Analytical and Bioanalytical Chemistry

, Volume 408, Issue 3, pp 671–676 | Cite as

Isothermal solid-phase amplification system for detection of Yersinia pestis

  • Olena Mayboroda
  • Angel Gonzalez Benito
  • Jonathan Sabaté del Rio
  • Marketa Svobodova
  • Sandra Julich
  • Herbert Tomaso
  • Ciara K. O’SullivanEmail author
  • Ioanis KatakisEmail author
Rapid Communication


DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin–HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10−13 and 3.14*10−16 M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets.


Recombinase polymerase amplification Yersinia pestis Solid-phase detection ELONA 



This work has been carried out with partial financial support from the Commission of the European Communities specific RTD programme (FP7-2010-SEC-261810) and the Ministerio de Economia y Competitividad, Ref. BIO2014-56024-C2-1-R. OM thanks the Generalitat de Catalunya for a FI pre-doctoral scholarship.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


  1. 1.
    Butler T (2014) Plague history: Yersin’s discovery of the causative bacterium in 1894 enabled, in the subsequent century, scientific progress in understanding the disease and the development of treatments and vaccines. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis 20(3):202–209. doi: 10.1111/1469-0691.12540 Google Scholar
  2. 2.
    Inglesby TV, Dennis DT, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, Friedlander AM, Hauer J, Koerner JF, Layton M, McDade J, Osterholm MT, O’Toole T, Parker G, Perl TM, Russell PK, Schoch-Spana M, Tonat K (2000) Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA 283(17):2281–2290CrossRefGoogle Scholar
  3. 3.
    Leal NC, Almeida AM (1999) Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR. Rev Inst Med Trop Sao Paulo 41(6):339–342CrossRefGoogle Scholar
  4. 4.
    Engelthaler DM, Gage KL, Montenieri JA, Chu M, Carter LG (1999) PCR detection of Yersinia pestis in fleas: comparison with mouse inoculation. J Clin Microbiol 37(6):1980–1984Google Scholar
  5. 5.
    Campbell J, Lowe J, Walz S, Ezzell J (1993) Rapid and specific identification of Yersinia pestis by using a nested polymerase chain reaction procedure. J Clin Microbiol 31(3):758–759Google Scholar
  6. 6.
    Tomaso H, Reisinger EC, Al Dahouk S, Frangoulidis D, Rakin A, Landt O, Neubauer H (2003) Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes. FEMS Immunol Med Microbiol 38(2):117–126CrossRefGoogle Scholar
  7. 7.
    Loiez C, Herwegh S, Wallet F, Armand S, Guinet F, Courcol RJ (2003) Detection of Yersinia pestis in sputum by real-time PCR. J Clin Microbiol 41(10):4873–4875CrossRefGoogle Scholar
  8. 8.
    Matero P, Pasanen T, Laukkanen R, Tissari P, Tarkka E, Vaara M, Skurnik M (2009) Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis. APMIS Acta Pathol Microbiol Immunol Scand 117(1):34–44. doi: 10.1111/j.1600-0463.2008.00013.x CrossRefGoogle Scholar
  9. 9.
    Yan Z, Zhou L, Zhao Y, Wang J, Huang L, Hu K, Liu H, Wang H, Guo Z, Song Y, Huang H, Yang R (2006) Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor. Sensors Actuators B Chem 119(2):656–663. doi: 10.1016/j.snb.2006.01.029 CrossRefGoogle Scholar
  10. 10.
    Amoako KK, Shields MJ, Goji N, Paquet C, Thomas MC, Janzen TW, Bin Kingombe CI, Kell AJ, Hahn KR (2012) Rapid detection and identification of Yersinia pestis from food using immunomagnetic separation and pyrosequencing. J Pathog 2012:781652. doi: 10.1155/2012/781652 Google Scholar
  11. 11.
    Jeon JW, Kim JH, Lee JM, Lee WH, Lee DY, Paek SH (2014) Rapid immuno-analytical system physically integrated with lens-free CMOS image sensor for food-borne pathogens. Biosens Bioelectron 52:384–390. doi: 10.1016/j.bios.2013.09.004 CrossRefGoogle Scholar
  12. 12.
    Zasada AA, Forminska K, Zacharczuk K, Jacob D, Grunow R (2015) Comparison of eleven commercially available rapid tests for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis. Lett Appl Microbiol 60(5):409–413. doi: 10.1111/lam.12392 CrossRefGoogle Scholar
  13. 13.
    Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA detection using recombination proteins. PLoS Biol 4(7):e204. doi: 10.1371/journal.pbio.0040204 CrossRefGoogle Scholar
  14. 14.
    Euler M, Wang Y, Heidenreich D, Patel P, Strohmeier O, Hakenberg S, Niedrig M, Hufert FT, Weidmann M (2013) Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents. J Clin Microbiol 51(4):1110–1117. doi: 10.1128/jcm.02704-12 CrossRefGoogle Scholar
  15. 15.
    Kersting S, Rausch V, Bier FF, von Nickisch-Rosenegk M (2014) Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens. Mikrochimica Acta 181(13–14):1715–1723. doi: 10.1007/s00604-014-1198-5 CrossRefGoogle Scholar
  16. 16.
    Santiago-Felipe S, Tortajada-Genaro LA, Morais S, Puchades R, Maquieira A (2015) Isothermal DNA amplification strategies for duplex microorganism detection. Food Chem 174:509–515. doi: 10.1016/j.foodchem.2014.11.080 CrossRefGoogle Scholar
  17. 17.
    Simon S, Demeure C, Lamourette P, Filali S, Plaisance M, Creminon C, Volland H, Carniel E (2013) Fast and simple detection of Yersinia pestis applicable to field investigation of plague foci. PLoS One 8(1):e54947. doi: 10.1371/journal.pone.0054947 CrossRefGoogle Scholar
  18. 18.
    Sabate Del Rio JS, Yehia Adly N, Acero-Sanchez JL, Henry OY, O’Sullivan CK (2014) Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification. Biosens Bioelectron 54:674–678. doi: 10.1016/j.bios.2013.11.035 CrossRefGoogle Scholar
  19. 19.
    Sabate Del Rio JS, Steylaerts T, Henry OY, Bienstman P, Stakenborg T, Van Roy W, O’Sullivan CK (2015) Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification. Biosens Bioelectron 73:130–137. doi: 10.1016/j.bios.2015.05.063 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Olena Mayboroda
    • 1
  • Angel Gonzalez Benito
    • 1
  • Jonathan Sabaté del Rio
    • 1
  • Marketa Svobodova
    • 1
  • Sandra Julich
    • 3
  • Herbert Tomaso
    • 3
  • Ciara K. O’Sullivan
    • 1
    • 2
    Email author
  • Ioanis Katakis
    • 1
    Email author
  1. 1.Interfibio Research Group, Department of Chemical EngineeringUniversitat Rovira i VirgiliTarragonaSpain
  2. 2.ICREABarcelonaSpain
  3. 3.Friedrich-Loeffler-InstitutJenaGermany

Personalised recommendations