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Detection of Tularemia Agent DNA by Loop Mediated Isothermal Amplification

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Abstract

Tularemia is a natural focal zoonotic infection that can cause epidemic manifestations of an emergency nature. The purpose of the study is to develop a method for detecting DNA strains of the tularemia pathogen Francisella tularensis by loop mediated isothermal amplification (LAMP). Primers for the selected targets were calculated using the on-line Primer Explorer 5 program and tested for the specificity using the BLAST program. The primers were synthesized by Synthol, Moscow. Isolation of DNA from vaccine and virulent strains of epidemically significant subspecies of the tularemia microbe, as well as pathogens of other infectious diseases, was performed using the commercial DNA-sorb-B kit (InterLabService, Russia). The amplification reaction was carried out at a temperature of 63°C for 60 min (without loop primers) or 30 min (with loop primers) with preliminary heating at a temperature of 92°C for 2 min on a Tertsik amplifier (DNA-Technology, Russia) in the presence of the thermostable SD polymerase. Sequences of the genes encoding acid phosphatase A (acpA), outer membrane protein (fopA), and a region of the iglC gene of the pathogenicity island were chosen as DNA targets for the detection of the tularemia pathogen. Of the two sets of outer, inner, and loop original primers synthesized for each selected marker gene, the sets acpFt101, fopFt132, and iglCFt1 reproducibly and specifically detected DNA of 100–1000 F. tularensis microbial cells. The opportunity to reduce the analysis time by half appeared due to the introduction of loop primers and visual detection of amplification products stained with the SYTO 82 intercalating dye without subsequent electrophoresis and visualization of the gel after staining with ethidium bromide. An easy-to-use test that does not require sophisticated equipment is proposed for clinical and field diagnostics of the tularemia pathogen.

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Notes

  1. SanPiN 3.3686-21 “Sanitary and Epidemiological Requirements for the Prevention of Infectious Diseases.”

  2. MU 1.3.2569-09 “Organization of the Work of Laboratories that Utilize Nucleic Acid Amplification Methods when Working with Material Containing Microorganisms of Pathogenicity Groups I–IV: Guidelines.”

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ACKNOWLEDGMENTS

The authors are grateful to R.I. Mironova, researcher at the Department of Especially Dangerous Infections of the State Research Center for Applied Microbiology and Biotechnology, for selecting and providing strains of the tularemia microbe and heterologous microorganisms.

Funding

This work was carried out within the sectorial program of Rospotrebnadzor.

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Authors and Affiliations

  1. State Research Center for Applied Microbiology and Biotechnology of Rospotrebnadzor, 142279, Obolensk, Moscow oblast, Russia

    I. Yu. Shchit, T. Yu. Kudryavtseva, A. N. Mokrievich & S. F. Biketov

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  1. I. Yu. Shchit
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  2. T. Yu. Kudryavtseva
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  3. A. N. Mokrievich
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  4. S. F. Biketov
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Correspondence to A. N. Mokrievich.

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This article does not contain any studies using warm-blooded animals or human beings as subjects.

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The authors declare that they have no conflicts of interest.

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Translated by D. Novikova

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Shchit, I.Y., Kudryavtseva, T.Y., Mokrievich, A.N. et al. Detection of Tularemia Agent DNA by Loop Mediated Isothermal Amplification. Mol. Genet. Microbiol. Virol. 37, 202–208 (2022). https://doi.org/10.3103/S0891416822040085

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