目的利用实时荧光定量PCR (quantitative PCR，qPCR)建立支原体的快速检测方法。
方法选择欧洲药典10.0版2.6.7规定可用于验证的8种支原体，根据支原体16S RNA序列设计特异性引物，建立支原体qPCR检测方法，并验证其特异性、灵敏度和重复性。
结果成功筛选出采用qPCR检测支原体的合适引物，8种支原体在浓度为10 5~10 6 CFU/mL时，循环阈值(cycle threshold ,CT)在20~30，相同浓度的细菌对照无CT。除精氨酸支原体的可检测浓度为10~100 CFU/mL外，其他7种支原体可检测浓度均达到10 CFU/mL，10 CFU/mL的支原体CT在35~40之间。特异性检测实验重复3次，对照细菌全部未检出，10 5~10 6 CFU/mL的上述8种支原体CT均在20~30，每种支原体3次特异性数据变异系数均小于10%。灵敏度实验重复3次，每次实验各支原体不同稀释浓度CT维持在20~40，变异系数均小于10%。
结论建立的支原体检测qPCR方法特异性较好、灵敏度较高且重复性较好，可为生物制品中可能存在的支原体污染风险的及时控制提供帮助。
ObjectiveTo establish a rapid method for the detection of mycoplasma using real-time quantitative PCR (qPCR) technology.
MethodsEight mycoplasma species available for validation under European Pharmacopoeia（10.0 edition）2.6.7 were selected. Specific primers were designed based on mycoplasma 16S RNA sequence. A qPCR method for the detection of mycoplasma was established and validated for specificity, sensitivity and reproducibility.
ResultsSuitable primers for the detection of mycoplasma by qPCR were successfully screened. Eight mycoplasma species showed a cycle threshold (CT) between 20-30 at a concentration of 10 5-10 6 CFU/mL, and there was no CT for the bacterial control at same concentration. Except for Mycoplasma sperminum, which could be detected at a concentration of 10-100 CFU/mL, the other 7 mycoplasma species could be detected at a concentration of 10 CFU/mL. CTs were between 35-40 at 10 CFU/mL. In specificity experiment that repeated 3 times, the control bacteria were all undetected, CTs of 8 species within 10 5-10 6 CFU/mL were between 20-30, and for each species, the coefficient of variation (CV) for 3 times was less than 10%. In sensitivity experiment that repeated 3 times, the CTs of each species with different dilution concentrations were between 20-40 in each experiment, and the CVs were less than 10%.
ConclusionThe established method has good specificity, sensitivity and reproducibility, and can support timely control of possible mycoplasma contamination risks in biologics.
马鑫宇,蔡秋龙,肖飞,等. 支原体实时荧光定量PCR检测方法的建立[J]. 国际生物制品学杂志,2025,48(01)：39-45.
DOI:10.3760/cma.j.cn311962-20240620-00043版权归中华医学会所有。
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马鑫宇：实验设计、研究实施、数据采集、数据分析、论文撰写、统计分析；蔡秋龙：实验设计、研究实施、数据采集、数据分析、论文撰写、论文修改、统计分析、经费获取、技术支持、研究指导、支持性贡献；肖飞：实验设计、研究实施、数据采集、论文撰写、统计分析；舒聪妍：实验设计、论文修改、统计分析、经费获取、技术支持、研究指导；袁月、陈婕、宋杰：实验设计、论文修改、经费获取、技术支持、研究指导、支持性贡献；常亚军：实验设计，论文修改，经费获取，技术支持，研究指导；王艳萍：实验设计、研究实施、数据采集、论文撰写、统计分析、支持性贡献；吴爽靖、周建宏：实验设计、研究实施、论文撰写、统计分析、支持性贡献；樊海青、熊增平：实验设计、数据采集、论文修改、统计分析、支持性贡献；高有：实验设计、论文修改、统计分析、支持性贡献；王灿斌：研究实施、数据采集、论文修改、统计分析、支持性贡献
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