细胞污染物浅黄假单胞菌的鉴定及处理

Identification and treatment of cell contamination caused by Pseudomonas luteola

目的鉴定实验中导致细胞污染的病原微生物,筛选消除该类病原微生物的抗生素药物。方法收集污染细胞上清,离心沉淀后提取核酸,使用细菌鉴定用的16S rDNA通用引物进行PCR扩增,回收产物后连接至pMD-18T载体,筛选阳性菌落测序,通过序列比对确定细胞污染物的类别,根据病原物的类别进一步筛选抗生素并摸索合适的抗生素使用浓度,以期达到消除病原物且不影响细胞正常生长的目的。结果 16S rDNA测序结果表明,细胞受到支原体和浅黄假单胞菌双重感染。抗生素处理结果表明,10μg/m L环丙沙星、10μg/m L美罗培南和10μg/m L新诺明联用可消除浅黄假单胞菌的污染,但处理后的细胞生长机能变差,需要在正常培养条件下培养并传代2次以上才会恢复正常。结论环丙沙星、美罗培南和新诺明联用能很好地消除浅黄假单胞菌对实验细胞的污染。

Objective To identify the pathogens causing cell contamination in cell culture and screen antibiotics to eliminate the pathogen. Methods The supernatant of contaminated cells was collected and precipitated by centrifugation,from which nucleic acid was extracted and identified by PCR using universal primers of 16 S r DNA. The PCR products were inserted into vector p MD-18 T,and the positive colonies were identified by sequencing and classified by sequence alignment. According to the classification of cell pollutants,the corresponding antibiotics were further screened,of which the concentration was optimized so as to eliminate the pathogen without affecting the normal growth of the cells. Results The 16 s r DNA sequencing result showed that the cells were infected with Mycoplasma and Pseudomonas luteola. Antibiotic treatment result indicated that the contamination with Pseudomonas luteola was eliminated by 10 μg/m L ciprofloxacin,10 μg/m L meropenem and 10 μg/m L sulfamethoxazole. However,the growth function of cells after treatment was damaged,which was recovered to normality after culture and subculture for more than two passages under normal culture condition. Conclusion The combined use of ciprofloxacin,meropenem and sulfamethoxazole may eradicate the contamination of experimental cells with Pseudomonas luteola.