Introduction of a validation concept for a PCR-based Mycoplasma detection assay. 2006

I Bruchmüller, and E Pirkl, and R Herrmann, and M Stoermer, and H Eichler, and H Klüter, and P Bugert
Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessen, University of Heidelberg, Faculty of Clinical Medicine Mannheim, Mannheim, Germany.

BACKGROUND 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. METHODS 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. RESULTS 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 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale. CONCLUSIONS The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.

UI MeSH Term Description Entries
D009174 Mycoplasma A genus of gram-negative, mostly facultatively anaerobic bacteria in the family MYCOPLASMATACEAE. The cells are bounded by a PLASMA MEMBRANE and lack a true CELL WALL. Its organisms are pathogens found on the MUCOUS MEMBRANES of humans, ANIMALS, and BIRDS. Eperythrozoon,Haemobartonella,Mycoplasma putrefaciens,PPLO,Pleuropneumonia-Like Organisms,Pleuropneumonia Like Organisms
D012107 Research Design A plan for collecting and utilizing data so that desired information can be obtained with sufficient precision or so that an hypothesis can be tested properly. Experimental Design,Data Adjustment,Data Reporting,Design, Experimental,Designs, Experimental,Error Sources,Experimental Designs,Matched Groups,Methodology, Research,Problem Formulation,Research Methodology,Research Proposal,Research Strategy,Research Technics,Research Techniques,Scoring Methods,Adjustment, Data,Adjustments, Data,Data Adjustments,Design, Research,Designs, Research,Error Source,Formulation, Problem,Formulations, Problem,Group, Matched,Groups, Matched,Matched Group,Method, Scoring,Methods, Scoring,Problem Formulations,Proposal, Research,Proposals, Research,Reporting, Data,Research Designs,Research Proposals,Research Strategies,Research Technic,Research Technique,Scoring Method,Source, Error,Sources, Error,Strategies, Research,Strategy, Research,Technic, Research,Technics, Research,Technique, Research,Techniques, Research
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012336 RNA, Ribosomal, 16S Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis. 16S Ribosomal RNA,16S rRNA,RNA, 16S Ribosomal,Ribosomal RNA, 16S,rRNA, 16S
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D015203 Reproducibility of Results The statistical reproducibility of measurements (often in a clinical context), including the testing of instrumentation or techniques to obtain reproducible results. The concept includes reproducibility of physiological measurements, which may be used to develop rules to assess probability or prognosis, or response to a stimulus; reproducibility of occurrence of a condition; and reproducibility of experimental results. Reliability and Validity,Reliability of Result,Reproducibility Of Result,Reproducibility of Finding,Validity of Result,Validity of Results,Face Validity,Reliability (Epidemiology),Reliability of Results,Reproducibility of Findings,Test-Retest Reliability,Validity (Epidemiology),Finding Reproducibilities,Finding Reproducibility,Of Result, Reproducibility,Of Results, Reproducibility,Reliabilities, Test-Retest,Reliability, Test-Retest,Result Reliabilities,Result Reliability,Result Validities,Result Validity,Result, Reproducibility Of,Results, Reproducibility Of,Test Retest Reliability,Validity and Reliability,Validity, Face
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D021161 Self-Sustained Sequence Replication An isothermal in-vitro nucleotide amplification process. The process involves the concomitant action of a RNA-DIRECTED DNA POLYMERASE, a ribonuclease (RIBONUCLEASES), and DNA-DIRECTED RNA POLYMERASES to synthesize large quantities of sequence-specific RNA and DNA molecules. Nucleic Acid Sequence-Based Amplification,NASBA,NASBA Analysis,Analyses, NASBA,Analysis, NASBA,NASBA Analyses,Nucleic Acid Sequence Based Amplification,Replication, Self-Sustained Sequence,Replications, Self-Sustained Sequence,Self Sustained Sequence Replication,Self-Sustained Sequence Replications,Sequence Replication, Self-Sustained,Sequence Replications, Self-Sustained

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