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Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva
BACKGROUND: Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we prese...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9286268/ https://www.ncbi.nlm.nih.gov/pubmed/35839162 http://dx.doi.org/10.1371/journal.pone.0265554 |
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author | Hansen, Julia Kolbe, Katharina König, Inke R. Scherließ, Regina Hellfritzsch, Marie Malm, Sven Müller-Loennies, Sven Zallet, Julia Hillemann, Doris Wiesmüller, Karl-Heinz Herzmann, Christian Brandenburg, Julius Reiling, Norbert |
author_facet | Hansen, Julia Kolbe, Katharina König, Inke R. Scherließ, Regina Hellfritzsch, Marie Malm, Sven Müller-Loennies, Sven Zallet, Julia Hillemann, Doris Wiesmüller, Karl-Heinz Herzmann, Christian Brandenburg, Julius Reiling, Norbert |
author_sort | Hansen, Julia |
collection | PubMed |
description | BACKGROUND: Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. METHODS: Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. RESULTS: We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 10(2) bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. CONCLUSIONS: Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity. |
format | Online Article Text |
id | pubmed-9286268 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-92862682022-07-16 Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva Hansen, Julia Kolbe, Katharina König, Inke R. Scherließ, Regina Hellfritzsch, Marie Malm, Sven Müller-Loennies, Sven Zallet, Julia Hillemann, Doris Wiesmüller, Karl-Heinz Herzmann, Christian Brandenburg, Julius Reiling, Norbert PLoS One Research Article BACKGROUND: Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. METHODS: Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. RESULTS: We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 10(2) bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. CONCLUSIONS: Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity. Public Library of Science 2022-07-15 /pmc/articles/PMC9286268/ /pubmed/35839162 http://dx.doi.org/10.1371/journal.pone.0265554 Text en © 2022 Hansen et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Hansen, Julia Kolbe, Katharina König, Inke R. Scherließ, Regina Hellfritzsch, Marie Malm, Sven Müller-Loennies, Sven Zallet, Julia Hillemann, Doris Wiesmüller, Karl-Heinz Herzmann, Christian Brandenburg, Julius Reiling, Norbert Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva |
title | Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva |
title_full | Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva |
title_fullStr | Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva |
title_full_unstemmed | Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva |
title_short | Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva |
title_sort | lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of mycobacterium tuberculosis from saliva |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9286268/ https://www.ncbi.nlm.nih.gov/pubmed/35839162 http://dx.doi.org/10.1371/journal.pone.0265554 |
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