Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei
Objective of the study was to develop a real-time multiplex PCR assay for the detection and differentiation of B. mallei and B. pseudomallei, characterized by high sensitivity and specificity. Materials and Methods. The primers and probes were designed to detect the species-specific sequence of the...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | Russian |
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Federal Government Health Institution, Russian Research Anti-Plague Institute “Microbe”
2016-12-01
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| Series: | Проблемы особо опасных инфекций |
| Subjects: | |
| Online Access: | https://journal.microbe.ru/jour/article/view/348 |
| _version_ | 1797220858887929856 |
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| author | L. V. Lemasova G. A. Tkachenko S. S. Savchenko O. S. Bondareva V. A. Antonov |
| author_facet | L. V. Lemasova G. A. Tkachenko S. S. Savchenko O. S. Bondareva V. A. Antonov |
| author_sort | L. V. Lemasova |
| collection | DOAJ |
| description | Objective of the study was to develop a real-time multiplex PCR assay for the detection and differentiation of B. mallei and B. pseudomallei, characterized by high sensitivity and specificity. Materials and Methods. The primers and probes were designed to detect the species-specific sequence of the fliР gene of B. mallei and gp68 gene of B. pseudomallei, respectively. Species specificity was tested with a panel of 56 B. pseudomallei strains, 14 B. mallei strains and 34 strains of closely or distantly related species. To define the analytical sensitivity of the assay, the serially diluted bacterial suspension at concentrations of 109 –102 cells /ml was used. Conclusions. The multiplex PCR assay with two primer pairs and fluorescently-labeled probes, allowing for simultaneous detection and differentiation between B. mallei and B. pseudomallei was designed. Species-specific for glanders agent, B. mallei, fragment of fliP gene, which encodes protein of flagellin biosynthesis, and species-specific gene region of B. pseudomallei, encoding gp68 protein, were identified as DNA targets. Testing of Burkholderia and non-Burkholderia bacterial species revealed 100 % specificity of the amplification assay. The minimum detection limit of the designed multiplex PCR test-system was 1·103 cells/ml for B. mallei, and 1·104 cells/ml for B. pseudomallei. |
| first_indexed | 2024-03-08T13:42:11Z |
| format | Article |
| id | doaj.art-3ddef4c1b00f4eb1bf001d83a0c57b90 |
| institution | Directory Open Access Journal |
| issn | 0370-1069 2658-719X |
| language | Russian |
| last_indexed | 2024-04-24T12:56:14Z |
| publishDate | 2016-12-01 |
| publisher | Federal Government Health Institution, Russian Research Anti-Plague Institute “Microbe” |
| record_format | Article |
| series | Проблемы особо опасных инфекций |
| spelling | doaj.art-3ddef4c1b00f4eb1bf001d83a0c57b902024-04-05T16:47:12ZrusFederal Government Health Institution, Russian Research Anti-Plague Institute “Microbe”Проблемы особо опасных инфекций0370-10692658-719X2016-12-0104565910.21055/0370-1069-2016-4-56-59348Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomalleiL. V. Lemasova0G. A. Tkachenko1S. S. Savchenko2O. S. Bondareva3V. A. Antonov4Volgograd Research Anti-Plague Institute, VolgogradVolgograd Research Anti-Plague Institute, VolgogradVolgograd Research Anti-Plague Institute, VolgogradVolgograd Research Anti-Plague Institute, VolgogradVolgograd Research Anti-Plague Institute, VolgogradObjective of the study was to develop a real-time multiplex PCR assay for the detection and differentiation of B. mallei and B. pseudomallei, characterized by high sensitivity and specificity. Materials and Methods. The primers and probes were designed to detect the species-specific sequence of the fliР gene of B. mallei and gp68 gene of B. pseudomallei, respectively. Species specificity was tested with a panel of 56 B. pseudomallei strains, 14 B. mallei strains and 34 strains of closely or distantly related species. To define the analytical sensitivity of the assay, the serially diluted bacterial suspension at concentrations of 109 –102 cells /ml was used. Conclusions. The multiplex PCR assay with two primer pairs and fluorescently-labeled probes, allowing for simultaneous detection and differentiation between B. mallei and B. pseudomallei was designed. Species-specific for glanders agent, B. mallei, fragment of fliP gene, which encodes protein of flagellin biosynthesis, and species-specific gene region of B. pseudomallei, encoding gp68 protein, were identified as DNA targets. Testing of Burkholderia and non-Burkholderia bacterial species revealed 100 % specificity of the amplification assay. The minimum detection limit of the designed multiplex PCR test-system was 1·103 cells/ml for B. mallei, and 1·104 cells/ml for B. pseudomallei.https://journal.microbe.ru/jour/article/view/348glandersmelioidosispcr assayreal-time pcrburkholderia malleiburkholderia pseudomallei |
| spellingShingle | L. V. Lemasova G. A. Tkachenko S. S. Savchenko O. S. Bondareva V. A. Antonov Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei Проблемы особо опасных инфекций glanders melioidosis pcr assay real-time pcr burkholderia mallei burkholderia pseudomallei |
| title | Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei |
| title_full | Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei |
| title_fullStr | Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei |
| title_full_unstemmed | Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei |
| title_short | Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei |
| title_sort | development of real time multiplex pcr assay for the detection and differentiation of burkholderia mallei and burkholderia pseudomallei |
| topic | glanders melioidosis pcr assay real-time pcr burkholderia mallei burkholderia pseudomallei |
| url | https://journal.microbe.ru/jour/article/view/348 |
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