Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Pathog Dis. 2022 Jan 12;79(9). doi: 10.1093/femspd/ftab056.
Mycoplasma contamination of cell culture represents a serious problem in research and decontamination from cell-propagated obligate intracellular bacteria has proven challenging. Here, we presented an optimized protocol to remove Mycoplasma from contaminated Chlamydia trachomatis culture. A stepwise procedure of Mycoplasma removal entails (i) incubation in nonionic detergent-containing solution and (ii) separation of viable chlamydial organisms by fluorescence-activated cell sorting (FACS), followed by subcloning using a focus-forming assay. We also adapted a polymerase chain reaction (PCR) assay using paired universal and Mycoplasma-specific primers, which are distinguishable from the C. trachomatis counterparts, in combination with Sanger sequencing to determine the presence of mycoplasmas' 16S rRNA genes. These integrated approaches allow for full removal of Mycoplasma, as verified by the improved PCR assay, without compromising the capacity of viable C. trachomatis to adapt to new infection in epithelial cells. Some pitfalls during the Mycoplasma decontamination process are discussed.
支原体污染细胞培养是研究中的一个严重问题，从细胞传播的专性内寄生菌中去除支原体已被证明具有挑战性。在这里，我们提出了一种优化的方案，用于从污染的沙眼衣原体培养物中去除支原体。去除支原体的分步程序包括：（i）在含有非离子型洗涤剂的溶液中孵育；（ii）通过荧光激活细胞分选（FACS）分离存活的衣原体生物，然后使用焦点形成测定法进行亚克隆。我们还使用配对的通用和支原体特异性引物，与沙眼衣原体对应的引物区分开来，结合聚合酶链反应（PCR）检测，对改良的 PCR 检测进行了改编，以确定支原体 16S rRNA 基因的存在。这些综合方法可确保完全去除支原体，正如改良的 PCR 检测所证实的那样，同时不影响存活的沙眼衣原体适应上皮细胞新感染的能力。讨论了支原体去除过程中的一些注意事项。
