Research Paper 研究报告 Acta Microbiologica Sinica 48(11) 1526~1531; 4 November 2008 ISSN 0001-6209; CN11-1995/Q http://journals.im.ac.cn 变性高效液相色谱检测食品中致泻性大肠杆菌 徐君怡 1, 曹际娟 1*, 郑秋月 1, 赵昕 1 2, 闫平平 ( 1 116015) 2 116021 摘要 : polymerase chain reaction, PCR denaturing high-performance liquid chromatography, DHPLC 4 PCR 32 4 27 CFU/mL 33 CFU/mL 25 CFU/mL 42 CFU/mL 关键词 PCR DHPLC 中图分类号 Q93-3 文献标识码 A 文章编号 0001-6209 (2008) 11-1526-06 Escherichia coli E.coli [1] (enterotoxigenic Escherichia coli ETEC) (enteropathogenic Escherichia coli EPEC) (enterohemorrhagic Escherichia coli EHEC) (enteroinvasive Escherichia coli EIEC) (enteroaggregative Escherichia coli EAEC) (Diffusely adherent Escherichia coli DAEC) [1,2] 4 attaching and effacing lesions, AE [3] heat stable enterotoxin, ST / (heat labile enterotoxin, LT) [2] [4] Stx1 Shiga-like toxin 1, Stx1 Stx2 Shiga-like toxin 2, Stx2 [5] [6] (PCR) 基金项目 : (2006BAK02A13) * Tel/Fax: +86-411-82863963; Fax: +86-411-82867690; E-mail: cjj0909@sina.com 作者简介 : (1979 ),,,,, E-mail: skyxjy@yahoo.com 收稿日期 : 2008-05-19; 修回日期 : 2008-07-03
./ (2008) 48(11) 1527 DNA PCR 6~8 d DHPLC [7~9] DHPLC [10] DHPLC DNA PCR DHPLC 1 材料和方法 1.1 材料 1.1.1 DNA TakaRa MiniBEST Bacterial Genomic DNA Extraction kit Taq PCR TaKaRa PCR PE24000 PerkinElmer NAV-99-4500 (Transgenomic ) centrifuge 5804(Eppendorf ) 1.1.2 ATCC CMCC 1 表 1 试验菌种及其编号 Table 1 Tested strains and strains number Bacterial strain No.of strains showing positive PCR result/no.of strains tested bfp lt rfbe ial Enteropathogenic E.coli O111:nm ATCC 43887 1/1 0/1 0/1 0/1 O86:K61 CIQ a 1/1 0/1 0/1 0/1 O26:K60 CIQ 2/2 0/2 0/2 0/2 O44:K74 CIQ 1/1 0/1 0/1 0/1 O119:K69 CIQ 1/1 0/1 0/1 0/1 O114:K90 CIQ 2/2 0/2 0/2 0/2 Enterotoxigenic E.coli O78:H11 ATCC 35401 0/1 1/1 0/1 0/1 O78:K80 CIQ 0/2 2/2 0/2 0/2 O25:K19 CIQ 0/1 1/1 0/1 0/1 Enterohemorrhagic E.coli O157:H7 ATCC 35150 0/1 0/1 1/1 0/1 O157:H7 CIQ 0/1 0/1 1/1 0/1 Enteroinvasive E.coli O124:nm ATCC 43893 0/1 0/1 0/1 1/1 Escherichia coli ATCC 25922 0/1 0/1 0/1 0/1 Escherichia coli ATCC 8379 0/1 0/1 0/1 0/1 Escherichia coli ATCC 11775 0/1 0/1 0/1 0/1 Escherichia coli CIQ 0/5 0/5 0/5 0/5 Salmonella. typhimurium ATCC 49416 0/1 0/1 0/1 0/1 Salmonella. cholerae ATCC 10708 0/1 0/1 0/1 0/1 Salmonella. enteritidis ATCC 13076 0/1 0/1 0/1 0/1 Citrobacter freundii ATCC 8090 0/1 0/1 0/1 0/1 Shigella flexneri ATCC 12022 0/1 0/1 0/1 0/1 Proteus mirabilis ATCC 29245 0/1 0/1 0/1 0/1 Proteus vulgaris CMCC 49027 0/1 0/1 0/1 0/1 Campylobacter jejuni ATCC 33291 0/1 0/1 0/1 0/1 Yersinia enterocolitica ATCC 9610 0/1 0/1 0/1 0/1 a CIQ, Liaoning Entry-Exit Inspection and Quarantine Bureau of China.
1528 Junyi Xu et al. /Acta Microbiologica Sinica (2008) 48(11) 1.2 引物及目的基因的选择 [1] DNA PCR 2 1.3 反应体系优化 DNA Taq Mg 2+ Table 2 表 2 目的基因及其引物序列 Primers used in this study for amplification of diarrheagenic E.coli genes Strain Gene Primer sequence 5 3 Size of product/bp enterotoxigenic E.coli lt GCACACGCAGCTCCTCAGTC 218 TCCTTCATCCTTTCAATGGCTTT enteropathogenic E.coli bfp GGAAGTCAAATTCATGGGGGTAT 294 GGAATCAGACGCAGACTGGTAGT enterohemorrhagic E.coli rfbe ATTGCGCTGAAGCCTTTG 499 CGAGTACATTGGCATCGTG enteroinvasive E.coli ial CTGGATGGTATGGTGAGG GGAGGCCAACAATTATTTCC 320 1.4 特异性试验 1 DNA 1.4.1 PCR 1.4.2 DHPLC PCR-DHPLC 1.4.1 PCR PCR 25 μl 94 3 min 94 60 s 60 60 s 72 60 s 35 72 7 min 4 1.4.2 DHPLC PS-DVB & C18 DNASep 4.6 mm 50 mm 3 μm 50 A 50.2% B 49.8% 0.9 ml/min 150W Xenon 15 nm 15.3 nm 350 nm 2 s PCR 5 μl 1.5 灵敏度试验 ETEC ATCC 35401 (EPEC)ATCC 43887 (EHEC)ATCC 35150 (EIEC)ATCC 43893 36 24 h OD 10 1 10 2 10 3 2 DNA PCR-DHPLC 1.6 食品检测模型试验 PCR PCR 2 结果 2.1 优化的反应体系 10 PCR 2 μl 10 μmol/l 1 μl dntp 10 mmol/l 2 μl Taq DNA 5 U/μL 0.2 μl 16.8 μl DNA 2 μl dh 2 O 25 μl 94 3 min 94 60 s 60 60 s 72 60 s 35 72 7 min 4 2.2 特异性试验 1 32 DNA bfp lt rfbe ial PCR DHPLC 4 DHPLC 1A-D bfp 6 8 (EPEC) EPEC 1-A lt 3 4 (ETEC) ETEC 1-B rfbe 2 (EHEC)O157:H7 EHEC 1-C ial 1 (EIEC) EIEC 1-D 1 4 PCR-DHPLC
./ (2008) 48(11) 1529 Fig. 1 图 1 4 种不同类型的致泻性大肠杆菌 DHPLC 特异性检测图谱 DHPLC chart of enteroinvasive E.coli. A: enteropathogenic E.coli; B: enterotoxigenic E.coli; C: enterohemorrhagic E.coli; D: enteroinvasive E.coli. 2.3 灵敏度试验 ETEC ATCC 35401 EPEC ATCC 43887 EHEC ATCC 35150 EIEC ATCC 43893 36 24 h 1.5 DNA PCR-DHPLC 10 7
1530 Junyi Xu et al. /Acta Microbiologica Sinica (2008) 48(11) ETEC ATCC 35401 27 CFU/mL EPEC ATCC 43887 33 CFU/ ml EHEC ATCC 35150 25 CFU/ ml EIEC ATCC 43893 42 CFU/mL 2.4 食品检测试验 2007 10 2008 5 PCR-DHPLC 9 1256 GB/T 4789.6 2003 PCR-DHPLC 21 GB PCR-DHPLC GB PCR- DHPLC 100% PCR-DHPLC GB 3 表 3 采用 PCR-DHPLC 和 GB 方法对致泻性大肠杆菌检 测结果的比较 Table 3 Comparation of the results of diarrheagenic E.coli detection with PCR-DHPLC method and GB method Statistical result PCR-DHPLC method GB method ETEC 9 ETEC 9 Positive result EPEC 11 EPEC 11 EHEC 0 EHEC 0 EIEC 1 EIEC 1 Negative result 1235 1235 Positive% 1.67% 1.67% Total 1256 1256 3 讨论 VIDAS O157:H7 PCR PCR- PCR-DHPLC PCR DHPLC Franciosa [11] A B E F DHPLC Hurtle [8] DHPLC rrna (Yersinia pestis) Bacillus anthracis DHPLC [12] DHPLC 7 P53 PCR [13] DHPLC PER1 DHPLC DHPLC PCR-DHPLC PCR- DHPLC 21 GB PCR-DHPLC GB PCR-DHPLC 100% PCR-DHPLC GC 4 PCR PCR-DHPLC
./ (2008) 48(11) 1531 参考文献 [1] Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol, 1998, 11: 132 201. [2] Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteroinvasive, entero- hemorrhagic, and enteroadherent. J Infect Dis, 1987, 155: 377 389. [3] Jerse AE, Yu J, Tall BD, et al. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci USA, 1990, 87: 7839 7843. [4] Tesh V. Virulence of enterohemorrhagic Escherichia coli: role of molecular crosstalk. Trends Microbiol, 1998, 6: 228 233. [5] Tesh VL, O Brien AD. The pathogenic mechanisms of Shiga toxin and the Shiga-like toxins. Mol Microbiol, 1991, 5: 1817 1822. [6],,. O157: H7. (Chinese Journal of Nature), 2003, 25: 164 166. [7] Barlaan EA, Sugimori M, Furukawa S, et al. Profiling and monitoring of microbial populations by denaturing high-performance liquid chromatography. J Microbiol Methods, 2005, 61(3): 399 412. [8] Hurtle W, Shoemaker D, Henchal E, et al. Denaturing HPLC for identifying bacteria. Biotechniques, 2002, 33(2): 386 391. [9] Hurtle W, Bode E, Kaplan RS, et al. Use of denaturing high-performance liquid chromatography to identify Bacillus anthracis by analysis of the 16S-23S rrna interspacer region and gyra gene. J Clin Microbiol, 2003, 41(10): 4758 4766. [10] Colosimo A, Guida V, Flex E, et al. Use of DHPLC for rapid screening of recombinan clones. Biotechniques, 2003, 34: 706 708. [11] Franciosa G, Pourshaban M, De Luca A, et al. Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography. Appl Environ Microbiol, 2004, 70: 4170 4176. [12],,,.. (Environmental Chemistry), 2006, 25(3): 336 339. [13],,,. RT-PCR PER1. (Basic & Clinic Medicine), 2005, 25(5): 409 413. Denaturing high-performance liquid chromatography for identifying four categories of diarrheagenic Escherichia coli Junyi Xu, Jijuan Cao *, Qiuyue Zheng, Xin Zhao, Pingping Yan ( 1 Liaoning Entry-Exit Inspection and Quarantine Bureau of China, Dalian 116001, China) ( 2 College of Life Sciences, Liaoning Normal University, Dalian 116021, China) Abstract: [Objective] We report a new molecular technique for the analysis of four categories of diarrheagenic Escherichia coli based on the separation of PCR-amplified target fragments by denaturing high-performance liquid chromatography (DHPLC). [Methods] Enteropathogenic E.coli, enterotoxigenic E.coli, enterohemorrhagic E.coli and enteroinvasive E.coli were identified by analyzing four specific virulence genes. [Results] A total of 32 bacterial strains were tested, giving no false-positive or false negative results. The detection limits were as low as: ETEC 27 CFU/mL, EPEC 33 CFU/mL, EHEC 25 CFU/mL and EIEC 42 CFU/mL. [Conclusion] The data suggest that PCR-DHPLC will be useful for diarrheagenic Escherichia coli detection and identification. Keywords: diarrheagenic Escherichia coli; Polymerase Chain Reaction; PCR; denaturing high-performance liquid chromatography (DHPLC) Supported by the Key Projects in the National Science and Technology Pillar Program in the Eleventh Five-year Plan Period (2006BAK02A13) * Corresponding author. Tel/Fax: +86-411-82863963; Fax: +86-411-82867690; E-mail: cjj0909@sina.com Received: 19 May 2008/ Revised: 3 July 2008