Scientific Publications by FDA Staff

J Appl Microbiol 2014 Jan;116(1):100-8

Preparation of reference stocks suitable for evaluation of alternative NAT-based mycoplasma detection methods.

Dabrazhynetskaya A, Furtak V, Volokhov D, Beck B, Chizhikov V

AIMS: To optimize conditions for preparation and cryopreservation of mycoplasma reference materials suitable to evaluate alternative nucleic acid testing (NAT)-based assays and to compare their limits of detection (LOD) with those of conventional culture-based methods. METHODS AND RESULTS: Acholeplasma laidlawii, Mycoplasma gallisepticum and Mycoplasma arginini stocks with low ratios of genomic copies to colony forming units (12, 8 and 4, respectively) harvested in early stationary phases of growth were preserved with different cryoprotective agents (CPAs) under slow (1° min-1 ), moderate (8° min-1 ), fast (13° min-1 ), and "snap-shot" (60 ° min-1 ) cooling rates. Depending on mycoplasma species, increasing the cooling rate from slow to snap-shot enhanced cell survival up to 5-fold. The addition of 10% (v/v) dimethyl sulfoxide (DMSO) and 15% (v/v) glycerol significantly improved cell survival of all tested strains. Cryoprotected stocks maintained high and stable titers for at least one year during storage at -80°C. Sonication of cell cultures prior to cryopreservation enhanced cell dispersion and reduced of GC/CFU ratios. CONCLUSIONS: It is feasible to prepare stable reference stocks of cryopreserved mycoplasma cells suitable to reliably compare NAT- and culture-based mycoplasma testing methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes experimental results demonstrating the preparation and storage of highly viable and dispersed mycoplasma reference stocks suitable for comparing alternative NAT- and conventional culture-based mycoplasma detection methods.