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

目的：建立细菌16S核糖体RNA基因片段分析的规范化方法，探讨其在药品葡萄球菌污染控制中的应用。方法：对Genbank数据库中用于常见细菌污染物鉴定的核酸序列进行筛选、定位和分区比较分析，建立具有鉴定和分类意义的标准核酸序列片段；以葡萄球菌为例进行菌株的核酸测序鉴定，分析菌株间的聚类关系和核酸序列的多样性。结果：16S rRNA基因中的V1~V3可变区具有显著的鉴定和分类意义，可实现葡萄球菌属20个“种”水平的鉴定。结论：本文对16S rRNA基因核酸测序鉴定规范化方法及在制药行业中葡萄球菌污染控制的应用进行探讨，为细菌核酸测序鉴定标准化和标准核酸序列数据库建设提供研究思路和参考依据。

Objective: To analyze variable region segments of bacterial 16S ribosomal RNA gene,and optimize criteria nucleotide sequence for Staphylococcus identification in drug production and examination environment.Methods: Nucleotide sequences of critical bacterial contaminants in drug production from Genbank nucleotide database were screened,positioned and analyzed,and criteria sequence was established.Staphylococcus isolates were collected and identified using sequencing method,and its sequence was analyzed by gene clustering method and polymeric method.Results: V1-V3 region sequences of 16S ribosomal RNA gene had the perfect capability in bacterial identification,and this method could specifically classify 20 species of Staphylococcus.Conclusion: The criteria gene sequencing method for identifying bacterial contaminants were analyzed and discussed,it may provide a reference data in establishing criteria procedure and nucleotide sequence database for drug quality control.

[1] PAUL DV,GARRITY GM,JONES D,et al.Bergey's Manual of Systematic Bacteriology.Vol Ⅲ[M].Berlin:Springer Berlin Heidelberg,2011:1
[2] USP 40-NF 35[S].2017:1419
[3] JP 17[S].2017:2220
[4] 中华人民共和国药典2015年版.四部[S].2015:393 ChP 2015.Vol Ⅳ[S].2015:393
[5] CHAN ER,HESTER J,KALADY M,et al.A novel method for determining microflora composition using dynamic phylogenetic analysis of 16S ribosomal RNA deep sequencing data[J].Genomics,2011,98(4):253
[6] KATAOKA M,UEDA K,KUDO T,et al.Application of the variable region in 16S rDNA to create an index for rapid species identification in the genus Streptomyces[J].FEMS Microbiol Lett,1997,151(2):249
[7] BECKER K,HARMSEN D,MELLMANN A,et al.Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species[J].J Clin Microbiol,2004,42(11):4988
[8] ENGELBREKTSON A,KUNIN V,WRIGHTON KC,et al.Experimental factors affecting PCR-based estimates of microbial species richness and evenness[J].ISME J,2010,4(5):642
[9] KIM M,MORRISON M,YU ZT.Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes[J].J Microbiol Methods,2011,84(1):81
[10] CHAKRAVORTY S,HELB D,BURDAY M,et al.A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria[J].J Microbiol Methods,2007,69(2):330
[11] LU JJ,PERNG CL,LEE SY,et al.Use of PCR with universal primers and restriction endonuclease digestions for detection and identification of common bacterial pathogens in cerebrospinal fluid[J].J Clin Microbiol,2000,38(6):2076
[12] HEBERT PD,CYWINSKA A,BALL SL,et al.Biological identifications through DNA barcodes[J].Proc Biol Sci,2003,270(1512):313
[13] HEBERT PD,PENTON EH,BURNS JM,et al.Ten species in one:DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator[J].Proc Natl Acad Sci USA,2004,101(41):14812
[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
[16] CHEN SL,YAO H,HAN JP,et al.Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J].PloS One,2010,5(1):e8613
[17] 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
[18] 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
[19] 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