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Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures

In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact represents a signifi...

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Autores principales: Carrillo-Ávila, José A., de la Fuente, Amanda, Aguilar-Quesada, Rocío, Ligero, Gertrudis, del Río-Ortiz, Juan Manuel, Catalina, Purificación
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10453501/
https://www.ncbi.nlm.nih.gov/pubmed/37623254
http://dx.doi.org/10.3390/cimb45080435
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author Carrillo-Ávila, José A.
de la Fuente, Amanda
Aguilar-Quesada, Rocío
Ligero, Gertrudis
del Río-Ortiz, Juan Manuel
Catalina, Purificación
author_facet Carrillo-Ávila, José A.
de la Fuente, Amanda
Aguilar-Quesada, Rocío
Ligero, Gertrudis
del Río-Ortiz, Juan Manuel
Catalina, Purificación
author_sort Carrillo-Ávila, José A.
collection PubMed
description In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact represents a significant problem since such contamination can cause disastrous effects on eukaryotic cells by altering their cellular parameters, which, in turn, can lead to unreliable experimental results. For this reason, it is mandatory to carry out continuous testing for the presence of Mycoplasma in cell culture and the development of appropriate methodologies for this purpose. An ideal detection methodology should be fast, sensitive, and reliable. In this study, we propose an alternative detection method based on real-time PCR in conjunction with a novel combination of primers and probes that have been improved to increase their efficiency. The new PCR method demonstrates 100% sensitivity and specificity results in the detection of common Mycoplasma species that contaminate cell cultures. Whilst 11 of 45 tested supernatants were positive for Mycoplasma (24.4%) using the new PCR method (corresponding to 5 of the 14 lines tested (35.71%)), only 10 of 45 supernatants showed positive results with the commercial Venor(®)GeM qEP and Plasmotest(®) kit. In addition, the new PCR method exhibits a high capacity to detect less-frequent Mycoplasma species, such as those related to the M. mycoides cluster. The use of an alternative Mycoplasma-detection method in cell culture labs can guarantee the detection of Mycoplasma contamination, especially in cases when dubious results are recorded.
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spelling pubmed-104535012023-08-26 Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures Carrillo-Ávila, José A. de la Fuente, Amanda Aguilar-Quesada, Rocío Ligero, Gertrudis del Río-Ortiz, Juan Manuel Catalina, Purificación Curr Issues Mol Biol Article In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact represents a significant problem since such contamination can cause disastrous effects on eukaryotic cells by altering their cellular parameters, which, in turn, can lead to unreliable experimental results. For this reason, it is mandatory to carry out continuous testing for the presence of Mycoplasma in cell culture and the development of appropriate methodologies for this purpose. An ideal detection methodology should be fast, sensitive, and reliable. In this study, we propose an alternative detection method based on real-time PCR in conjunction with a novel combination of primers and probes that have been improved to increase their efficiency. The new PCR method demonstrates 100% sensitivity and specificity results in the detection of common Mycoplasma species that contaminate cell cultures. Whilst 11 of 45 tested supernatants were positive for Mycoplasma (24.4%) using the new PCR method (corresponding to 5 of the 14 lines tested (35.71%)), only 10 of 45 supernatants showed positive results with the commercial Venor(®)GeM qEP and Plasmotest(®) kit. In addition, the new PCR method exhibits a high capacity to detect less-frequent Mycoplasma species, such as those related to the M. mycoides cluster. The use of an alternative Mycoplasma-detection method in cell culture labs can guarantee the detection of Mycoplasma contamination, especially in cases when dubious results are recorded. MDPI 2023-08-18 /pmc/articles/PMC10453501/ /pubmed/37623254 http://dx.doi.org/10.3390/cimb45080435 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Carrillo-Ávila, José A.
de la Fuente, Amanda
Aguilar-Quesada, Rocío
Ligero, Gertrudis
del Río-Ortiz, Juan Manuel
Catalina, Purificación
Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures
title Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures
title_full Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures
title_fullStr Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures
title_full_unstemmed Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures
title_short Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures
title_sort development and evaluation of a new qpcr assay for the detection of mycoplasma in cell cultures
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10453501/
https://www.ncbi.nlm.nih.gov/pubmed/37623254
http://dx.doi.org/10.3390/cimb45080435
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