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1 微生物学通报 JUN 20, 2009, 36(6): 923~928 Microbiology 2009 by Institute of Microbiology, CAS 生物实验室空肠弯曲菌 LAMP 快速检测方法的建立 * 林超梁成珠徐彪孙敏刘彩霞高宏伟 ( ) 摘要 : 本研究使用恒温环介导技术, 以空肠弯曲菌的促旋酶基因 A(gyrA) 设计引物, 建立空肠弯曲菌的 LAMP 快速检测方法不同来源的 4 株空肠弯曲菌 LAMP 检测均显示阳性, 其他 14 种细菌 LAMP 检测显示阴性实验结果表明, 设计的引物具有良好的特异性本研究进行了 LAMP 检测方法与细菌平板计数法和 PCR 法的灵敏度比较, 结果 LAMP 检测与 PCR 法有相近的灵敏度, 比细菌平板计数法灵敏度高 3 个数量级我们还研究发现提取核酸前加入 DNase 可以有效地减少死菌 DNA 对 LAMP 结果的影响使用 LAMP 方法对鸡法氏囊的检测表明, 结合核酸提取步骤中的 DNase 处理步骤, 可以准确的检测出鸡法氏囊中的空肠弯曲菌关键词 : 恒温环介导, 空肠弯曲菌, 检测 Establishment of Loop-mediated Isothermal Amplification (LAMP) Method for Campylobacter jejuni Detection LIN Chao LIANG Cheng-Zhu XU Biao SUN Min LIU Cai-Xia GAO Hong-Wei * (Technical Center for Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao, Shandong , China) Abstract: A rapid LAMP detection method with primers designed on genus-specific region identified in the gyra gene was established in this assay. All four Campylobacter jejuni from different sources were detected positive and fourteen non Campylobacter bacteria were negative, which shows excellent specificity of the primers. Compared with plate count and PCR method, the LAMP method and the PCR method had equal sensitivity, which were three orders of magnitude higher than plate count. In this assay, we also found out that the treatment of DNase could reduce the dead bacteria DNA effectively. The LAMP detection on chicken indicated relatively good result on detection of Campylobacter jejuni combining with treatment of DNase. Keywords: Loop-mediated isothermal amplification, Campylobacter jejuni, Detection (Campylobacter jejuni), [1], (Viable but non-culturable, VNC) [2],,, 基金项目 : (No. 2006BAD05A06); (No. 2007IK167) * 通讯作者 : Tel: ; : ghw75@126.com 收稿日期 : ; 接受日期 :

2 924 微生物学通报 2009, Vol.36, No.6 [3], ,, ( 240 [4],,,,,, 90%, 10 9 CFU/g [5],,,, [6], ; [7] (VNC), [8],,, Pearson [9] PCR(Polymerase chain reaction) DNA [10] PCR DNA PCR, PCR, PCR PCR,, Novga [11] 5 PCR, PCR PCR,, (Loop-mediated isothermal amplification, LAMP) [12] PCR, LAMP,, PCR,, LAMP, gyra LAMP,, 1 材料与方法 1.1 材料主要仪器 : (Infinity, EU) PCR (Eppendorf, )(Eppendorf, ) 主要试剂与试剂盒 : Taq dntps (, ), No. 2 (OXOID ), mccda (OXOID ), (OXOID ), (OXOID ), DNA (, ) 引物设计 : LAMP gyra(genbank access number: DQ , AJ CJE567822) PrimerExploerV4 ( LAMP (, ) B3: 5 -TTCCAAATTATGATGGTTCAGA-3, F3: 5 -TGGACCTTTA(G)ATAAACTGCATAA- 3, BIP: 5 -ACAGCTATACCACTTGAACCATTTA- AAGTGAACCTGATGTTTTACCT-3, FIP: 5 -ACAAACATCCCGCCTCATAGTTTAA- TCTCTTCTAAGCTTGCATCT 菌株与样品 : 4 14 ( IQCC13603) 1.2 方法 LAMP 法与细菌培养计数法和 PCR 方法的灵敏度比较 : (IQCC13603) 1 ml,, 9 ml, 10 1 ml10 9 ml, 100,, ml mccda

3 : LAMP 925 Table 1 表 1 空肠弯曲菌 LAMP 法特异性实验中使用的菌株与其来源 The bacteria strains and their sources in Campylobacter jejuni LAMP species-specific experiment Name Serial Number Campylobacter jejuni Campylobacter jejuni Campylobacter jejuni Campylobacter jejuni Salmonella enteritidis Vibrio parahaemolyticus Escherichia coli Salmonella typhi Listeria monocytogenes Listeria ivanovii Yersinia enterocolitica Listeria welshimeri Shigella flexneri Staphylococcus aureus ATCC33560 IQCC13603 IQCC10528 IQCC12310 IQCC10102 IQCC10593 IQCC22201 IQCC22227 IQCC10901 IQCC22261 IQCC10130 IQCC22045 Raoultella planticola Klebsiella oxytoca Citrobacter freundii Citrobacter braakii Source c ATCC : a: ; b: ; c:. Note: a: CAIQ, Chinese Academy of Inspection and Quarantine; b: SDCIQ, Shandong Entry-Exit Inspection and Quarantine Bureau; c: ATCC, American Type Culture Collection. 30 C, ; 1 ml 30 C, DNA(, )DNA DNA 10, , LAMPPCR LAMP 25 µl 1.2 mmol/l gyra-fip gyra-bip, 0.2 mmol/l gyra-f3 gyra-b3, 2.5 µl 10 buffer (Biolabs, ), 12 mmol/l MgSO 4, 1.0 mol/l, 1.0 mmol/l(dgtp, datp, dctp), 2.0 mmol/l dutp, 3 µl DNA, U/µL N (UNG) 50 C 3 min, 95 C 5 min, 0 C 1 min, 2000 r/min 1 min 1 µl (8 U) Bst DNA (New England Biolabs),, 65 C 60 min, 80 C 3 min 4 µl, GelRed (Biotium, USA) 2.0%, PCR 25 µl 10 pmol/l gyra F3 gyra B3, 1 mmol/l dntps, 2.5 µl 10 buffer (Biolabs, ), 2 mmol/l MgCl 2, 2 mmol/l, 1.5 U Taq DNA, 1 µl DNA : 94 C 3 min; 94 C 30 s, 56 C 30 s, 72 C 30 s, 35 ; 72 C 10 min; 4 C [11] PCR 6 µl, GelRed (Biotium, USA) 2.0%, LAMP 法检测 Campylobacter jejuni 的特异性 : 4 14 DNA LAMP, LAMP,, 2.0%, GelRed (Biotium, ), 4 µl, LAMP DNA, LAMP

926 微生物学通报 2009, Vol.36, No.6 1.2.3 用不同方法检测 LAMP 产物 : LAMP PicoGreen (Invitrogen, Carlsbad, USA), [12] LAMP 2 µl PicoGreen, PicoGreen 1.2.4 空肠弯曲菌死菌 DNA 与活菌 DNA 的稳定性比较 : 2.

0 10 7 CFU/mL 1 ml 50 ng/µl DNA 100 µl, C 1 ml 50 ng/µl DNA 100 µl 10 DNase 1 DNase buffer, 1 h DNase, 1 h 8000 r/min 5 min, 100 µl, 95 C 5 min, DNA LAMP 1.2.5 鸡肉样品中空肠弯曲菌检测 : 4 120, 4 2, 10 ml, 41.

4 926 微生物学通报 2009, Vol.36, No 用不同方法检测 LAMP 产物 : LAMP PicoGreen (Invitrogen, Carlsbad, USA), [12] LAMP 2 µl PicoGreen, PicoGreen 空肠弯曲菌死菌 DNA 与活菌 DNA 的稳定性比较 : CFU/mL 50 ng/µl DNA 3 A B C 3 A CFU/mL 1 ml, B CFU/mL 1 ml 50 ng/µl DNA 100 µl, C 1 ml 50 ng/µl DNA 100 µl 10 DNase 1 DNase buffer, 1 h DNase, 1 h 8000 r/min 5 min, 100 µl, 95 C 5 min, DNA LAMP 鸡肉样品中空肠弯曲菌检测 : 4 120, 4 2, 10 ml, 41.5 C 48 h [13], r/min 5 min,, DNA 41.5 C 48 h,, 10, mccda, DNA LAMP ( 2) 图 1 LAMP 法 (a) 与 PCR 法 (b) 检测空肠弯曲菌 grya 基因的灵敏度比较 Fig. 1 Comparison of sensitivity between the LAMP reaction (a) and PCR reaction (b) for detection of the Campylobacter jejuni gyra gene : 1~14 DNA : 7.6 ng 7.6 ng 760 pg 760 pg 76 pg 76 pg 7.6 pg 7.6 pg 760 fg 760 fg 76 fg 76 fg 7.6 fg 7.6 fg; 15, 16: ; M: Marker DL 2000 (TaKaRa, ). Note: 1~14: Campylobacter jejuni DNA: 7.6 ng, 7.6 ng, 760 pg, 760 pg, 76 pg, 76 pg, 7.6 pg, 7.6 pg, 760 fg, 760 fg, 76 fg, 76 fg, 7.6 fg, 7.6 fg; 15, 16: Negative control; M: Marker DL 2000 (Ta- KaRa, China). 2 实验结果 2.1 LAMP 法与细菌培养计数法和 PCR 方法的灵敏度比较 1,, LAMP PCRDNA, 7.6 fg(10 5 ); 10 2, LAMPPCR LAMP 反应检测空肠弯曲菌的特异性 : 4 14 DNA, 图 2 LAMP 法检测不同细菌中的空肠弯曲菌 Fig. 2 Detection of Campylobacter jejuni samples from non-campylobacter jejuni samples by LAMP methods : 1~4: ; 5: ; 6-11 : ; M: Marker DL 2000 (TaKaRa, ). Note: 1~4: Campylobacter jejuni; Lane 5: Negative control; Lanes 6 to 11 from left to right: Salmonella enteritidis, Yersinia enterocolitica, Vibrio parahaemolyticus, Escherichia coli, Shigella flexneri, Listeria monocytogenes; M: DL 2000 (TaKaRa, China).

5 : LAMP 927 LAMP PicoGreen,, ( 3), LAMP 图 3 扩增产物中加入 2 μl PicoGreen dsdna 试剂的颜色比较 Fig. 3 Amplified products visualized by adding 2 µl PicoGreen dsdna reagent at the end of the reaction : A: 76 fg Campylobacter jejuni DNA ; B:. Note: A: The LAMP products of 76 fg Campylobacter jejuni -DNA; B: Negative control. 2.3 DNase 处理对细菌 DNA 扩增的影响, DNase A B C LAMP, DNase A B, C A LAMP A, B B 3 讨论,, gyra gyra(gyrase A), gyra LAMP, NCBI %, LAMP DNA,,,, LAMP DNA, LAMP,, LAMP, LAMPPCR, [12], DNA, LAMP 3,, LAMP,, 4 LAMP, [14] VNC,, [15], DNA PCR, DNA, DNA,, DNA, DNA, DNA [16], DNase DNA, DNA, DNaseDNA LAMP, LAMP LAMP 65 C,, LAMP, 参考文献 [1] Solomon, EB, Hoover DG. Campylobacter jejuni: a bacterial paradox. J Food Safety, 1999, 19(2): [2] Rollins DM, Colwell RR. Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl Environ Microbiol, 1986, 52(3): [3] Sahin O, Kobalka P, Zhang Q. Detection and survival of Campylobacter jejuni in chicken eggs. Journal of Applied Microbiology, 2003, 95(5): [4] Mead PS, Slutsker L, Dietz V, et al. Food-related illness and death in the United States. Emerg Infect Dis, 1999, 5(5): [5] Berndtson E, Danielsson-Tham ML, Engvall A. Campy-

6 928 微生物学通报 2009, Vol.36, No.6 lobacter incidence on a chicken farm and the spread of Campylobacter during the slaughter process. Int J Food Microbiol, 1996, 32(1): [6],,,. PCR., 2007, 36(1): [7],,,.., 2002, 29(5): [8] Jones DM, Sutcliffe EM, Curry A. Recovery of viable but non-culturable Campylobacter jejuni. J Gen Microbiol, 1991, 137(10): [9] Pearson AD, Greenwood M, Healing TD, et al. Colonization of broiler chickens by waterborne Campylobacter jejuni. Appl Environ Microbiol, 1993, 59(4): [10] Winters DK, O Leary AE, Slavik MF. Rapid PCR with nested primers for direct detection of in chicken washes. Mol Cell Probes, 1997, 11(4): [11] Nogva HK, Bergh A, Holck A, et al. Application of the 5 -nuclease PCR assay in evaluation and development of method for quantitative detection of Campylobacter jejuni. Applied and Environmental Microbiology, 2000, 66(9): [12] Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 2000, 28(12): E63 e63. [13],,,. PCR., 2007, 28(1): 5 8. [14] ISO : 2006 Microbiology of food and animal feeding stuffs -- Horizontal method for detection and enumeration of Campylobacter spp. -- Part 1: Detection method. [15] Tomlinson JA, Boonham NK, Hughes JD, et al. On-Site DNA extraction and Real-Time PCR for detection of Phytophthora ramorum in the field. Applied and Environmental Microbiology, 2005, 12(71): [16] Elvers KT, Helps CR, Wassenaar TM, et al. Development of a strain-specific molecular method for quantitating individual Campylobacter strains in mixed population. Applied and Environmental Microbiology, 2008, 8(74): 年中国微生物学会及各专业委员会学术活动计划表 (2-2)

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Microsoft Word 高琳-二校1 出210 8 1 Vol. 8 No. 1 2017 1 Journal of Food Safety and Quality Jan., 2016 * 高琳, 王淞, 刘国红, 温华蔚, 王禹, 刘掘茫, 张霞 (, 300461) 摘要 : 目的, 方法 19, 35 ; DNA, ; 6 18 ISO 5725-2:1994, 2 5 结果,, ISO 结论,, 关键词 : ; ; Application