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Updated: Jun 23, 2026
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
Comparison of Five Commercial Molecular Assays for Mycoplasma Testing of Cellular Therapy Products
Arthur H Totten1, Anna Julia Adams2, Hyunmi K Halas3
1Clinical Microbiology Service, Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland, USA.
Abstract:
Testing of cellular therapy products for Mycoplasma is a regulatory requirement by the United States Food and Drug Administration (FDA) to ensure the sterility and safety of the product prior to release for patient infusion. The risk of Mycoplasma contamination in cell culture is high. Gold standard testing follows USP 63 which requires a 28-day agar and broth cultivation method that is impractical for short shelf-life biologics. Several commercial molecular platforms have been marketed for faster raw material and product release testing; however, little performance data are available in the literature. In this study, we performed a proof-of-principle analysis to evaluate the performance of five commercial molecular assays, including the MycoSEQ Mycoplasma detection kit (Life Technologies), the MycoTOOL Mycoplasma real-time detection kit (Roche), the VenorGEM qOneStep kit (Minerva Biolabs), the ATCC universal Mycoplasma detection kit, and the Biofire Mycoplasma assay (bioMérieux Industry) using 10 cultured Mollicutes spp., with each at four log-fold dilutions (1,000 CFU/mL to 1 CFU/mL) in biological duplicates with three replicates per condition (n = 6) to assess limit of detection (LOD) and repeatability. Additional testing was performed in the presence of tumor infiltrating lymphocytes (TILs). Based on LOD alone, the Biofire Mycoplasma assay was most sensitive followed by the MycoSEQ and MycoTOOL which were comparable. We showed that not all assays were capable of meeting the ≤10 CFU/mL LOD to replace culture-based methods according to European and Japanese pharmacopeia standards. No assay interference was observed when testing in the presence of TILs.
Insights
Regulatory testing for Mycoplasma contamination in cellular therapies is crucial. This study evaluated five commercial molecular assays, finding the Biofire assay most sensitive, but not all met pharmacopeia standards for replacing traditional culture methods.
Area of Science:
- Microbiology
- Cellular Therapy
- Molecular Diagnostics
Background:
- Mycoplasma contamination poses a significant risk to cellular therapy products, necessitating rigorous testing for sterility and safety.
- Current gold standard testing methods (USP 63) involve 28-day cultivation, which is impractical for short shelf-life biologics.
- Faster molecular assays are available, but limited performance data hinders their adoption for raw material and product release.
Purpose of the Study:
- To evaluate the performance, specifically the limit of detection (LOD) and repeatability, of five commercial molecular assays for Mycoplasma detection.
- To assess the suitability of these assays for replacing traditional culture-based methods based on pharmacopeial standards.
- To investigate potential interference from tumor-infiltrating lymphocytes (TILs) during testing.
Main Methods:
- Proof-of-principle analysis of five commercial Mycoplasma detection kits: MycoSEQ, MycoTOOL, VenorGEM qOneStep, ATCC universal, and Biofire.
- Testing involved serial dilutions (1,000 CFU/mL to 1 CFU/mL) of 10 cultured Mollicutes species in biological duplicates (n=6).
- Assays were also tested in the presence of tumor-infiltrating lymphocytes (TILs) to assess interference.
Main Results:
- The Biofire Mycoplasma assay demonstrated the highest sensitivity (lowest LOD), followed by MycoSEQ and MycoTOOL, which showed comparable performance.
- Not all evaluated molecular assays met the ≤10 CFU/mL LOD requirement specified by European and Japanese pharmacopeias for replacing culture methods.
- No significant assay interference was observed when testing Mycoplasma in the presence of TILs.
Conclusions:
- Commercial molecular assays offer faster alternatives for Mycoplasma detection in cellular therapies compared to traditional culture methods.
- Assay sensitivity varies, with Biofire, MycoSEQ, and MycoTOOL showing promising performance, though not all meet stringent pharmacopeial LOD requirements.
- The tested molecular assays are suitable for use with tumor-infiltrating lymphocytes, indicating their potential utility in clinical settings.
