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

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....

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Autores principales:	S. S. Vetchinin, I. Yu. Shchit, A. G. Shevyakov, S. F. Biketov
Formato:	article
Lenguaje:	RU
Publicado:	Sankt-Peterburg : NIIÈM imeni Pastera 2019
Materias:	glanders melioidosis monoclonal antibody rt-pcr dot-blot Infectious and parasitic diseases RC109-216
Acceso en línea:	https://doaj.org/article/bfc3082c3cae420ab3dd161ea4acc334
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spelling	oai:doaj.org-article:bfc3082c3cae420ab3dd161ea4acc3342021-11-22T07:09:53ZThe possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods2220-76192313-739810.15789/2220-7619-2019-2-404-408https://doaj.org/article/bfc3082c3cae420ab3dd161ea4acc3342019-07-01T00:00:00Zhttps://www.iimmun.ru/iimm/article/view/704https://doaj.org/toc/2220-7619https://doaj.org/toc/2313-7398Causative 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.S. S. VetchininI. Yu. ShchitA. G. ShevyakovS. F. BiketovSankt-Peterburg : NIIÈM imeni Pasteraarticleglandersmelioidosismonoclonal antibodyrt-pcrdot-blotInfectious and parasitic diseasesRC109-216RUInfekciâ i Immunitet, Vol 9, Iss 2, Pp 404-408 (2019)
institution	DOAJ
collection	DOAJ
language	RU
topic	glanders melioidosis monoclonal antibody rt-pcr dot-blot Infectious and parasitic diseases RC109-216
spellingShingle	glanders melioidosis monoclonal antibody rt-pcr dot-blot Infectious and parasitic diseases RC109-216 S. S. Vetchinin I. Yu. Shchit A. G. Shevyakov S. F. Biketov The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods
description	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.
format	article
author	S. S. Vetchinin I. Yu. Shchit A. G. Shevyakov S. F. Biketov
author_facet	S. S. Vetchinin I. Yu. Shchit A. G. Shevyakov S. F. Biketov
author_sort	S. S. Vetchinin
title	The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods
title_short	The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods
title_full	The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods
title_fullStr	The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods
title_full_unstemmed	The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods
title_sort	possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and dna amplification methods
publisher	Sankt-Peterburg : NIIÈM imeni Pastera
publishDate	2019
url	https://doaj.org/article/bfc3082c3cae420ab3dd161ea4acc334
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The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods

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