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. 2017 Sep 20;32(2):100–108. doi: 10.3103/S0891416817020094

The use of loop-mediated isothermal DNA amplification for the detection and identification of the anthrax pathogen

I Yu Shchit 1, K B Ignatov 2,3, T Yu Kudryavtseva 1, N A Shishkova 1, R I Mironova 1, L I Marinin 1, A N Mokrievich 1,, V M Kramarov 2,3, S F Biketov 1, I A Dyatlov 1
PMCID: PMC7088587  PMID: 32214650

Abstract

The results of detection and identification of Bacillus anthracis strains in loop-mediated isothermal DNA amplification (LAMP) reaction performed under optimized conditions with original primers and thermostable DNA polymerase are presented. Reproducible LAMP-based detection of chromosomal and plasmid DNA targets specific for B. anthracis strains has been demonstrated. No cross reactions with DNA from bacterial strains of other species of the B. cereus group were detected. The development of tests for anthrax-pathogen detection based on the optimized reaction of loop isothermal DNA amplification is planned. These tests will be convenient for clinical studies and field diagnostics due to the absence of requirements for sophisticated equipment.

Keywords: loop isothermal amplification of DNA, LAMP, Bacillus anthracis

Footnotes

Original Russian Text © I.Yu. Shchit, K.B. Ignatov, T.Yu. Kudryavtseva, N.A. Shishkova, R.I. Mironova, L.I. Marinin, A.N. Mokrievich, V.M. Kramarov, S.F. Biketov, I.A. Dyatlov, 2017, published in Molekulyarnaya Genetika, Mikrobiologiya i Virusologiya, 2017, No. 2, pp. 69–76.

References

  • 1.Daffonchio D., Cherif A., Brusetti L., Rizzi A., Mora D., Boudabous A., et al. Nature of polymorphisms in 16S–23S rRNA gene intergenic transcribed spacer fingerprinting of bacillus and related genera. Appl. Environ. Microbiol. 2003;69:5128–5137. doi: 10.1128/AEM.69.9.5128-5137.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Priest F., Barker M., Baillie L., Holmes E., Maiden M. Population structure and evolution of the Bacillus cereus group. J. Bacteriol. 2004;186:7959–7970. doi: 10.1128/JB.186.23.7959-7970.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Van Ert M., Easterday W., Huynh L., Okinaka R., Hugh-Jones M., Ravel J., et al. Global genetic population structure of Bacillus anthracis. PloS One. 2007;2:e461. doi: 10.1371/journal.pone.0000461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Van Ert M., Easterday W., Simonson T., U’Ren J., Pearson T., Kenefic L., et al. Strain-specific singlenucleotide polymorphism assays for the Bacillus anthracis Ames strain. J. Clin. Microbiol. 2007;45:47–53. doi: 10.1128/JCM.01233-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Luna V., King D., Peak K., Reeves F., Heberlein-Larson L., Veguilla W. Bacillus anthracis virulent plasmid pX02 genes found in large plasmids of two other Bacillus species. J. Clin. Microbiol. 2006;44(7):2367–2377. doi: 10.1128/JCM.00154-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Han C., Xie G., Challacombe J., Altherr M., Bhotika S., Brown N., et al. Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis. J. Bacteriol. 2006;188:3382–3390. doi: 10.1128/JB.188.9.3382-3390.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Hoffinaster A., Ravel J., Rasko D., Chapman G., Chute M., Marston C., et al. Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. Proc. Natl. Acad. Sci. U. S. A. 2004;101(22):8449–8454. doi: 10.1073/pnas.0402414101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Hoffinaster A., Hill K., Gee J., Marston C., De B., Popovic T., et al. Characterization of Bacillus cereus isolates associated with fatal pneumonias: Strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes. J. Clin. Microbiol. 2006;44(9):3352–3360. doi: 10.1128/JCM.00561-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hoffinaster A., Novak R., Marston C., Gee J., Helsel L., Pruckler J., Wilkins P. Genetic diversity of clinical isolates of Bacillus cereus using multilocus sequence typing. BMC Microbiol. 2008;8:191. doi: 10.1186/1471-2180-8-191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Oh S.Y., Budzik J.M., Garufi G., Schneewind O. Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthraxlike disease. Mol. Microbiol. 2011;80:455–470. doi: 10.1111/j.1365-2958.2011.07582.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Klee S., Brzuszkiewicz E., Nattermann H., Bruggemann H., Dupke S., Wollherr A., et al. The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids. PloS One. 2010;5:e10986. doi: 10.1371/journal.pone.0010986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Hu X., Van der Auwera G., Timmery S., Zhu L., Mahillon J. Distribution, diversity, and potential mobility of extrachromosomal elements related to the Bacillus anthracis pXO1 and pXO2 virulence plasmids. Appl. Environ. Microbiol. 2009;75:3016–3028. doi: 10.1128/AEM.02709-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Van der Auwera G., Andrup L., Mahillon J. Conjugative plasmid pAw63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727. BMC Genomics. 2005;6:103. doi: 10.1186/1471-2164-6-103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63. doi: 10.1093/nar/28.12.e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Notomi T., Kanda H., Taguch F., Minekaw H., Itamura S., Odagiri T., Tashiro M. RT-LAMP method provides a simple, rapid and specific detection system for SARS-CoV RNA, Proc. Int. Conference on SARS. Lübeck: German Medical Science; 2004. [Google Scholar]
  • 16.Parida M., Sannarangaiah S., Dash P.K., Rao P.V.L., Morita K. Loop mediated isothermal amplification (LAMP): A new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev. Med. Virol. 2008;18:407–421. doi: 10.1002/rmv.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Pandey B.D., Poudel A., Yoda T., Tamaru A., Oda N., Fukushima Y., et al. Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients. J. Med. Microbiol. 2008;57:439–443. doi: 10.1099/jmm.0.47499-0. [DOI] [PubMed] [Google Scholar]
  • 18.Geojith G., Dhanasekaran S., Chandran S.P., Kenneth J. Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting. J. Microbiol. Methods. 2011;84:71–73. doi: 10.1016/j.mimet.2010.10.015. [DOI] [PubMed] [Google Scholar]
  • 19.Sonthayanon P., Chierakul W., Wuthiekanun V., Thaipadungpanit J., Kalambaheti T., Boonsilp S., et al. Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand. Am. J. Trop. Med. Hyg. 2011;84:614–620. doi: 10.4269/ajtmh.2011.10-0473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lim K.T., Teh C.S.J., Thong K.L. Loop mediated isothermal amplification assay for the rapid detection of Staphylococcus aureus. Bio. Med. Res. Int. 2013;2013:895816. doi: 10.1155/2013/895816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Tang M.J., Zhou S., Zhang X.Y., Pu J.H., Ge Q.L., Tang X.J., Gao Y.S. Rapid and sensitive detection of Listeria monocytogenes by loop-mediated isothermal amplification. Curr. Microbiol. 2011;63:511–516. doi: 10.1007/s00284-011-0013-3. [DOI] [PubMed] [Google Scholar]
  • 22.Yamazaki W., Seto K., Taguchi M., Ishibashi M., Inoue K. Sensitive and rapid detection of cholera toxin producing Vibrio cholerae using a loop mediated isothermal amplification. BMC Microbiol. 2008;8:94. doi: 10.1186/1471-2180-8-94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kawai Y., Kimura Y., Lezhava A., Kanamori H., Usui K., Hanami T., et al. One-step detection of the 2009 pandemic influenza A(H1N1) virus by the RTSmartAmp Assay and its clinical validation. PloS One. 2012;7(1):e30236. doi: 10.1371/journal.pone.0030236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Moslemi E., Shahhosseiny M.H., Javadi G., Praivar K., Sattari T.N., Amini H.K. Loop mediated isothermal amplification (LAMP) for rapid detection of HBV in Iran. Afr. J. Microbiol. Res. 2009;3:439–445. [Google Scholar]
  • 25.Qiao Y.-M., Guo Y.-C., Zhang X.-En., Zhou Y.-F., Zhang Z.-P., Wei H.-P., et al. Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis spores. Biotechnol. Lett. 2007;29:1939–1946. doi: 10.1007/s10529-007-9472-9. [DOI] [PubMed] [Google Scholar]
  • 26.Hatano B., Maki T., Obara T., Fukumoto H., Hagisawa K., Matsushita Y., et al. LAMP using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism. Jpn. J. Infect. Dis. 2010;63:36–40. [PubMed] [Google Scholar]
  • 27.Dugan L., Bearinger J., Hinckley A., Strout C., Souza B. Detection of Bacillus anthracis from spores and cells by loop-mediated isothermal amplification without sample preparation. J. Microbiol. Methods. 2012;90(3):280–284. doi: 10.1016/j.mimet.2012.05.022. [DOI] [PubMed] [Google Scholar]
  • 28.Jain N., Kumar J.S., Parida M.M., Merwyn S., Rai G.P., Agarwal G.S. Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder. J. Microbiol. Biotechnol. 2011;27(6):1407–1413. doi: 10.1007/s11274-010-0592-3. [DOI] [PubMed] [Google Scholar]
  • 29.Kurosaki Y., Sakuma T., Fukuma A., Fujinami Y., Kawamoto K., Kamo N., et al. A simple and sensitive method for detection of Bacillus anthracis by loopmediated isothermal amplification. J. Appl. Microbiol. 2009;107(6):1947–1956. doi: 10.1111/j.1365-2672.2009.04379.x. [DOI] [PubMed] [Google Scholar]
  • 30.Radnedge L., Agron P.G., Hill K.K., Jackson P.J., Ticknor L.O., Keim P., Andersen G.L. Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. Appl. Environ. Microbiol. 2003;69(5):2755–2764. doi: 10.1128/AEM.69.5.2755-2764.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Nagamine K., Watanabe K., Ohtsuka K., Hase T., Notomi T. Loop-mediated isothermal amplification reaction using a nondenatured template. Clin. Chem. 2001;47:1742–1743. [PubMed] [Google Scholar]
  • 32.Wozniakowski G., Kozdrun W., Samorek-Salamonowicz E. Loop-mediated isothermal amplification for the detection of goose circovirus. Virol. J. 2012;9:110. doi: 10.1186/1743-422X-9-110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ignatov K.B., Barsova E.V., Fradkov A.F., Blagodatskich K.A., Kramarova T.V., Kramarov V.M. A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification. BioTechniques. 2014;57:81–87. doi: 10.2144/000114198. [DOI] [PubMed] [Google Scholar]
  • 34.Shishkova N.A., Mokrievich A.N., Pavlov V.M., Marinin L.I., Vakhrameeva G.M., Kudryavtseva T.Yu., Dyatlov I.A. Usage of chi-sequence for differentiating the strains of anthrax from closely related bacilli. Probl. Osobo Opasnykh Infekts. 2014;1:97–100. [Google Scholar]
  • 35.Shishkova N.A., Mokrievich A.N., Platonov M.E., Svetoch T.E., Marinin L.I. The way for studying genetic diversity of anthrax strains from the collection of State Research Center for Applied Microbiology and Biotechnology. Probl. Osobo Opasnykh Infekts. 2010;2(104):60–66. [Google Scholar]

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