The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods

Cover Page

Cite item

Abstract

Causative agents of melioidosis and glanders are among the most dangerous bacterial pathogens for human. Moreover, Burkholderia pseudomallei and Burkholderia mallei are considered to be potential bioterrorism agents. In connection with this, timely diagnostics of such bacteria is of high importance. In our study, we made an attempt to develop an approach for detecting pathogenic Burkholderia spp. by combining species-specific amplification and strain-specific dot blotting assay with monoclonal antibodies. The following pathogenic Burkholderia strains were used in experiments: B. mallei (C-4, C-5, t-12, B-120, P-1, Мuksuwar-11, Z-12,Zagreb, Ivanovich, 5534), and B. pseudomallei (100, 102, 115, 116, 132, 135, 301, 51274, 60913, 61503). Real-Time PCR (RT-PCR) and dot blotting with monoclonal antibodies against surface Burkholderia epitopes were used to detect such pathogens. RT-PCR was carried out by using primers designed to recognize DNA fragments in B. mallei IS407A-fliP and the gene Orf12 from B. pseudomallei. For this, DNA was isolated from bacterial cells suspended at 1 × 104 microbial cells/ml. accumulation of the end reaction products was visualized by staining with dye SYBR Green I. Specificity of amplification reaction was determined by measuring melting temperature (Tm) for end products followed by running gel electrophoresis. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. Importantly, no signals specific to heterologous microbial DNA (isolated from bacterial cell suspension at concentration of 1 × 107 microbial cells/ml) were detected by using RT-PCR. Thus, RT-PCR provides an opportunity for assessing an inter-species diversity among pathogenic Burkholderia species. A genus-specificity was observed by using monoclonal antibodies 3D3 which bind to both Burkholderia strains, whereas antibodies 2D11 exhibited no selective binding to strain Р1 B. mallei and strain 100 B. pseudomallei, thereby displaying a strain-specific interaction. Thus, it allowed to conclude that combining a species-specific DNA amplification particularly RT-PCR together with immune-based assay such as dot blotting by using a panel of monoclonal antibodies seems to be a promising approach for assessing intra-species diversity among pathogenic Burkholderia. 

About the authors

S. S. Vetchinin

State Research Center for Applied Microbiology and Biotechnology

Email: vetchinin@obolensk.org
Vetchinin Sergey Sergeevich, PhD (Biology), Senior Researcher, Leading Researcher, Lime-Borreliosis Sector, Department for Immunobiochemistry of Pathogenic Microorganisms Russian Federation

I. Yu. Shchit

State Research Center for Applied Microbiology and Biotechnology

Email: schchit@obolensk.org
Shchit IrinaYurievna, PhD (Biology), Senior Researcher, Lime-Borreliosis Sector, Department for Immunobiochemistry of Pathogenic Microorganisms Russian Federation

A. G. Shevyakov

State Research Center for Applied Microbiology and Biotechnology

Author for correspondence.
Email: shevyakov@obolensk.org

Shevyakov Anton Georgievich, Junior Researcher, Lime-Borreliosis Sector, Department for Immunobiochemistry of Pathogenic Microorganisms

Contacts: Anton G. Shevyakov 142279, Russian Federation, Moscow Region, Serpukhov District, Obolensk, State Research Center for Applied Microbiology and Biotechnology. Phone: +7 (4967) 36-07-73 (office).

Russian Federation

S. F. Biketov

State Research Center for Applied Microbiology and Biotechnology

Email: biketov@obolensk.org
Biketov Sergey Fedorovich, PhD (Biology), Head of the Department for Immunobiochemistry of Pathogenic Microorganisms Russian Federation

References

  1. Антонов В.А., Илюхин В.И., Храпова Н.П., Прохватилова Е.В., Викторов Д.В., Сенина Т.В., Будченко А.А., Ткаченко Г.А., Алексеева В.В., Захарова И.Б., Савченко С.С., Зинченко О.В., Сорокина Ю.И., Алексеев В.В. Современные подходы к диагностике сапа и мелиоидоза. Идентификация и типирование B. mallei и B. pseudomallei //Проблемы особо опасных инфекций. 2012. № 2 (112). С. 46–50.
  2. Безопасность работы с микроорганизмами I–II групп патогенности (опасности): санитарные правила СП 1.3.1285-03. [Safety of work with microorganisms of the I–II pathogenicity (hazard) groups: sanitary rules SP 1.3.1285-03]
  3. Лемасова Л.В., Ткаченко Г.А., Савченко С.С., Бондарева О.С., Антонов В.А. Разработка мультиплексной тест-системы для обнаружения дифференциации Burkholderia mallei и Burkholderia pseudomallei методом ПЦР в режиме реального времени //Проблемы особо опасных инфекций. 2016. Вып. 4. С. 56–59.
  4. Организация работы лабораторий, использующих методы амплификации нуклеиновых кислот при работе с материалом, содержащим микроорганизмы I–IV групп патогенности: методические указания МУ 1.3.2569-09.
  5. Федюкина Г.Н., Ветчинин С.С., Баранова Е.В., Рудницкий С.Ю., Соловьев П.В., Колосова Н.В., Бикетов С.Ф. Получение компонентов иммунохроматографического теста для выявления возбудителей сапа и мелиоидоза //Биотехнология. 2015. № 1. С. 85–93.
  6. Andersen K., Dargis R., Kemp M., Christensen J.J. Detection of Burkholderia pseudomallei by SYBR green real time PCR. Open Pathol. J., 2009, vol. 3, pp. 30–32.
  7. Esters D.M., Dow S.W., Schweizer H.P., Torres A.G. Present and future strategies for melioidosis and glanders. Expert Rev. Anti Infect. Ther., 2010, vol. 8, no. 3. pp. 325–338.
  8. Foong Y.C., Tan M.R., Bradbury R.S. Melioidosis: a review. Rural Remote Health., 2014, vol. 14, p. 2763.
  9. Gregory B.C., Waag D.M. Glanders. In: Medical aspects of biological warfare. Ed. Dembek Z.F. Washington, DC: Borden Institute Walter Reed Army Medical Center, 2007, pp. 121–146.
  10. Houghton R.L., Reed D.E., Hubbard M.A., Dillon M.J., Chen H., Currie B.J., Mayo M., Sarovich D.S., Theobald V., Limmathurot sakul D., Wongsuvan G., Chantratita N., Peacock S.J., Hoffmaster A.R., Duval B., Brett P.J., Burtnick M.N., Aucoin D.P. Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis. PLOS Neglected Tropical Dis., 2014. vol. 8, no. 3, pp. 1–10.
  11. Lee M.A., Wang D., Yap E.H. Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR. FEMS Immunol. Med. Microbiol., 2005, vol. 43, pp. 413–417.
  12. Lowe W., March J.K., Bannell A.J., O’Noill R.L., Robinson R.A. PCR-based methodologies used to detect and differentiate the Burkholderia pseudomallei complex: B. mallei and B. pseudomallei and B. thailandensis. Curr. Issues Mol. Biol., 2013, vol. 22, no. 16 (2), pp. 23–54.
  13. Sanford J.P. Pseudomonas species (including melioidosis and glanders). In: Principles and practice of infectious diseases. Eds. Mandell G.L., Bennett J.E., Dolin R. 8th ed. New York, N.Y.: Churchill Livingstone, 1995, pp. 2003–2009. 1
  14. Tomaso H., Scholz H.C., Al Dahouk S., Eickhoff M., Treu T.M., Wernery R., Wernery U., Neubauer H. Development of a 5’-nuclease real-time PCR assay targeting fliP for the rapid identification of Burkholderia mallei in clinical samples. Clin. Chem., 2006, vol. 52, no. 2, pp. 307–310.
  15. Tomaso H., Scholz H.C., Al Dahouk S., Pitt T.L., Treu T.M., Neubauer H. Development of 5′ nuclease real-time PCR assays for the rapid identification of the Burkholderia mallei/Burkholderia pseudomallei complex. Diagn. Mol. Pathol., 2004, vol. 13, pp. 247–253.

Copyright (c) 2019 Vetchinin S.S., Shchit I.Y., Shevyakov A.G., Biketov S.F.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies