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1 Research Paper 研究报告 Acta Microbiologica Sinica 48(11) 1526~1531; 4 November 2008 ISSN ; CN /Q 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 ( ) 摘要 : polymerase chain reaction, PCR denaturing high-performance liquid chromatography, DHPLC 4 PCR 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 (2008) 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: ; Fax: ; cjj0909@sina.com 作者简介 : (1979 ),,,,, skyxjy@yahoo.com 收稿日期 : ; 修回日期 :

2 ./ (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 DNA TakaRa MiniBEST Bacterial Genomic DNA Extraction kit Taq PCR TaKaRa PCR PE24000 PerkinElmer NAV (Transgenomic ) centrifuge 5804(Eppendorf ) 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 /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 /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 /1 0/1 1/1 0/1 O157:H7 CIQ 0/1 0/1 1/1 0/1 Enteroinvasive E.coli O124:nm ATCC /1 0/1 0/1 1/1 Escherichia coli ATCC /1 0/1 0/1 0/1 Escherichia coli ATCC /1 0/1 0/1 0/1 Escherichia coli ATCC /1 0/1 0/1 0/1 Escherichia coli CIQ 0/5 0/5 0/5 0/5 Salmonella. typhimurium ATCC /1 0/1 0/1 0/1 Salmonella. cholerae ATCC /1 0/1 0/1 0/1 Salmonella. enteritidis ATCC /1 0/1 0/1 0/1 Citrobacter freundii ATCC /1 0/1 0/1 0/1 Shigella flexneri ATCC /1 0/1 0/1 0/1 Proteus mirabilis ATCC /1 0/1 0/1 0/1 Proteus vulgaris CMCC /1 0/1 0/1 0/1 Campylobacter jejuni ATCC /1 0/1 0/1 0/1 Yersinia enterocolitica ATCC /1 0/1 0/1 0/1 a CIQ, Liaoning Entry-Exit Inspection and Quarantine Bureau of China.

3 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 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 特异性试验 1 DNA PCR DHPLC PCR-DHPLC PCR PCR 25 μl 94 3 min s s s min 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 (EPEC)ATCC (EHEC)ATCC (EIEC)ATCC h OD 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 s s s min 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

4 ./ (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 EPEC ATCC EHEC ATCC EIEC ATCC h 1.5 DNA PCR-DHPLC 10 7

5 1530 Junyi Xu et al. /Acta Microbiologica Sinica (2008) 48(11) ETEC ATCC CFU/mL EPEC ATCC CFU/ ml EHEC ATCC CFU/ ml EIEC ATCC CFU/mL 2.4 食品检测试验 PCR-DHPLC GB/T 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 Positive% 1.67% 1.67% Total 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

6 ./ (2008) 48(11) 1531 参考文献 [1] Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol, 1998, 11: [2] Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteroinvasive, entero- hemorrhagic, and enteroadherent. J Infect Dis, 1987, 155: [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: [4] Tesh V. Virulence of enterohemorrhagic Escherichia coli: role of molecular crosstalk. Trends Microbiol, 1998, 6: [5] Tesh VL, O Brien AD. The pathogenic mechanisms of Shiga toxin and the Shiga-like toxins. Mol Microbiol, 1991, 5: [6],,. O157: H7. (Chinese Journal of Nature), 2003, 25: [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): [8] Hurtle W, Shoemaker D, Henchal E, et al. Denaturing HPLC for identifying bacteria. Biotechniques, 2002, 33(2): [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): [10] Colosimo A, Guida V, Flex E, et al. Use of DHPLC for rapid screening of recombinan clones. Biotechniques, 2003, 34: [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: [12],,,.. (Environmental Chemistry), 2006, 25(3): [13],,,. RT-PCR PER1. (Basic & Clinic Medicine), 2005, 25(5): 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 , China) ( 2 College of Life Sciences, Liaoning Normal University, Dalian , 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: ; Fax: ; cjj0909@sina.com Received: 19 May 2008/ Revised: 3 July 2008

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus