摘要
      任何分子生物学实验室的日常工作都包括日常使用微生物，包括用多种表达至少一种抗生素抗性基因（ARG）的质粒转化的大肠杆菌菌株。
为了验证消毒方法对实验室液体废物的有效性，通过16S核糖体RNA测序鉴定了从实验室和研究所排水沟中分离的细菌，并测试了实验室质粒中常见的复制来源和几种ARG的存在。令人惊讶的是，在非肠杆菌科细菌菌株中检测到肠杆菌科质粒的复制来源，这表明实验室质粒已经发生了种间转移。
使用定量聚合酶链式反应，我们测定了几种去污方法对用卡那霉素抗性质粒（pET28A + 或pEGFP-C2）。硫酸的D值估计值为0.7 M，商用P3消毒剂的D值为6.3%，121°C蒸汽灭菌25分钟，紫外线灭菌49分钟。用最终浓度为1-10%的次氯酸钠处理细菌培养物20分钟，发现在完全破坏细菌质粒基因标记物（编码pBR322复制起源）方面无效。来自HClO处理的细胞的残余DNA为60%，而使用稀释为5%的商用消毒剂P3，其减少到10%以下。由于这两种情况下的降解都不完全，为了防止实验室ARGs水平转移到环境细菌中，如果没有额外的质粒破坏处理，消毒后的液体废物不应释放到污水中。
Abstract
The routine work of any molecular biology laboratory includes the daily use of microorganisms, including strains of E. coli, transformed with a variety of plasmids expressing at least one antibiotic resistance gene (ARG).

To verify the effectiveness of disinfection methods on laboratory liquid waste, bacteria isolated from laboratory and research institute drains were identified by 16S ribosomal RNA sequencing and tested for the presence of an origin of replication and several ARGs frequently found in laboratory plasmids. Surprisingly, the origin of replication of Enterobacteriaceae plasmids was detected in strains of non-Enterobacteriaceae bacteria suggesting that interspecific transfer of laboratory plasmids had occurred.

Using quantitative Polymerase Chain Reaction, we determined the Decimal reduction value (D-value, expressed as concentration of disinfectant or length of physical treatment) of several decontamination methods for their DNA degradation effect on cultures of E. coli Top10 transformed with a kanamycin resistant plasmid (pET28A + or pEGFP-C2). The estimated D-values were 0,7 M for Sulfuric, 6,3% for a commercial P3 disinfectant, 25 minutes for steam sterilization at 121°C and 49 minutes for disinfection by UVC. A 20-minute treatment of bacteria cultures with a final concentration of 1–10% sodium hypochlorite was found to be ineffective in completely destroying a bacteria plasmid gene marker (coding for the pBR322 origin of replication). Residual DNA from HClO treated cells was 60%, while it decreased under 10% using the commercial disinfectant P3 diluted at 5%. As the degradation was uncomplete in both cases, to prevent the horizontal transfer of laboratory ARGs to environmental bacteria, disinfected liquid waste should not be released in sewage without additional plasmid destruction treatment.

https://www.researchsquare.com/article/rs-2609208/v1