de Freitas Michelon Nathalia, Júnior José Valter Joaquim Silva, Weiblen Rudi, Flores Eduardo Furtado
Programa de Pós-Graduação Em Medicina Veterinária, Universidade Federal de Santa Maria, Rio Grande Do Sul, Brazil.
Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Av. Roraima, 1000, Prédio 63A, Camobi, Santa Maria, Rio Grande Do Sul, Brazil.
Folia Microbiol (Praha). 2025 Jul 11. doi: 10.1007/s12223-025-01297-x.
Mycoplasma spp. contamination is a major concern in laboratories handling cell cultures, and routine detection methods are usually time-consuming, laborious and lack sensitivity. This study presents a streamlined workflow integrating rapid thermal DNA extraction (99 °C min) with a SYBR Green-based qPCR for Mycoplasma detection. High-coverage primers targeting an 86-bp region of the 16S rDNA were designed using 109 Mycoplasma spp. sequences from GeneBank. In silico analysis confirmed full primer annealing to major cell culture contaminants (M. arginini, M. hominis, M. orale, and M. hyorhinis). Upon thermal lysis and qPCR optimization, the yield of the protocol was equivalent to that of phenol-chloroform extraction plus qPCR, with a detection limit of 64 bacterial cells. Finally, the performance of the protocol was confirmed in cell cultures with known Mycoplasma spp. contamination, accurately reproducing the contamination status. Thus, the developed protocol provides a simple, rapid, cost-effective, and sensitive method for monitoring Mycoplasma spp. in cell cultures.
支原体属污染是细胞培养实验室的一个主要问题，常规检测方法通常耗时、费力且缺乏灵敏度。本研究提出了一种简化的工作流程，将快速热DNA提取（99°C 分钟）与基于SYBR Green的qPCR相结合用于支原体检测。使用来自基因库的109个支原体属序列设计了靶向16S rDNA 86bp区域的高覆盖引物。计算机分析证实引物能与主要的细胞培养污染物（精氨酸支原体、人型支原体、口腔支原体和猪鼻支原体）完全退火。经过热裂解和qPCR优化后，该方法的产量与酚-氯仿提取加qPCR相当，检测限为64个细菌细胞。最后，在已知支原体属污染的细胞培养物中证实了该方法的性能，准确再现了污染状况。因此，所开发的方法为监测细胞培养物中的支原体属提供了一种简单、快速、经济高效且灵敏的方法。
