O, Shelby T, Banerjee T, Santra S. A comparison of optical, electrochemical, magnetic, and colorimetric point-of-care biosensors for infectious disease diagnosis. ACS Infect Dis. 2018;4:1162–78.

    Article  CAS  Google Scholar 

  15. Zhou H, Liu J, Xu JJ, Zhang SS, Chen HY. Optical nano-biosensing interface: via nucleic acid amplification strategy: construction and application. Chem Soc Rev. 2018;47:1996–2019.

    Article  CAS  Google Scholar 

  16. Ma Q, Gao Z. A simple and ultrasensitive fluorescence assay for single-nucleotide polymorphism. Anal Bioanal Chem. 2018;410:3093–100.

    Article  CAS  Google Scholar 

  17. Moyano A, Salvador M, Martínez-García JC, Socoliuc V, Vékás L, Peddis D, et al. Magnetic immunochromatographic test for histamine detection in wine. Anal Bioanal Chem. 2019;411:6615–24.

    Article  CAS  Google Scholar 

  18. Fock J, Jonasson C, Johansson C, Hansen MF. Characterization of fine particles using optomagnetic measurements. Phys Chem Chem Phys. 2017;19:8802–14.

    Article  CAS  Google Scholar 

  19. Minero GAS, Cangiano V, Garbarino F, Fock J, Hansen MF. Integration of microbead DNA handling with optomagnetic detection in rolling circle amplification assays. Microchim Acta. 2019;186:528.

    Article  CAS  Google Scholar 

  20. Garbarino F, Minero GAS, Rizzi G, Fock J, Hansen MF. Integration of rolling circle amplification and optomagnetic detection on a polymer chip. Biosens Bioelectron. 2019;142:111485.

    Article  CAS  Google Scholar 

  21. Stepanauskas R, Fergusson EA, Brown J, et al. Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles. Nat Commun. 2017;8:84.

    Article  CAS  Google Scholar 

  22. Engström A, Morcillo N, Imperiale B, Hoffner SE, Juréen P. Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing. J Clin Microbiol. 2012;50:2026–33.

    Article  CAS  Google Scholar 

  23. Minero GAS, Nogueira C, Rizzi G, Tian B, Fock J, Donolato M, et al. Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA. Analyst. 2017;142:3441–50.

    Article  CAS  Google Scholar 

  24. Minero GAS, Cangiano V, Fock J, Garbarino F, Hansen MF. Optomagnetic detection of rolling circle amplification products. In: Nucleic acid detect. Struct Investig; 2020. pp 3–15.

  25. Bejhed RS, Zardán Gómez De La Torre T, Donolato M, Hansen MF, Svedlindh P, Strömberg M. Turn-on optomagnetic bacterial DNA sequence detection using volume-amplified magnetic nanobeads. Biosens Bioelectron. 2015;66:405–11.

    Article  CAS  Google Scholar 

  26. Zhou W, Chen Q, Huang PJJ, Ding J, Liu J. DNAzyme hybridization, cleavage, degradation, and sensing in undiluted human blood serum. Anal Chem. 2015;87:4001–7.

    Article  CAS  Google Scholar 

  27. Gao Z, Wu C, Lv S, et al. Nicking-enhanced rolling circle amplification for sensitive fluorescent detection of cancer-related microRNAs. Anal Bioanal Chem. 2018;410:6819–26.

    Article  CAS  Google Scholar 

  28. Hernández-Neuta I, Pereiro I, Ahlford A, Ferraro D, Zhang Q, Viovy JL, et al. Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode. Biosens Bioelectron. 2018;102:531–9.

    Article  CAS  Google Scholar 

  29. Strömberg M, Göransson J, Gunnarsson K, Nilsson M, Svedlindh P, Strømme M. Sensitive molecular diagnostics using volume-amplified magnetic nanobeads. Nano Lett. 2008;8:816–21.

    Article  CAS  Google Scholar 

  30. Donolato M, Antunes P, Bejhed RS, et al. Novel readout method for molecular diagnostic assays based on optical measurements of magnetic nanobead dynamics. Anal Chem. 2015;87:1622–9.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

MB thanks Erasmus+ Program (key action 1 A. Y. 2019) and Roberto Raiteri for sponsoring of the Erasmus program. We thank Prof. Mats Nilsson for discussions and for directing our attention to Equiphi29 polymerase.

Funding

The work was supported by DFF project (#4184-00121B). JF received support from MUDP (MST-141-01415).

Author information

Author notes

  1. Gabriel Antonio S. Minero and Martina Bagnasco contributed equally to this work.

Authors and Affiliations

  1. Department of Health Technology, DTU Health Tech, Technical University of Denmark, Building 345C, 2800, Kongens Lyngby, Denmark

    Gabriel Antonio S. Minero, Martina Bagnasco, Bo Tian, Francesca Garbarino & Mikkel F. Hansen

  2. BluSense Diagnostics ApS, Fruebjergvej 3, DK-2100, Copenhagen, Denmark

    Jeppe Fock

Authors
  1. Gabriel Antonio S. Minero
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martina Bagnasco
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeppe Fock
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bo Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Francesca Garbarino
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mikkel F. Hansen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Gabriel Antonio S. Minero or Mikkel F. Hansen.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 751 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Minero, G.A.S., Bagnasco, M., Fock, J. et al. Automated on-chip analysis of tuberculosis drug-resistance mutation with integrated DNA ligation and amplification. Anal Bioanal Chem 412, 2705–2710 (2020). https://doi.org/10.1007/s00216-020-02568-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-020-02568-x

Keywords

Search

Navigation