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Modern Approaches to Differentiation of Live and Dead Bacteria Using Selective Amplification of Nucleic Acids

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Abstract

Specific amplification of nucleic acids is a convenient and quick alternative to the culture-based method of detecting bacterial cells. However, conventional PCR and other amplification reactions can not differentiate between live bacteria and dead or dormant ones, and are also capable of amplifying DNA that persists for a long time and in a cell-free state. Several methods have been developed in order to establish the viability of microorganisms by amplification of specific sequences of nucleic acids, both those controlled by changing temperatures and isothermal ones. For some of them, DNA modified by phenanthridine dyes serves as a target, and simultaneous use of monoazides of ethidium and propidium was shown to be preferable for the purpose. For other methods, the targets are directly RNA molecules or their cDNA copies. Pre-rRNA detection seems to be the most preferable approach, due to the presence of these types of RNA exclusively in living cells.

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Funding

An.Kh. Baymiev, Al.Kh. Baymiev, B.R. Kuluev, K.Yu. Shvets, R.S. Yamidanov, Ya.A. Ivanenkov, and A.V. Chemeris were supported by the Russian Science Foundation, project no. 17-74-30012.

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Correspondence to An. Kh. Baymiev.

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Translated by D. Timchenko

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Baymiev, A.K., Baymiev, A.K., Kuluev, B.R. et al. Modern Approaches to Differentiation of Live and Dead Bacteria Using Selective Amplification of Nucleic Acids. Microbiology 89, 13–27 (2020). https://doi.org/10.1134/S0026261720010038

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Keywords:

  • microbial viability
  • live and dead bacteria
  • PCR
  • RT–PCR
  • NASBA (Nucleic Acid Sequence Based Amplification)
  • Molecular Viability Testing (MVT)
  • ethidium monoazide (EMA)
  • propidium monoazide (PMA)
  • pre-rRNA