细菌DNA特征序列鉴定法在常见种属中的鉴定水平研究

目的：开展细菌DNA特征序列鉴定法在药品质量控制中常见种属的鉴定水平研究，为药品全生命周期质量控制中的菌株鉴定与污染风险识别提供方法依据。方法：针对性地选择芽孢杆菌、库克氏菌、微球菌、柠檬酸杆菌、肠杆菌、肠球菌、假单胞菌、不动杆菌、鞘氨醇单胞菌和罗尔斯通氏菌共10个常见属中41个种72株的典型菌株，依据细菌DNA特征序列鉴定法，建立菌株鉴定的DNA特征序列，分析属内种间的菌株聚类关系，评价菌株DNA特征序列的鉴定水平。结果：10个属41个种典型菌株的DNA特征序列均具有特异性。结论：细菌DNA特征序列可满足"种"水平的鉴定，为药品质量控制中常见细菌的鉴定、分类与溯源提供研究思路和方法依据。

Objective: To study on the identification level of common bacteria in the drug quality control by DNA signature sequence, and to provide reference for the identification of bacterial strains and contamination risk investigation of the whole life cycle quality control of drugs. Methods: 72 strains in 41 species in 10 common genus were selected for study, including Bacillus, Kocuria, Micrococcus, Citrobacter, Enterobacter, Enterococcus, Pseudomonas, Acinetobacter, Sphingomonas and Rolstonia. According to the bacterial DNA signature sequence identification method, the bacterial DNA signature sequences were established, the cluster relationship between the species within the genus were analyzed, and the strain identification level was evaluated. Results: The DNA signature sequences which identified by 72 strains of 14 species in 10 genus were respectively specificity in the level of species. Conclusion: The bacterial DNA signature sequence can identify bacterial in the level of species. The results may provide research ideas and methodological basis for the identification, classification and traceability of common bacteria in drug quality control.

[1] PAUL DV,GARRITY GM,JONES D,et al.Bergey's Manual of Systematic Bacteriology.VolⅡ[M].Berlin:Springer Berlin Heidelberg,2011:587
[2] USP 41-NF 36[S].2019:1419
[3] 中华人民共和国药典2015年版.四部[S].2015:393 ChP 2015.Vol Ⅳ[S].2015:393
[4] FERGUSON A,PATEL A,BAIRD RM.A comparison of two incubation temperatures for the isolation of gram-negative contaminants from raw materials and non-sterile pharmaceuticals[J].J Clin Pharm Ther,1987,12(4):249
[5] HUGHES KA,CORNWALL J,THEIS JC,et al.Bacterial contamination of unused,disposable non-sterile gloves on a hospital orthopaedic ward[J].Australas Med J,2013,30(6):331
[6] COBLEY M,ATKINS M,JONES PL.Environmental contamination during tracheal suction.A comparison of disposable conventional catheters with a multiple-use closed system device[J].Anaesthesia,1991,46(11):957
[7] KREMER T,PATEL R.Correlation between environmental monitoring and product bioburden[J].Biomed Instrum Technol,2019,53(s2):32
[8] 冯震,李芳,宋明辉,等.16S核糖体RNA基因片段分析及在药品葡萄球菌污染控制应用中的探讨[J].药物分析杂志,2018,38(4):690 FENG Z,LI F,SONG MH,et al.A detailed analysis of 16S ribosomal RNA gene segments for identification of Staphylococcus contaminants in pharmaceutical quality control[J].Chin J Pharm Anal,2018,38(4):690
[9] TOYAMA T,KURODA M,OGATA Y,et al.Enhanced biomass production of duckweeds by inoculating a plant growth-promoting bacterium,Acinetobacter calcoaceticus P23,in sterile medium and non-sterile environmental waters[J].Water Sci Technol,2017,76(6):1418
[10] MORENO-FORERO SK,van der MEER JR.Genome-wide analysis of Sphingomonas wittichii RW1 behaviour during inoculation and growth in contaminated sand[J].ISME J,2015,9(1):150
[11] 洪小栩,冯震,许华玉,等.分子生物学技术用于药品质量控制国家标准核酸数据库的建设规划研究[J].中国医药工业杂志,2016,47(5):651 HONG XX,FENG Z,XU HY,et al.Program of national standard nucleotide sequence database used in quality control of pharmaceutical products by molecular biology technology[J].Chin J Pharm,2016,47(5):651
[12] KIM M,HS O,PARK SC,et al.Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[J].Int J Syst Evol Microbiol,2014,64(2):346
[13] PETTI CA,POLAGE CR,SCHRECKENBERGER P.The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods[J].J Clin Microbiol,2005,43(12):6123
[14] NILSSON RH,RYBERG M,KRISTIANSSON E,et al.Taxonomic reliability of DNA sequences in public sequence databases:a fungal perspective[J].PLoS One,2006,1(1):e59
[15] MILLER SE.DNA barcoding and the renaissance of taxonomy[J].Proc Natl Acad Sci USA,2007,104(12):4775