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Updated: May 9, 2026
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
Collaborative study report: evaluation of the ATCC experimental mycoplasma reference strains panel prepared for
Alena Dabrazhynetskaya1, Dmitriy V Volokhov, Tsai-Lien Lin
1CBER, FDA, Division of Viral Products, 1401 Rockville Pike, Rockville, MD 20852-1448, USA.
Abstract:
The main goal of this collaborative study was to evaluate the experimental panel of cryopreserved mycoplasma reference strains recently prepared by the American Type Culture Collection (ATCC(®)) in order to assess the viability and dispersion of cells in the mycoplasma stocks by measuring the ratio between the number of genomic copies (GC) and the number of colony forming units (CFU) in the reference preparations. The employment of microbial reference cultures with low GC/CFU ratios is critical for unbiased and reliable comparison of mycoplasma testing methods based on different methodological approaches, i.e., Nucleic Acid Testing (NAT) and compendial culture-based techniques. The experimental panel included ten different mycoplasma species known to represent potential human and animal pathogens as well as common contaminants of mammalian and avian cell substrates used in research, development, and manufacture of biological products. Fifteen laboratories with expertise in field of mycoplasma titration and quantification of mycoplasmal genomic DNA participated in the study conducted from February to October of 2012. The results of this study demonstrated the feasibility of preparing highly viable and dispersed (possessing low GC/CFU ratios) frozen stocks of mycoplasma reference materials, required for reliable comparison of NAT-based and conventional mycoplasma detection methods.
Insights
This study evaluated cryopreserved mycoplasma reference strains, crucial for comparing Nucleic Acid Testing (NAT) and culture-based methods. Results show these reference materials have low genomic copy (GC) to colony forming unit (CFU) ratios, ensuring reliable mycoplasma detection.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Mycoplasma contamination poses risks in biological products.
- Accurate mycoplasma detection methods are essential for quality control.
- Standardized reference materials are needed to compare Nucleic Acid Testing (NAT) and culture-based techniques.
Purpose of the Study:
- To evaluate cryopreserved mycoplasma reference strains from ATCC.
- To assess cell viability and dispersion using the genomic copy (GC) to colony forming unit (CFU) ratio.
- To determine the suitability of these strains for validating mycoplasma testing methods.
Main Methods:
- Preparation of cryopreserved mycoplasma reference strains.
- Quantification of genomic copies (GC) and colony forming units (CFU) for ten mycoplasma species.
- Multi-laboratory collaborative study involving mycoplasma titration and DNA quantification expertise.
Main Results:
- Demonstrated feasibility of preparing viable and dispersed mycoplasma reference stocks.
- Achieved low GC/CFU ratios in the experimental panel.
- Confirmed the utility of these reference materials for method comparison.
Conclusions:
- Cryopreserved mycoplasma reference materials with low GC/CFU ratios can be reliably prepared.
- These reference materials are critical for the unbiased comparison of NAT and culture-based mycoplasma detection methods.
- The study supports the use of these ATCC reference strains for mycoplasma testing validation.
