Microsoft Word 高琳-二校1 出210

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1 8 1 Vol. 8 No 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 (, ) 摘要 : 目的, 方法 19, 35 ; DNA, ; 6 18 ISO :1994, 2 5 结果,, ISO 结论,, 关键词 : ; ; Application of isothermal amplification technique in the detection of Salmonella in food GAO Lin, WANG Song, LIU Guo-Hong, WEN Hua-Wei, WANG Yu, LIU Jue-Mang, ZHANG Xia * (Tianjin Entry-Exit Inspection and Quarantine Bureau, Tianjin , China) ABSTRACT: Objective To design the specific primers and probes on the basis of sequence analysis of Salmonella, and evaluate the application of isothermal amplification methods (cross-priming amplification and loop-mediated isothermal amplification, CPA and LAMP) in the detection of Salmonella in food. Method The specificity of the method was evaluated by 19 strains of Salmonella and 35 negative strains of Enterobacteriaceae. The sensitivity of the CPA method was evaluated by testing the quantitative DNA and pure bacteria count compared with the LAMP method. According to the ISO standard, 2 isothermal amplification methods were confirmed from the aspects of sensitivity, specificity, false positive rate, false negative rate and relative accuracy with the current national standard as the reference method. Result The CPA method had the same specificity as the LAMP method, was more sensitive than the LAMP method. The performance of methods could meet the requirements of ISO which compared with the reference method. Conclusion This method is highly sensitive, specific, and does not require any complex equipment, which can be used as a rapid qualitative detection method for Salmonella in food. It has practical significance for the rapid screening in the ports, and the detection in grassroots areas or the economically underdeveloped areas in remote areas. KEY WORDS: Salmonella; cross-priming amplification; loop-mediated isothermal amplification 基金项目 : (2011IK241) Fund: Supported by the AQSIQ Projects (2011IK241) * 通讯作者 :,, zhangxia_75@126.com *Corresponding author: ZHANG Xia, Senior Engineer, Technical Center of Tianjin Entry-Exit Inspection & Quarantine Bureau, No.158, Jingmen Road, Free Trade Zone, Tianjin Port, Tianjin , China. zhangxia_75@126.com

2 1, : 引言,,, [1-4],,, PCR PCR (loop-mediated isothermal amplification, LAMP) [5-7], 5 d [8],,, (cross-priming amplification, CPA) [9],,,, PCR, [10-13],,,, 2 材料与方法 2.1 主要材料与试剂 10 Bst buffer(new England Biolabs ); MgSO 4 (Sigma ); Bst DNA polymerase large fragment (New England Biolabs ); dntps (Fermentas); betaine (Sigma ); 10 loading buffer( ); (Sigma ); ( ); DNA ladder II( ); 5 TBE( ) 19 (4 CMCC 15 ), 35, 1 表 1 试验菌株 Table 1 Experimental bacterial strains CMCC CMCC CMCC CMCC CMCC CMCC CMCC L.mon6801 S.senf ATCC S.kent CMCC S.abo CMCC SAL CMCC SAL CMCC SAL CMCC SAL CMCC SAL CMCC SAL ATCC SAL CMCC SAL70216 ENF5329 SAL70307 O157: H7 PTS001 SAL ATCC SAL ATCC SAL Serliq5123 E coli0856 entagg5624 pesaer CMCC E.clo CMCC CMCC CMCC CMCC E.int51231

3 仪器与设备 PCR (T gradient, Biometra ); (5417R, Eppendorf ); (UVmini-1240, ); (PS3000, Hoefer ); (T2A, Bio-Rad ) 2.3 等温扩增引物及探针 invasion protein (inva) [14] 交叉引物等温扩增法引物及探针 SALF: 5 -GCGGAAGTCGCGGCCCG; SALB: 5 -TCGCACCGTCAAAGGAACC; SALCPR: 5 -AGATGAGTATTGATGCCGATTTGTCAGTACGCTTCG CCGTTCGC; SALDF: 5 -FITC-AGGCCGGTATTATTGATGC; SALDB: 5 -BIOTIN-TTTCTCTGGATGGTATGC 环介导等温扩增法引物序列 FIP BIP F3 B3 Loop F Loop B 2.4 检测方法 5 -GACGACTGGTACTGATCGATAGTTTTT CAACGTTTCCTGCGG 5 -CCGGTGAAATTATCGCCACACAAAAC CCACCGCCAGG 5 -GGCGATATTGGTGTTTATGGGG 5 -AACGATAAACTGGACCACGG 5 -GACGAAAGAGCGTGGTAATTAAC 5 -GGGCAATTCGTTATTGGCGATAG 反应体系 (1) 50 µl dntp 0.16 mmol/l, MgSO mmol/l, Betaine 0.2 mol/l, Bst DNA pol 0.16 U/µL, SALF/SALB 40 pmol/l, SALCPR 160 pmol/l, SALD/SALDB 320 pmol/l, 1 µl : min (2) 50 µl, : dntp 0.8 mmol/l, MgSO 4 2 mmol/l, Betaine 1 mol/l, Bst DNA pol 0.16 U/µL, FIP/BIP 160 pmol/l, F3/B µmol/l, Loop F/Loop B 0.64 µmol/l, 2 μl : min 特异性试验 1 19, ; 35 DNA 检测低限试验 CMCC50041DNA DNA CMCC50041, CFU/mL,, 检测方法评价选择样品基质 6, 3, 样品检测 3, GB ,,, 1 ml DNA, 方法参数 (p-) (p+) (pf+) (pf-)5, 2 GB/T ( ) 2 : [15],, 2 3 结果与分析 3.1 特异性试验 54, 19, 1 2 ; 35, 3( ),, 3.2 检测低限试验 DNA 检测 (CMCC 50041)DNA μg/ml,, 2, 3, 2,,, 1 Fig.1 Detection of Salmonella strains by CPA : N: ; P: ; 1~9:

4 1, : Fig. 2 Detection of Salmonella strains by LAMP : CK: ; 1~6 表 2 沙门氏菌 DNA 灵敏度试验结果 Table 2 Sensitivity of detecting Salmonella DNA DNA 6.84 ng 684 pg 684 pg 6.84 pg 684 fg fg 6.84 fg fg CPA/LAMP CPA/LAMP CPA/LAMP CPA/LAMP CPA/LAMP CPA/LAMP CPA/LAMP CPA/LAMP 1 +/+ +/+ +/+ +/+ +/+ +/ /+ +/+ +/+ +/+ +/+ +/+ +/ /+ +/+ +/+ +/+ +/+ +/ /+ +/+ +/+ +/+ +/+ +/(+/-) +/- - : + ; - 表 3 沙门氏菌液灵敏度试验 Table 3 Sensitivity of detecting Salmonella CFU/mL LAMP CPA : + ; - CFU/mL, PBS CFU/mL, 1 ml DNA, 10 1 CFU/mL, 方法参数评价 3 Fig. 3 Detection of negative control strains by CPA : N: ; P: ; 1-9: 10 1 fg/, 10 1 fg/, 纯菌液检测 (CMCC 50041) ,, ( ) ( ) ( )3,,,,, 22, 27, 26, 4 5

5 214 8 表 4 性能指标结果 ( 交叉引物等温扩增法 ) Table 4 Results of the performance (CPA) b a 22 0 N1= N2=158 N1= 27 N2= 153 N=180 (χ 2 ) χ 2 =3.20, (df)=1 (p+) 100% (p-) 96.8% (pf-) 0 (pf+) 3.2% 97.2% : a b 表 5 性能指标结果 ( 环介导等温扩增法 ) Table 5 Results of the performance (LAMP) b a 22 0 N1= N2=158 N1= 26 N2= 154 N=180 (χ 2 ) χ 2 =2.25, (df)=1 (p+) 100% (p-) 97.5% (pf-) 0 (pf+) 2.5% 97.8% : a b 4 结论,,,,, PCR,,,,, 2, 19 35, DNA, 10 1 CFU/mL, ; DNA,, 10 1 fg/, 10 1 fg/, 3 2, χ 2 <3.84, ;,,,, DNA,, ; 3, ISO,, 参考文献 [1] Bean NH, Griffin PM. Foodborne disease outbreaks in the United States, : pathogens, vehicles, and trends [J]. J Food Prot,1990, 53: [2] Dargatz DA, Wells SA, Fedorka-Cray PJ, et al. The veterinarian s role in diagnosis, treatment, and prevention of multidrug resistant Salmonella typhimurium DT104 [J]. Bovine Pract, 1998, 32: 1 6. [3] Murray MJ. Salmonellosis in horses [J]. J Am Vet Med Ass, 1996, 209: [4] Wells SJ, Ott SL, Seitzinger AH. Key health issues for dairy cattle-new and old [J]. J Dairy Sci, 1998, 81: [5] Bej AK, Mahbubani MH, Boyce MJ, et al. Detection of Salmonella spp. in oysters by PCR [J]. Appl Environ Microbiol 1994, 60: [6] Rasmussen SR, Rasmussen HB, Larsen MR, et al. Combined polymerase chain reaction-hybridization microplate assay used to detect bovine leukemia virus and Salmonella [J]. Clin Chem, 1994, 40: [7] Widjojoatmodjo MN, Fluit AC, Torensma R, et al. Evaluation of the magnetic immuno PCR assay for rapid detection of Salmonella [J]. Eur J Clin Microbiol Infect Dis, 1991, 10: [8] GB/T [S]. GB/T Food microbiological examination: Salmonella [S]. [9] Fang R, Li X, Hu L, et al. Cross-priming amplification for rapid detection of Mycobacterium tuberculosis in sputum specimsal [J]. J Clin Microbiol, 2009, 47: [10] Zhao Y, Zhang X, Zhang H, et al. Rapid and sensitive detection of Enterobacter sakazakii by cross-priming amplification combined with immuno-blotting analysis [J]. Mol Cell Prob, 2010, 24(2): [11],,,. [J].

6 1, : 215, 2013, 34(2): Zhang X, Wu DX, Qu P, et al. A new isothermal amplification detection of Enterobacter sakazakii [J]. Food Sci, 2013, 34(2): [12] 旸,,,. [J]..2012, 33(9): Liu Y, Zhang HY, Liu GH, et al. Rapid detection of Listeria monocytogenes [J]. Food Res Devel, 2012, 33(9): [13],,,. [J]..2013, 34(11): Qi J, Zhang X, Jiang GQ, et al. A cross prime amplification detection of Shigella [J]. Food Res Dev, 2013, 34(11): [14] Liu C, Zheng W, Zhang H, et al. Selective and rapid detection of Salmonella in infant formula by loop-mediated isothermal amplification method [J]. J Food Saf, 2009, 29: 作者简介 ( 责任编辑 : 杨翠娜 ) 高琳, 工程师, 主要研究方向为食品安全 gaol@tjciq.gov.cn 张霞, 高级工程师, 主要研究方向为食品安全检测 zhangxia_75@126.com

Microsoft Word 聂雪梅.doc

Microsoft Word 聂雪梅.doc 4 4 Vol. 4 No. 4 2013 8 Journal of Food Safety and Quality Aug., 2013 聂雪梅 *, 李立, 孙利, 高飞 (, 100123) 摘要 : 2011 12,, 2007~2011,,,, 关键词 : ; ; ; ; Trade and notification situation on food import and export

Microsoft Word 高 琳-二校1 出210

Microsoft Word 高琳-二校1 出210