Advanced therapy medicinal products (ATMP) are required to maintain their quality and safety throughout the production cycle, and they must be free of microbial contaminations. Among them, mycoplasma contaminations are difficult to detect and undesirable in ATMP, especially for immunosuppressed patients. Mycoplasma detection tests suggested by European Pharmacopoeia are the “culture method” and “indicator cell culture method” which, despite their effectiveness, are time consuming and laborious. Alternative methods are accepted, provided they are adequate and their results are comparable with those of the standard methods.

To validate a novel in-house method, we performed and optimized, a real time PCR protocol, using a commercial kit and an automatic extraction system, in which we tested different volumes of matrix, maximizing the detection sensitivity. The results were compared with those obtained with the gold standard methods.

From a volume of 10 ml, we were able to recognize all the mycoplasmas specified by the European Pharmacopoeia, defined as genomic copies per colony forming unit ratio (GC/CFU).

Our strategy allows to achieve faster and reproducible results when compared with conventional methods and meets the sensitivity and robustness criteria required for an alternative approach to mycoplasmas detection for in-process and product-release testing of ATMP.

需要高级治疗药物（ATMP）来在整个生产周期中保持其质量和安全性，并且它们必须没有微生物污染。其中，支原体污染难以检测，在ATMP中是不可取的，特别是对于免疫抑制患者。欧洲药典建议的支原体检测方法是“培养方法”和“指示细胞培养方法”，尽管它们有效，但既费时又费力。可以接受其他方法，只要它们足够且结果与标准方法相当即可。

为了验证一种新颖的内部方法，我们使用商业试剂盒和自动提取系统执行并优化了实时PCR方案，在其中我们测试了不同体积的基质，从而最大程度地提高了检测灵敏度。将结果与通过金标准方法获得的结果进行比较。

从10 ml的体积中，我们能够识别出欧洲药典规定的所有支原体，定义为每菌落形成单位比（GC / CFU）的基因组拷贝。

与常规方法相比，我们的策略可实现更快，可重现的结果，并满足ATMP进行过程中和产品释放测试的支原体检测替代方法所需的灵敏度和鲁棒性标准。