title: Preparation of reference strains for validation and comparison of mycoplasma testing methods.
abstract: OBJECTIVE
  To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)-based methods in comparison to the conventional microbiological methods.
  
  
  RESULTS
  A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤ 10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co-cultured with suspension of Chinese hamster ovary (CHO) cells.
  
  
  CONCLUSIONS
  Tested mycoplasma strains harvested at the exponential-early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study.
  
  
  CONCLUSIONS
  This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT-based mycoplasma testing methods.
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20852, USA. alena.dabrazhynetskaya@fda.hhs.gov
authors.forename: A
authors.initials: A
authors.lastname: Dabrazhynetskaya
authors.forename: D V
authors.initials: DV
authors.lastname: Volokhov
authors.forename: S W
authors.initials: SW
authors.lastname: David
authors.forename: P
authors.initials: P
authors.lastname: Ikonomi
authors.forename: A
authors.initials: A
authors.lastname: Brewer
authors.forename: A
authors.initials: A
authors.lastname: Chang
authors.forename: V
authors.initials: V
authors.lastname: Chizhikov
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D016466:CHO Cells; D018920:Coculture Techniques; D015169:Colony Count, Microbial / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D004269:DNA, Bacterial / Q000235:genetics; D005069:Evaluation Studies as Topic; D018628:Gene Dosage; D057230:Limit of Detection; D009174:Mycoplasma / Q000235:genetics / Q000254:growth & development*; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D054928:Validation Studies as Topic
chemical_list: D004269:DNA, Bacterial