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A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy

Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the dire...

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Autores principales: Ligasová, Anna, Vydržalová, Markéta, Buriánová, Renata, Brůčková, Lenka, Večeřová, Renata, Janošťáková, Anna, Koberna, Karel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952905/
https://www.ncbi.nlm.nih.gov/pubmed/31775352
http://dx.doi.org/10.3390/cells8121510
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author Ligasová, Anna
Vydržalová, Markéta
Buriánová, Renata
Brůčková, Lenka
Večeřová, Renata
Janošťáková, Anna
Koberna, Karel
author_facet Ligasová, Anna
Vydržalová, Markéta
Buriánová, Renata
Brůčková, Lenka
Večeřová, Renata
Janošťáková, Anna
Koberna, Karel
author_sort Ligasová, Anna
collection PubMed
description Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas’ DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas. The method is based on the enzymatic labeling using DNA polymerase I and modified nucleotides utilizing nicks in the mycoplasmas’ DNA. Modified nucleotides are incorporated into mycoplasmas’ DNA and subsequently visualized by immunofluorescence microscopy. The developed approach is independent of the mycoplasma strain, does not intensely stain nuclear DNA, does not stain other bacteria, and provides higher sensitivity than the approach based on the direct labeling using DAPI or Hoechst dyes.
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spelling pubmed-69529052020-01-23 A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy Ligasová, Anna Vydržalová, Markéta Buriánová, Renata Brůčková, Lenka Večeřová, Renata Janošťáková, Anna Koberna, Karel Cells Article Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas’ DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas. The method is based on the enzymatic labeling using DNA polymerase I and modified nucleotides utilizing nicks in the mycoplasmas’ DNA. Modified nucleotides are incorporated into mycoplasmas’ DNA and subsequently visualized by immunofluorescence microscopy. The developed approach is independent of the mycoplasma strain, does not intensely stain nuclear DNA, does not stain other bacteria, and provides higher sensitivity than the approach based on the direct labeling using DAPI or Hoechst dyes. MDPI 2019-11-25 /pmc/articles/PMC6952905/ /pubmed/31775352 http://dx.doi.org/10.3390/cells8121510 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ligasová, Anna
Vydržalová, Markéta
Buriánová, Renata
Brůčková, Lenka
Večeřová, Renata
Janošťáková, Anna
Koberna, Karel
A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
title A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
title_full A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
title_fullStr A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
title_full_unstemmed A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
title_short A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
title_sort new sensitive method for the detection of mycoplasmas using fluorescence microscopy
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952905/
https://www.ncbi.nlm.nih.gov/pubmed/31775352
http://dx.doi.org/10.3390/cells8121510
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