AIMS: The aim of this study was 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 (LODs) 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°C min(-1)), moderate (8°C min(-1)), fast (13°C min(-1)) and 'snapshot' (60°C min(-1)) cooling rates. Depending on mycoplasma species, increasing the cooling rate from slow to snapshot 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 titres for at least 1 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.
AIMS: The aim of this study was 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 (LODs) 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°C min(-1)), moderate (8°C min(-1)), fast (13°C min(-1)) and 'snapshot' (60°C min(-1)) cooling rates. Depending on mycoplasma species, increasing the cooling rate from slow to snapshot 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 titres for at least 1 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.
Authors: Lisa Dreolini; Mark Cullen; Eric Yung; Lawrence Laird; John R Webb; Brad H Nelson; Kevin A Hay; Miruna Balasundaram; Natasha Kekre; Robert A Holt Journal: Mol Ther Methods Clin Dev Date: 2020-01-30 Impact factor: 6.698