Introduction of a validation concept for a PCR-based Mycoplasma detection assay

Cytotherapy
I BruchmüllerP Bugert

Abstract

Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept. The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values. The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-184...Continue Reading

References

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Citations

Sep 2, 2010·Journal of Basic Microbiology·Kyung Ah CheongAi-Young Lee
Dec 21, 2006·Veterinary Microbiology·Agata BaczynskaGunna Christiansen
Oct 12, 2013·Journal of Applied Microbiology·A DabrazhynetskayaV Chizhikov

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