Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members
Abstract The possibility of introducing a reliable assay for a quick identification and differentiation of the main species of Mycobacterium tuberculosis complex (MTBC) supports the improvement of efficient tuberculosis combating strategies worldwide. Commercially available assays are often based on...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2019-12-01
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| Series: | MicrobiologyOpen |
| Online Access: | https://doi.org/10.1002/mbo3.919 |
| _version_ | 1819110855072022528 |
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| author | Patricia Landolt Roger Stephan Marc J. A. Stevens Simone Scherrer |
| author_facet | Patricia Landolt Roger Stephan Marc J. A. Stevens Simone Scherrer |
| author_sort | Patricia Landolt |
| collection | DOAJ |
| description | Abstract The possibility of introducing a reliable assay for a quick identification and differentiation of the main species of Mycobacterium tuberculosis complex (MTBC) supports the improvement of efficient tuberculosis combating strategies worldwide. Commercially available assays are often based on cultured samples; however, due to the long cultivation time of mycobacteria, results are delayed. Developed PCR approaches have been published previously, though, when testing intricate veterinary samples, the complex composition of multiplex qPCRs frequently leads to assay failure. In order to overcome those limits, a paradigm of a three‐reaction high‐resolution melting (HRM) assay for the simultaneous identification and differentiation of the main members of MTBC was established. The assay is based on single nucleotide polymorphisms within gyrB and gyrA, which have been used as target for the establishment of two highly specific HRM assays (HRM assays 1 and 2) discriminating M. tuberculosis/ Mycobacterium canetti, Mycobacterium bovis/M. bovis BCG, Mycobacterium caprae/rare M. caprae/M. bovis ecotypes, Mycobacterium africanum/Mycobacterium orygis/ Mycobacterium pinnipedii/Clade A1, Mycobacterium microti, and a rare subtype of M. canettii followed by a third HRM assay (HRM assay 3) allowing a further differentiation of M. bovis, M. bovis BCG, and a rare subtype of M. caprae/M. bovis, which is considered to be a novel ecotype. High‐resolution melting assay 1 is described in a previously published report. High‐resolution melting assay 2 showed 100% correlation of all 39 examined isolates with the results of a commercial identification kit. 96% of the clinical samples tested demonstrated concordant results. High‐resolution melting assay 3 showed an accordance of 100% with the results of the commercially available identification kit of all 22 samples analyzed. The proposed strategy of the three‐reaction HRM assay can be used for an accurate differentiation of up to seven groups of MTBC and potentially to identify a rare subtype of M. canettii either on isolates or on clinical samples. |
| first_indexed | 2024-12-22T03:48:21Z |
| format | Article |
| id | doaj.art-fe5cfe2c3fc544f1b86f630fbbe19f84 |
| institution | Directory Open Access Journal |
| issn | 2045-8827 |
| language | English |
| last_indexed | 2024-12-22T03:48:21Z |
| publishDate | 2019-12-01 |
| publisher | Wiley |
| record_format | Article |
| series | MicrobiologyOpen |
| spelling | doaj.art-fe5cfe2c3fc544f1b86f630fbbe19f842022-12-21T18:40:05ZengWileyMicrobiologyOpen2045-88272019-12-01812n/an/a10.1002/mbo3.919Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex membersPatricia Landolt0Roger Stephan1Marc J. A. Stevens2Simone Scherrer3Section of Veterinary Bacteriology, Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich Zurich SwitzerlandSection of Veterinary Bacteriology, Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich Zurich SwitzerlandSection of Veterinary Bacteriology, Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich Zurich SwitzerlandSection of Veterinary Bacteriology, Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich Zurich SwitzerlandAbstract The possibility of introducing a reliable assay for a quick identification and differentiation of the main species of Mycobacterium tuberculosis complex (MTBC) supports the improvement of efficient tuberculosis combating strategies worldwide. Commercially available assays are often based on cultured samples; however, due to the long cultivation time of mycobacteria, results are delayed. Developed PCR approaches have been published previously, though, when testing intricate veterinary samples, the complex composition of multiplex qPCRs frequently leads to assay failure. In order to overcome those limits, a paradigm of a three‐reaction high‐resolution melting (HRM) assay for the simultaneous identification and differentiation of the main members of MTBC was established. The assay is based on single nucleotide polymorphisms within gyrB and gyrA, which have been used as target for the establishment of two highly specific HRM assays (HRM assays 1 and 2) discriminating M. tuberculosis/ Mycobacterium canetti, Mycobacterium bovis/M. bovis BCG, Mycobacterium caprae/rare M. caprae/M. bovis ecotypes, Mycobacterium africanum/Mycobacterium orygis/ Mycobacterium pinnipedii/Clade A1, Mycobacterium microti, and a rare subtype of M. canettii followed by a third HRM assay (HRM assay 3) allowing a further differentiation of M. bovis, M. bovis BCG, and a rare subtype of M. caprae/M. bovis, which is considered to be a novel ecotype. High‐resolution melting assay 1 is described in a previously published report. High‐resolution melting assay 2 showed 100% correlation of all 39 examined isolates with the results of a commercial identification kit. 96% of the clinical samples tested demonstrated concordant results. High‐resolution melting assay 3 showed an accordance of 100% with the results of the commercially available identification kit of all 22 samples analyzed. The proposed strategy of the three‐reaction HRM assay can be used for an accurate differentiation of up to seven groups of MTBC and potentially to identify a rare subtype of M. canettii either on isolates or on clinical samples.https://doi.org/10.1002/mbo3.919 |
| spellingShingle | Patricia Landolt Roger Stephan Marc J. A. Stevens Simone Scherrer Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members MicrobiologyOpen |
| title | Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members |
| title_full | Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members |
| title_fullStr | Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members |
| title_full_unstemmed | Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members |
| title_short | Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members |
| title_sort | three reaction high resolution melting assay for rapid differentiation of mycobacterium tuberculosis complex members |
| url | https://doi.org/10.1002/mbo3.919 |
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