7 6 Vol. 7 No. 6 2016 6 Journal of Food Safety and Quality Jun., 2016 * 石伟雄, 王羽, 杨粤, 张伟 (, 071000) 摘要 : 目的 (real-time fluorescence loop-mediated isothermal amplification, RT-LAMP) 方法 ( AY702093)16S~23S rrna, dntps Mg 2+, 结果 LAMP : 1.6 μl FIP 1.6 μl BIP, : 0.5 μl F3 0.5 μl B3, 2.5 μl2.5 mmol/l dntp, 2 μl 4 mmol/l MgSO 4, 3 μl Buffer, 1.6 μl 10 Bst DNA, 3 μl DNA, 0.5 μl 100, 25 μl, 63 60 min LAMP 8 10-2 CFU/mL 结论, 关键词 : ; ; ; Detection of Cronobacters akazakii in infant formula by real-time fluorescence loop-mediated isothermal amplification SHI Wei-Xiong, WANG Yu, YANG Yue, ZHANG Wei * (College of Food Science and Technology, Agricultural University of Hebei, Baoding 071000, China) ABSTRACT: Objective To establish a method for the determination of Cronobacters akazakii by real-time fluorescence loop-mediated isothermal amplification (RT-LAMP). Methods Using 16S~23S rrna conserved sequence of Cronobacter sakazakii as target sequences for primer design, the reaction conditions including dntps, Mg 2+, template concentration.etc. were optimized, and then the specificity and sensitivity of the method were investigated. Results The optimal reaction conditions of RT-LAMP was as follows: 1.6 μl FIP and 1.6 μl FIP (inner primer), 0.5 μl F3 and B3 (outer primer), 2.5 μl2.5 mmol/l dntp, 2 μl 4 mmol/l MgSO 4, 3 μl Buffer, 1.6 μl 10 Bst DNA polymerase, 3 μl DNA templates, 0.5 μl 100 SYBR, adding sterilized double-distilled water to make up 25 μl LAMP reaction system mixtures, and the reaction was under 63 for 60 min. The results verified that only Cronobacter sakazakii displayed the typical fluorescence curve, and the sensitivity of the real-time fluorescence LAMP for the detection of Cronobacter sakazakii was 8 10-2 CFU/mL. Conclusion The method has the advantages of less time-consuming, strong specificity and high sensitivity, which can provide references for the 基金项目 : (201510086003); (2015043) Fund: Supported by College students' innovative entrepreneurial training projects at the provincial level(201510086003)and Field project(2015043). * 通讯作者 :,,, E-mail: 1294202936@qq.com *Corresponding author: ZHANG Wei, Professor, Agricultural University of Hebei College of Food Science and Technology, Baoding 071000, China. E-mail: zhangwei631126@yahoo.com.cn
2264 7 fast detection of Cronobacter sakazakii in infant formula. KEY WORDS: real-time fluorescence; loop-mediated isothermal amplification; Cronobacters akazakii; infant formula 1 引言 (Cronobacters akazakii), 2008, 5, [1-3],, (Enterobacters akazakii) [4],,,,, (International Commission on Microbiological Specifications for Foods, ICMSF), [5] 2002, 1 1/100000, 8.7/100000, 20%~50% [6,7], (Food and Drug Administration, FDA) (polymerase chain reaction, PCR), ; [8,9] ; PCR, (loop-mediated isothermal amplification, LAMP) LAMP, LAMP, LAMP [10], 2 材料与方法 [11-13] 2.1 试验菌株 1 表 1 试验用菌株 Table 1 Strains for experiment ATCC29544 (Cronobactersakazakii) ATCC51007 (Cronobactersakazakii) ATCC51024 (Cronobactersakazakii) NK (Cronobactersakazakii) CMCC44102 (Escherichia coli) (CMCC) CMCC44752 (Escherichia coli) (CMCC) CMCC50001 (Salmonella schottmuelleri) (CMCC) CMCC50004 (Salmonella. pafatyphi B) (CMCC) CMCC64609 (Clostridium perfringens) (CMCC) CMCC51334 (Shigellasonnei) (CMCC) CMCC51522 (Shigellaboydii) (CMCC) CMCC44828 O157: H7 (E.coli O157: H7) (CMCC) CMCC49027 (Proteus vu Lgaris) (CMCC) CMCC50115 (Salmonella typhimurium) (CMCC) CMCC50041 (Salmonella enteritidis) (CMCC) CMCC51571 (Shigellaflexnei) (CMCC) CMCC51135 (Shigelladysenteriae) (CMCC) CMCC63302 (Bacilus cereus) (CMCC) CMCC52302 (Enterobacteryersinia) (CMCC) CMCC54001 (Listeria monocytogenes) (CMCC)
6, : 2265 2.2 仪器与试剂 ESEQuant TS-LAMP ( ); ( ); DLCJ-1N ( ); WH-861 ( ); TGL-16B ( ); SPX-150B-Z ( ); ( ) Bst, NEB ; dntps MgSO 4, ; Betaine Solution PCR Grade ; SuperSYBR EG, : 2.3 培养基制备 (1) (nutrient agar, NA): 3 g, 10 g, 5 g, 20 g 1 L, ph, 7.2, 121 15 min [14] (2) (nutrient broth, NB): 10 g, 3 g, 5 g 1 L, ph 7.4, 121 15 min 2.4 实验方法 2.4.1 引物设计 GenBank (ATCC29544)( AY702093)16S~23S rrna, DNAman Primer premier 5.0 RNA structure LAMP, 4 : 2 (F3 B3), 2 (FIP BIP), 125~327 203 bp 2 2.4.2 阪崎克罗诺杆菌的培养, 37 24 h, 2, 37 12 h 2.4.3 提取细菌的模板 DNA DNA [15,16] 1 1 ml 1.5 ml, 12000 r/min 1 min, 2 500 µl, 12000 r/min 1 min, ; 100 µl 3 l0 min, 12000 r/min 1 min, 1.5 ml, -20 2.4.4 反应体系优化 (1) Mg 2+ MgSO 4, 0 0.5 1.0 1.5 2.0 2.5 3 μl, Mg 2+ (2) dntps dntps, 0 3 2.5 2 1.5 1 0.5μL, dntps (3) Bst Bst 0 1 1.6 2 2.5 3 3.5 μl, (4) DNA DNA, 4 3.5 3 2.5 2 1.5 1 0.5 μl, DNA 2.4.5 反应程序 DNA 55~65 90 min,, LAMP 25 µl LAMP : (FIP BIP) 1.6 μl, (F3 B3) 0.5 μl, 2.5 mmol/l dntp 2.5 μl, 4 mmol/l MgSO 4 2 μl, Buffer 3 μl, 10 Bst DNA 1.6 µl, DNA 3 µl, 100 0.5 µl, 63 60 min 表 2 LAMP 引物 Table 2 LAMP primers (5 ~3 ) (F3) (B3) (FIP) (BIP) CGGTTGGATCACCTCCTTAC CGGTAACGCCTTTCACTCAT CTTCGCTTTCGCAGACAACCCTTCGCGTGCTCACACAGAT TTCGTCTAGAGGCCCAGGACACTTAGGGGATTCGAACC
2266 7 2.4.6 特异性的检测 (1) LAMP 20, LAMP, 4 16 ( 1) 2.4.3 DNA, LAMP, LAMP, LAMP (2)PCR, (ATCC29544) 2.4.3 DNA PCR PCR, PCR, NCBI BLAST, LAMP PCR 2.4.7 阪崎克罗诺杆菌纯培养的灵敏度检测 (1) LAMP :, 37 12 h 10, [17] 8 10 6 CFU/mL; 10 ml, 12000 r/min 1 min,, 1 ml 0.9%, 1.5 ml,, 2.4.3 DNA, LAMP LAMP LAMP, 37 12 h, 10, 1 ml 2.4.3 DNA, LAMP, (2)PCR LAMP, PCR, PCR LAMP 2, 2 3 结果与分析 3.1 实时荧光 LAMP 体系中组分的优化 3.1.1 体系中 Mg2+ 添加量的优化 1, Mg 2+ 2 μl,, ;, Mg 2+ 2 μl 3.1.2 体系中 dntps 添加量的优化 2, dntps 2.5 µl, ; ; dntps 2.5 μl 1 Mg 2+ Fig. 1 Real-time fluorescence spectrogram of different additive amounts of Mg 2+ 1: 0 μl; 2: 0.5 μl; 3: 2 μl; 4: 1 μl; 5: 1.5 μl; 6: 2.5 μl; 7: 3 μl; 8: 1: 0 μl; 2: 0.5 μl; 3: 2 μl; 4: 1 μl; 5: 1.5 μl; 6: 2.5 μl; 7: 3 μl; 8: negative control
第6期 3.1.3 石伟雄, 等: 实时荧光环介导等温扩增技术检测婴儿配方奶粉中阪崎克罗诺杆菌的研究 Bst 酶添加量的优化 Bst DNA 聚合酶添加量优化结果如图 3 所示, 其中 3 4 6 和 7 管出现扩增峰, 其他管没有出现扩增峰, 但是从 横坐标的时间来看可以得出第 3 管出峰时间较早, 因此最 2267 终确定最适的 Bst DNA 聚合酶添加量是 1.6 μl 3.1.4 模板 DNA 添加量的优化 结果如图 4 所示, 出现扩增峰的只有第 2 管, 进而确 定本试验体系中最适模板 DNA 添加量是 3 μl 图 2 不同 dntps 添加量的实时荧光谱图 Fig. 2 Real-time fluorescence spectrogram of different additive amounts of dntps 1: 0 μl; 2: 3 μl; 3: 2.5 μl; 4: 2 μl; 5: 1.5 μl; 6: 1 μl; 7: 0.5 μl; 8:阴性对照 1: 0 μl; 2: 3 μl; 3: 2.5 μl; 4: 2 μl; 5: 1.5 μl; 6: 1 μl; 7: 0.5 μl; 8: negative control 图 3 不同 Bst DNA 聚合酶添加量的实时荧光谱图 Fig. 3 Real-time fluorescence spectrogram of different additive amounts of Bst DNA polyase 1: 0 μl; 2: 1.0 μl; 3: 1.6 μl; 4:2.0 μl; 5: 2.5 μl; 6: 3.5 μl; 7: 3.5 μl; 8: 阴性对照 1: 0 μl; 2: 1.0μL; 3: 1.6 μl; 4: 2 μl; 5: 2.5 μl; 6:3 5 μl; 7: 3.5 μl; 8: negative control
2268 7 4 Fig. 4 Real-time fluorescence spectrogram of different additive amounts of template 1: 4.0 μl; 2: 3.0 μl; 3: 2.5 μl; 4: 2.0 μl; 5: 1.5 μl; 6: 1.0 μl; 7: 0.0 μl; 8: 1: 4.0 μl; 2: 3.0 μl; 3: 2.5 μl; 4: 2.0 μl; 5: 1.5 μl; 6: 1.0 μl; 7: 0.0 μl; 8: negative control 3.2 引物特异性的研究 3.2.1 实时荧光 LAMP 引物特异性的研究 5(A)(B),, 16,, 3.2.2 PCR 法的引物特异性研究, LAMP 2 PCR, 2 PCR, 2% (182 bp), 6, GeneBank, 7, 3.3 阪崎克罗诺杆菌纯培养灵敏度的研究 3.3.1 实时荧光 LAMP 检测阪崎克罗诺杆菌的灵敏度 (8 10 6 CFU/mL)10, LAMP 7, 10 8 (8 10-2 CFU/mL) 30, 10 9 (8 10-3 CFU/mL) LAMP 8 10-2 CFU/mL 3.3.2 PCR 检测阪崎克罗诺杆菌的灵敏度 (1.01 10 9 CFU/mL)10, PCR, 8 10 7 (1.01 10 2 CFU/mL), PCR 1.01 10 2 CFU/mL LAMP PCR, LAMP, PCR 1250 4 结论 LAMP, dntp Mg 2+ Bst, DNA
第6期 石伟雄, 等: 实时荧光环介导等温扩增技术检测婴儿配方奶粉中阪崎克罗诺杆菌的研究 2269 (A) 1-阪崎克罗诺杆菌; 2-阴性对照; 3-大肠杆菌; 4-鼠伤寒沙门氏菌; 5-痢疾志贺氏菌; 6-福氏志贺氏菌; 7-鲍氏志贺氏菌; 8-宋内志贺氏菌 1-Cronobacter sakazakii; 2-negative control; 3-Escherichia coli; 4-Salmonella typhimurium; 5-Shigella dysenteriae; 6-Shigella flexnei; 7-Shigella boydii; 8-Shigella sonnei (B) 1-阪崎克罗诺杆菌; 2-阴性对照; 3-小肠结肠耶尔森氏菌; 4-大肠杆菌 O157:H7; 5-甲型副伤寒沙门氏菌; 6-乙型副伤寒沙门氏菌; 7-肠炎沙 门氏菌; 8-普通变形杆菌 1-Cronobacter sakazakii; 2-negative control; 3-Enterobacter yersinia; 4-E.coli O157:H7; 5-Salmonella schottmuelleri; 6-Salmonella pafatyphi B; 7-Salmonella enteritidis; 8-common Proteus vulgaris Fig. 5 图 5 实时荧光 LAMP 引物特异性的检测结果 Results of the specificity of real-time fluorescence LAMP primers
2270 食品安全质量检测学报 Fig. 6 Fig. 7 第7卷 图 6 PCR 产物测序结果 Sequencing result of PCR products 图 7 PCR 产物测定的序列与 GenBank 中目的基因序列的比对结果 Comparison of objective gene sequences between sequencing in PCR and GenBank 图 8 实时荧光 LAMP 检测阪崎克罗诺杆菌的灵敏度 Fig. 8 The sensitivity of Cronobacter sakazakii by real-time fluorescence LAMP 1-阳性对照; 2-8 102 CFU/m L; 3-8 101 CFU/mL; 4-8 100 CFU/mL; 5-8 10-1 CFU/mL; 6-8 10-2 CFU/mL; 7-8 10-3 CFU/mL; 8-阴性对照 1-positive control; 2-8 102 CFU/mL; 3-8 101 CFU/mL; 4-8 100 CFU/mL; 5-8 10-1 CFU/mL; 6-8 10-2 CFU/mL; 7-8 10-3 CFU/mL; 8-negative control
6, : 2271 [7] Himelright I, Harris E. Enterobacter sakazakii infections associated with the use of powdered infant formula-tennessee, 2001 [J]. Morb Mortal Wkly Rep, 2002, 51(14): 297 300. [8],,,. [J]., 2015, 27(05): 524 530. Duan YS, Wang JC, Sun XX, et al. Establishment of the real-time fluorescence single primer isothermal amplification for the detection of Cronobacter sakazakii [J]. Chin J Food Hyg, 2015, 27(05): 524 530. [9]. PCR [J]., 2013, 01: 69 70. 9 Fig. 9 The sensitivity of Cronobacter sakazakii by electrophoresis M-100 bp DNA marker; 1- ; 2- ; 3-1.01 10 8 CFU/mL; 4-1.01 10 7 CFU/mL; 5-1.01 10 6 CFU/mL; 6-1.01 10 5 CFU/mL; 7-1.01 10 4 CFU/mL; 8-1.01 10 3 CFU/mL; 9-1.01 10 2 CFU/mL; 10-1.01 10 1 CFU/mL; 11-1.01 10 0 CFU/mL LAMP,,, LAMP PCR, 参考文献 [1],,,. PCR(ERIC-PCR) [J]. ( ), 2013, 34(2): 56 61. Chen WY, Ai LZ, Ren J, et al. Molecular typing of Cronobacter sakazkii by using enterobacterial repetitive intergenic consensus sequence PCR [J]. J Henan Univ Technol (Nat Sci Ed), 2013, 34(2): 56 61. [2],,,. recn [J]., 2015, 31(01): 63 66. Tian X, Wang P, Zhao YS, et al. Species identification of Cronobacter spp. based on recn gene [J]. Chin J Public Health, 2015, 31(01): 63 66. [3],,. gyrb [J]., 2013, 38(06): 40 44. Chen WY, Ai LZ, Ren J, et al. The gyrb gene sequence analysis for phylogenetic analysis of Cronobacter sakazakii from infant formula powder [J]. Food Sci Technol, 2013, 38(06): 40 44. [4] YAN QQ, Conndell O, Power K, et al. Cronobacter species (formerly known as Enterobacer sakazaki) in powdered infant formula: a review of our current understanding of the biology ofthis bacterium [J]. J Appl Microbiol, 2012, 113(1): 1 15 [5] ICMSF. Microorganisms in foods number 7: Microbiological testing in food safety management [M]. Netherlands: Kluwer Academic/Plenum Publishers, 2002. [6]. - [J]., 2004, 16(5): 385 387. Liu XM. Enterobactersakazakii-The new target of food safety control [J]. Chin J Food Hyg, 2004, 16(5): 385 387. Deng ZJ. Multiple PCR detection technology application in the detection of food microorganism [J]. Biotech World, 2013, 01: 69 70. [10],,,. LAMP [J]., 2011, 36(09): 340 346. Meng XZ, Ma XY, Zhang HY, et al. Detection of enterotoxigenic Escherichia coli in food by LAMP [J] Food Sci Technol, 2011, 36(09): 340 346. [11],,,. PCR [J]., 2012, 33(11): 180 183. Di H, WangY, Zhang XZ, et al. Study on the detection of Shigella Spp., Salmonella Spp. and Proteus vulgaris by multiple PCR [J]. Food Ind, 2012, 33(11): 180 183. [12],,,. [J]., 2012, 28(06): 703 706. Lu YX, Meng ZX, Ma XY, et al. Rapid detection of Listeria monocytogenes in milk powder by loop-mediated isothermal amplification [J]. Mod Food Sci Technol, 2012, 28(06): 703 706. [13],,,. O157: H7 [J]., 2010, 31(24): 355 359. Huang FL, DuanX, Chen Y, et al. Preparation of colloidal gold strip for the detection of Escherichia coli O157:H7 [J]. Food Sci, 2010, 31(24): 355 359. [14],,,. [J]., 2011, 36(6): 416 425. Liu ZX, Huang K, Xiao HD, et al. Screening and preliminary identification of the antimicrobial strains associated with Anthopleura xanthogrammica [J].Chin J Antibiotics, 2011, 36(6): 416 425. [15] Sreenivas K Detection of Listeria monocytogenes in pigs and pork [J]. J Food Prot, 2002, 65(9): 1470 1474 [16]. PCR [D]. :, 2008 Chen SS. Study on polymerase chain reaction assay for detection of Enterobacter sakazakii in powder infant formula milk [D]. Baoding: Agricultural University of Hebei, 2008. [17],,,. LAMP [J]., 2011, 38(1): 43 47. Tang MJ, Zhou S, Zhang XY, et al. Loop-mediated isothermal amplification assay for rapid detection of Salmonella spp. [J]. J Anhui Agric Univ, 2011, 38(1): 43 47. ( 责任编辑 : 姚菲 )
2272 7 作者简介 石伟雄, 主要研究方向为微生物检测 E-mail: shiweixiong@foxmail.com 张伟, 教授, 主要研究方向为食品加工与安全 E-mail: zhangwei631126@126.com 食品包装材料 专题征稿函,,,,,, (, ), 2016 8,, 2016 7 10 E-mail 投稿方式 : : www.chinafoodj.com E-mail: jfoodsq@126.com 食品安全质量检测学报 编辑部