An integrated workflow for Mycoplasma detection and removal from cell cultures and viral stocks.
5 affiliations
- 1Grupo de Pesquisa em Imunologia Celular e Molecular, Instituto René Rachou-Fiocruz Minas, 30190-002, Belo Horizonte, Minas Gerais, Brazil.
- 2Instituto Nacional de Ciência e Tecnologia em Poxvírus, Brazil.
- 3Rede Mineira de Antivirais, MG, Brazil.
- 4These authors contributed equally to this work.
- 5Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte 31270-901, Belo Horizonte, MG, Brazil.
Abstract
Contamination of cell cultures and viral stocks with Mycoplasma is one of the most frequent and underdiagnosed problems in laboratories working with these materials, severely affecting experimental reproducibility, data reliability, and biosafety. This article presents an integrated set of robust, sensitive, and broad applicable protocols for the detection, treatment, and mitigation of Mycoplasma contamination in cellular and viral systems. Basic Protocol 1 describes a sensitive qPCR-based method for Mycoplasma detection using DNA extracted from cell cultures and viral suspensions, including clear interpretation criteria based on amplification and melting curve analyses. Basic Protocol 2 outlines a sequential antibiotic treatment strategy for eliminating Mycoplasma from contaminated cell cultures, combining different antimicrobial classes and continuous monitoring of cell viability and morphology. Basic Protocol 3 presents a complementary approach to reduce Mycoplasma burden in viral stocks through serial dilution and controlled replication, aiming to preserve viral infectivity while minimizing bacterial contaminants. Together, these protocols provide a practical and reproducible workflow that enables rapid identification of contamination, implementation of effective corrective measures, and molecular validation of decontamination. The procedures are suitable for virology, cell biology, and experimental virus production laboratories, thereby improving experimental quality and data reliability. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sensitive detection of Mycoplasma contamination in cell cultures and viral stocks by qPCR Basic Protocol 2: Treatment of cell cultures for Mycoplasma decontamination Basic Protocol 3: Elimination of Mycoplasma contamination in viral stocks using serial dilution.
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BibTeX
@article{floressouza2026integrated,
title = {{An integrated workflow for Mycoplasma detection and removal from cell cultures and viral stocks.}},
author = {Eliza Flores-Souza and Carolina Colombelli Pacca-Mazaro and Naiara Clemente Tavares and Camila Salles Nascimento and Tamiris Vanessa Miguel de Souza and Anna Beatriz de Oliveira Mendes and Erna Geessien Kroon and Carlos Eduardo Calzavara-Silva and Jaquelline Germano de Oliveira},
journal = {Current protocols},
year = {2026},
month = oct,
doi = {10.1002/cpz1.70496},
url = {https://europepmc.org/article/MED/42961168},
note = {CC BY},
}
RIS
TY - JOUR
TI - An integrated workflow for Mycoplasma detection and removal from cell cultures and viral stocks.
AU - Eliza Flores-Souza
AU - Carolina Colombelli Pacca-Mazaro
AU - Naiara Clemente Tavares
AU - Camila Salles Nascimento
AU - Tamiris Vanessa Miguel de Souza
AU - Anna Beatriz de Oliveira Mendes
AU - Erna Geessien Kroon
AU - Carlos Eduardo Calzavara-Silva
AU - Jaquelline Germano de Oliveira
PY - 2026
DA - 2026/10/30
JO - Current protocols
DO - 10.1002/cpz1.70496
UR - https://europepmc.org/article/MED/42961168
AB - Contamination of cell cultures and viral stocks with Mycoplasma is one of the most frequent and underdiagnosed problems in laboratories working with these materials, severely affecting experimental reproducibility, data reliability, and biosafety. This article presents an integrated set of robust, sensitive, and broad applicable protocols for the detection, treatment, and mitigation of Mycoplasma contamination in cellular and viral systems. Basic Protocol 1 describes a sensitive qPCR-based method for Mycoplasma detection using DNA extracted from cell cultures and viral suspensions, including clear interpretation criteria based on amplification and melting curve analyses. Basic Protocol 2 outlines a sequential antibiotic treatment strategy for eliminating Mycoplasma from contaminated cell cultures, combining different antimicrobial classes and continuous monitoring of cell viability and morphology. Basic Protocol 3 presents a complementary approach to reduce Mycoplasma burden in viral stocks through serial dilution and controlled replication, aiming to preserve viral infectivity while minimizing bacterial contaminants. Together, these protocols provide a practical and reproducible workflow that enables rapid identification of contamination, implementation of effective corrective measures, and molecular validation of decontamination. The procedures are suitable for virology, cell biology, and experimental virus production laboratories, thereby improving experimental quality and data reliability. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sensitive detection of Mycoplasma contamination in cell cultures and viral stocks by qPCR Basic Protocol 2: Treatment of cell cultures for Mycoplasma decontamination Basic Protocol 3: Elimination of Mycoplasma contamination in viral stocks using serial dilution.
SN - 2691-1299
ER -