Skip to main content
SpringerLink
Log in

Multiplex PCR for joint amplification of carbapenemase genes of molecular classes A, B, and D

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

Here we present a method for joint amplification of genes of carbapenemases of molecular classes A, B, and D for hybridization analysis on DNA microarrays. Using new-generation DNA polymerase KAPA2G Fast (KAPA Biosystems, USA) together with optimization of the conditions for the multiplex PCR with 20 primer pairs allowed us to carry out joint amplification of full-length genes of seven different types of carbapenemases (KPC, VIM, IMP, SPM, SIM, GIM, and OXA) with simultaneous inclusion of biotin as a label. Yield of the labeled PCR product sufficient for further analysis by microarray hybridization was achieved 40 min after the start of the reaction. This reduced the total duration of DNA identification techniques, including sample preparation stage, to 4 h. The method for gene identification by DNA microarrays with the improved stage of amplification of specific carbapenemase genes was tested with clinical strains of gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae spp. with different sensitivity towards carbapenems according to phenotyping tests. All clinical strains of A. baumannii resistant to carbapenems were found to have genes of OXA-type carbapenemases (subtypes OXA-51, OXA-23, OXA-40, and OXA-58), and clinical strains of P. aeruginosa resistant to carbapenems were found to possess the gene of VIM-type metallo-beta-lactamase (subtype VIM-2). When testing clinical strains sensitive to carbapenems, carbapenemase genes were not detected. Thus, the method of identifying carbapenemase genes on DNA microarrays is characterized by high accuracy and can be used in clinical microbiology laboratories for express diagnostics of resistance to carbapenems.

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

Similar content being viewed by others

References

  1. Livermore, D. M. (2001) J. Antimicrob. Chemother., 47, 247–250.

    Article  CAS  PubMed  Google Scholar 

  2. Ziha-Zarifi, I., Llanes, C., Kohler, T., Pechere, J. C., and Plesiat, P. (1999) Antimicrob. Agents Chemother., 43, 287–291.

    CAS  PubMed Central  PubMed  Google Scholar 

  3. Poole, K. (2004) Cell. Mol. Life Sci., 61, 2200–2223.

    Article  CAS  PubMed  Google Scholar 

  4. Bush, K. (2013) Ann. N. Y. Acad. Sci., 1277, 84–90.

    Article  CAS  PubMed  Google Scholar 

  5. Queenan, A. M., and Bush, K. (2007) Clin. Microbiol. Rev., 20, 440–458.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Miriagou, V., Cornaglia, G., Edelstein, M., Galani, I., Giske, C. G., Gniadkowski, M., Malamou-Lada, E., Martinez-Martinez, L., Navarro, F., Nordmann, P., Peixe, L., Pournaras, S., Rossolini, G. M., Tsakris, A., Vatopoulos, A., and Canton, R. (2010) Clin. Microbiol. Infect., 16, 112–122.

    Article  CAS  PubMed  Google Scholar 

  7. Semina, N. A., Sidorenko, S. V., and Rezvan, S. P. (2004) Klin. Mikrobiol. Antimikrob. Khimioter., 4, 306–359.

    Google Scholar 

  8. Shevchenko, O. V., Edelstein, M. V., and Stepanova, M. N. (2007) Klin. Mikrobiol. Antimikrob. Khimioter., 3, 211–218.

    Google Scholar 

  9. Stuart, C. J., Dierikx, C., Al Naiemi, N., Karczmarek, A., Van Hoek, A. H. A. M., Vos, P., Fluit, A. C., Scharringa, J., Duim, B., Mevius, D., and Leverstein-Van Hall, M. A. (2010) J. Antimicrob. Chemother., 65, 1377–1381.

    Article  Google Scholar 

  10. Peter, H., Berggrav, K., Thomas, P., Pfeifer, Y., Witte, W., Templeton, K., and Bachmann, T. T. (2012) J. Clin. Microbiol., 50, 3990–3998.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Ulyashova, M. M., Pobolelova, Yu. I., Rubtsova, M. Yu., Pisarev, V. V., and Egorov, A. M. (2013) FEBS J., 280,Suppl. 1, SW04, S16–35, 284.

    Google Scholar 

  12. Ulyashova, M. M., Khalilova, Y. I., Rubtsova, M. Y., Edelstein, M. V., Alexandrova, I. A., and Egorov, A. M. (2010) Acta Naturae, 2, 101–109.

    PubMed  Google Scholar 

  13. Tanzer, L. R., Hu, Y., Cripe, L., and Moore, R. E. (1999) Anal. Biochem., 273, 307–310.

    Article  CAS  PubMed  Google Scholar 

  14. Dang, C., and Jayasena, S. D. (1996) J. Mol. Biol., 264, 268–278.

    Article  CAS  PubMed  Google Scholar 

  15. Mizuguchi, H., Nakatsuji, M., Fujiwara, S., Takagi, M., and Imanaka, T. (1999) J. Biochem., 126, 762–768.

    Article  CAS  PubMed  Google Scholar 

  16. Koukhareva, I., Haoqiang, H., Yee, J., Shum, J., Paul, N., Hogrefe, R. I., and Lebedev, A. V. (2008) Nucleic Acids Symp. Ser. (Oxf.), 259–260.

    Google Scholar 

  17. Lebedev, A. V., Paul, N., Yee, J., Timoshchuk, V. A., Shum, J., Miyagi, K., Kellum, J., Hogrefe, R. I., and Zon, G. (2008) Nucleic Acids Res., 36, e131.

    Article  PubMed Central  PubMed  Google Scholar 

  18. Ashrafi, E. H., and Paul, N. (2009) Curr. Protoc. Mol. Biol., Chap. 15, Unit 15.9.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Chemical Faculty, Lomonosov Moscow State University, 119991, Moscow, Russia

    Yu. I. Pobolelova, M. M. Ulyashova, M. Yu. Rubtsova & A. M. Egorov

  2. Russian Medical Academy of Postgraduate Education, ul. Barrikadnaya 2/1, 123995, Moscow, Russia

    A. M. Egorov

Authors
  1. Yu. I. Pobolelova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. M. Ulyashova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Yu. Rubtsova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. M. Egorov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Yu. Rubtsova.

Additional information

Original Russian Text © Yu. I. Pobolelova, M. M. Ulyashova, M. Yu. Rubtsova, A. M. Egorov, 2014, published in Biokhimiya, 2014, Vol. 79, No. 6, pp. 718–723.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pobolelova, Y.I., Ulyashova, M.M., Rubtsova, M.Y. et al. Multiplex PCR for joint amplification of carbapenemase genes of molecular classes A, B, and D. Biochemistry Moscow 79, 566–570 (2014). https://doi.org/10.1134/S0006297914060108

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297914060108

Key words

Search

Navigation