8 5 Vol. 8 No. 5 2017 5 Journal of Food Safety and Quality May., 2017 - 李可, 张晓峰 *, 吴姗, 方莹, 帅江冰, 杨兰花 (, 310016) 摘要 : 目的 (Pseudomonas aeruginosa) 方法 16S rrna (peptide nucleic acid, PNA) PA-16S-1,, (peptide nucleic acid-fluorescence in situ hybridization, PNA-FISH) 结果 PNA-FISH 300, 2 1, 结论 PNA-FISH, 关键词 : -; ; ; Rapid identification of Pseudomonas aeruginosa by peptide nucleic acid fluorescence in-situ hybridization LI Ke, ZHANG Xiao-Feng *, WU Shan, FANG Ying, SHUAI Jiang-Bing, YANG Lan-Hua (Zhejiang Entry-Exit Inspection and Quarantine Bureau, Hangzhou 310016, China) ABSTRACT: Objective To establish a method for rapid detection of Pseudomonas aeruginosa in cosmetics. Methods The Pseudomonas aeruginosa species-specific peptide nucleic acid (PNA) probe PA-16S-1 was designed based on the conserved regions in 16S rrna sequences, and its specificity and sensitivity were experimentally verified. The peptide nucleic acid fluorescence in-situ hybridization (PNA-FISH) method was developed for specific detection of Pseudomonas aeruginosa. Results Three hundred cosmetic samples were detected by PNA-FISH, 2 strains of Pseudomonas aeruginosa and 1 strain of Staphylococcus aureus positive samples were identified, which were exactly the same as the results of traditional biochemical identification. Conclusion The established PNA-FISH identification method provides a fast, sensitive, and specific detection of pathogenic bacteria in cosmetics, which has an important significance to improve the sanitary quality of cosmetics. KEY WORDS: peptide nucleic acid-fluorescence in situ hybridization; identification; Pseudomonas aeruginosa; cosmetics 基金项目 : (2015IK301, 2016IK284)(2015C33042) Fund: Supported by the General Administration of Quality Supervision, Inspection and Quarantine (2015IK301, 2016IK284) and Science and Technology Program of Zhejiang Province (2015C33042) * 通讯作者 :,,,. E-mail: zxf@ ziq.gov.cn *Corresponding author: ZHANG Xiao-Feng, Ph.D, Professor, Zhejiang Entry-Exit Inspection and Quarantine Bureau, 126 Fuchun Rd, Hangzhou 310016, China. E-mail: zxf@ ziq.gov.cn
1710 8 1 引言,, [1],,,,,,,, (2015 ) [2],,, [3,4], Anelich [5] 58, 30% 2005 2008 5, 24, 42% [6],,,,,,, [7] (peptide nucleic acid, PNA) (DNA), rrna, DNA,, DNA,, DNA, [8,9] (fluorescent in situ hybridization, FISH),, RNA,,, PNA-FISH, FISH PNA-FISH PNA, 2 材料与方法 2.1 材料 3 (ATCC 27853 ATCC 9027 ATCC 15442) 9 ( ATCC 1388328 ATCC 29930 ATCC 51329 ATCC 25922 O157:H7 NCTC 12900 ATCC14028 EGD ATCC 6538 ATCC 25923) 300 ( ) 2.2 仪器与试剂 Friocell 222 ( MMM ); BX61 ( Olympus ); (Tryptic Soy Agar, TSA, : 140424) SCDLP (: 160507)(: 150504) (: 150625) (: 151112), ; (: 20140219-00, ) 2.3 实验方法 2.3.1 PNA 探针设计 PNA NCBI (http://www.ncbi.hlm.nih.gov/blast/) 16S rrna, MegAlign (version 5.0; DNASTAR, Madison, WI) Clustal V PA-16S-1(5 -CAC CTC GGG TGG GCA CT-3 ) BLAST, PA-16S-1 BacUni(5 -CTG CCT CCC GTA GGA-3 ) [10], BacUni 16S rrna 5 CY 3, Panagene 2.3.2 PNA FISH 方法的建立 (1) TSA, 37 24 h,
5, : - 1711 TSA, 18~24 h (2) [11], BacUni,, (3) PNA 3 (ATCC 27853 ATCC 9027 ATCC 15442)(ATCC 1388328) (ATCC 29930)(ATCC 51329) (ATCC 25922) O157:H7 (NCTC 12900) (ATCC14028) (EGD)(ATCC 6538 ATCC 25923) 12 PNA 2.3.3 PNA FISH 检测化妆品中的铜绿假单胞菌 300, (2015 ), PNA-FISH, A B 3 结果与分析 3.1 PNA FISH 方法优化 OD 600 =0.1~2.0, OD 600 =0.8~1.2, Triton X-100 0.125% 0.15% 0.2% 0.3%, TritonX-100, TritonX-100 0.3%,, TritonX-100 0.2%, PNA, PNA 300~500 pmol/ml,, 15 min 90 min, 60 min 90 min,, 20 min 10 min, (ATCC 27853) PNA-FISH 1 3.2 PNA 探针灵敏度和特异性验证 3 (ATCC 27853 ATCC 9027 ATCC 15442), 9 ( 1) 3.3 化妆品样品中检测绿假单胞菌 300, PNA-FISH 2 1 GB, PA-16S-1, 2 1 (ATCC 27853) PNA-FISH Fig. 1 PNA-FISH hybridization graphs of Pseudomonas aeruginosa ATCC 27853 A. PA-16S-1; B. BacUni 表 1 铜绿假单胞菌 PNA 探针灵敏度和特异性验证 Table 1 Sensitivity and specificity evaluation of PNA probe of Pseudomonas aeruginosa BacUni PA-16S-1 ATCC 27853 + + ATCC 9027 + + ATCC 15442 + + ATCC 1388328 + - ATCC 29930 + - ATCC 51329 + - ATCC 25922 + - O157:H7 NCTC 12900 + - ATCC14028 + - EGD + - ATCC 6538 + - ATCC 25923 + - : +; -
1712 8 表 2 PAN-FISH 法和生化方法比较 Table 2 Comparison between PNA-FISH and biochemical methods PNA-FISH ( ) 80 2 2 80 0 0 50 0 0 30 0 0 60 1 1 300 3 3 4 结论, (2015 ), ISO : 22717 ISO 22718 [12,13],,, 16S rrna PNA PA-16S-1,, PNA,, 9, 3 h,,, DNA, PNA ( ), DNA K, DNA, PNA ph [14], PNA ph,,, PNA rrna [15],, PNA-FISH [16-18],,,, ;,,, ;,,,,,, PNA, 参考文献 [1] [EB/OL]. http://www.doc88.com/p-697588320365.html. 2012-09-14. Hygienic standard for cosmetics-microbiological examination methods. [EB/OL]. http://www.doc88.com/p-697588320365.html. 2012-09-14. [2]. [S]. State Food and Drug Supervision and Administration Bureau. Safety and technical standards for cosmetics [S]. [3] Lundov MD, Moesby L, Zachariae C, et al. Contamination versus preservation of cosmetics: a review on legislation, usage, infections and contact allergy [J]. Contact Dermatitis, 2009, 60: 70 78. [4] Becks VE, Lorenzoni NM. Pseudomonas aeruginosa out-break in a neonatal intensive care unit: a possible link to contaminated hand lotion [J]. Am J Infect Control, 1995: 23: 396 398. [5] Anelich LE, Korsten L. Survey of micro-organisms associated with spoilage of cosmetic creams manufactured in South Africa [J]. Int J Cosmet Sci, 1996: 18: 25 40. [6] Lundov MD, Zachariae C. Recalls of microbiological contaminated cosmetics in EU from 2005 to May 2008 [J]. Int J Cosmet Sci, 2008, 130: 471 474. [7] Miller MJ. Breaking the rapid microbiological method financial barrier: A case study in return on investment and economic justification [J]. Bio Pharm Int, 2009, 22(9): 44 63. [8] Zhang XF, Wu S, Li K, et al. Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii [J]. Int J Food Microbiol, 2012, 157(2): 309 313. [9],,,. [J]., 2012, 39(7): 1 7. LI K, Zhang XF, WANG ZC, et al. Application of peptide nucleic acid probes in microbiological diagnostication [J]. Microbiol China, 2012, 39(7): 1 7. [10] Perry-O'Keefe H, Stender H, Broomer A, et al. Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms [J]. J Appl Microbiol, 2001, 90(2): 180 189. [11] Zhang XF, Li K, Wu S, et al. Peptide nucleic acid fluorescence in-situ hybridization for identification of Vibrio spp. in aquatic products and environments [J]. Int J Food Microbiol, 2015, 206: 39 44. [12] ISO 22717:2006. Cosmetics-Microbiology-Detection of Pseudomonas aeruginosa [S]. [13] ISO 22718:2006 Cosmetics-Microbiology-Detection of Staphylococcus aureus [S]. [14] Bohnert J, Hübner B, Botzenhart K. Rapid identification of Enterobacteriaceae using a novel 23S rrna-targeted oligonucleotide probe [J]. Int J Hyg Environ Health, 2000, 203(1): 77 82.
5, : - 1713 [15] Sforza S, Corradini R, Tedeschi T, et al. Food analysis and food authentication by peptide nucleic acid (PNA)-based technologies [J]. Chem Inform, 2011, 42(17): 221 32. [16] Peltroche-Llacsahuanga H, Fiandaca MJ, Oy SV, et al. Rapid detection of Streptococcus agalactiae from swabs by peptide nucleic acid fluorescence in situ hybridization [J]. J Med Microbiol, 2010, 59(2): 179 184. [17] Almeida C, Azevedo NF, Fernandes RM, et al. Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples [J]. Appl Environ Microbiol, 2010, 76(13): 4476 4485. [18] Almeida C, Azevedo NF, Santos S, et al. Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization, (PNA FISH) [J]. PLoS One, 2013, 8(6): e14786. 作者简介 ( 责任编辑 : 姚菲 ) 李可, 高级工程师, 主要研究方向为事食源性致病微生物检测 分子分型溯源 E-mail: lk@ziq.gov.cn 张晓峰, 博士, 研究员, 主要研究方向为食品安全 E-mail: zxf@ ziq.gov.cn 粮油产品质量安全专题 征稿函,,,,,,, (1); (2) ; (3) ; (4); (5) ; (6), 2017 10,,, 2017 9 30 Email 投稿方式 : : www.chinafoodj.com Email: jfoodsq@126.com 食品安全质量检测学报 编辑部