由于新的基因编辑工具,DNA和异种元素在水道上的传播受到科学和公众的关注,例如自己动手(DIY)可在厨房桌子上部署的CRISPR-Cas套件。几十年来,防止转基因生物(GMOs)的传播,抗微生物耐药性(AMR),来自转基因系统的病原体集中在微生物灭活上。然而,灭菌方法尚未评估DNA释放和完整性。这里,我们研究了传统上用作微生物底盘进行遗传修饰的模型原核(大肠杆菌)和真核(酿酒酵母)细胞培养物中细胞内DNA的命运。在这些培养物暴露于常规灭菌方法期间跟踪DNA释放。高压灭菌,用戊二醛消毒,微波炉用来灭活肉汤,医疗保健设备,和在厨房桌子上生产的转基因生物。在细胞活力和形态之上测量DNA片段化和PCR能力。在游离λDNA的模板上验证了这些方法对DNA完整性的影响。剧烈的常规高压灭菌(121°C,20分钟)导致最严重的DNA降解和最低的家庭基因扩增能力:与未处理的对照相比,从大肠杆菌测得的对数差异为1.28±0.11、2.08±0.03和4.96±0.28,酿酒酵母,和λDNA,分别。当游离λDNA在溶液中时,微波在暴露100s后产生强烈的DNA片段化(3.23±0.06log差异),但当DNA从大肠杆菌和酿酒酵母中释放时观察到较小的影响(0.24±0.14和1.32±0.02log差异与对照,分别)。戊二醛通过保留细胞结构来防止DNA泄漏,而DNA完整性没有改变。结果表明,目前的灭菌方法对微生物灭活是有效的,但不能保护水性残留物免除生物可重复使用的异种材料,定期高压灭菌是影响DNA最严重的方法。需要对灭菌方法进行重新评估,并对城市系统和自然界中DNA片段的排放进行风险评估。
The dissemination of DNA and xenogenic elements across waterways is under scientific and public spotlight due to new gene-editing tools, such as do-it-yourself (DIY) CRISPR-Cas kits deployable at kitchen table. Over decades, prevention of spread of genetically modified organisms (GMOs), antimicrobial resistances (AMR), and pathogens from transgenic systems has focused on microbial inactivation. However, sterilization methods have not been assessed for DNA release and integrity. Here, we investigated the fate of intracellular DNA from cultures of model prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) cells that are traditionally used as microbial chassis for genetic modifications. DNA release was tracked during exposure of these cultures to conventional sterilization methods. Autoclaving, disinfection with glutaraldehyde, and microwaving are used to inactivate broths, healthcare equipment, and GMOs produced at kitchen table. DNA fragmentation and PCR-ability were measured on top of cell viability and morphology. Impact of these methods on DNA integrity was verified on a template of free λ DNA. Intense regular autoclaving (121°C, 20 min) resulted in the most severe DNA degradation and lowest household gene amplification capacity: 1.28 ± 0.11, 2.08 ± 0.03, and 4.96 ± 0.28 logs differences to the non-treated controls were measured from E. coli, S. cerevisiae, and λ DNA, respectively. Microwaving exerted strong DNA fragmentation after 100 s of exposure when free λ DNA was in solution (3.23 ± 0.06 logs difference) but a minor effect was observed when DNA was released from E. coli and S. cerevisiae (0.24 ± 0.14 and 1.32 ± 0.02 logs differences with the control, respectively). Glutaraldehyde prevented DNA leakage by preserving cell structures, while DNA integrity was not altered. The results show that current sterilization methods are effective on microorganism inactivation but do not safeguard an aqueous residue exempt of biologically reusable xenogenic material, being regular autoclaving the most severe DNA-affecting method. Reappraisal of sterilization methods is required along with risk assessment on the emission of DNA fragments in urban systems and nature.