Abstract
A multiplex LAMP-melting curve method for simultaneous detection of Salmonella spp. and Staphylococcus aureus in food was developed. The primers were designed according to the sequences of invA gene of Salmonella spp. and nuc gene of Staphylococcus aureus, and the reaction system was optimized. The amplification products were analyzed by melting curves, electrophoresis, precipitation observation and fluorescence observation as well. No false positive and false negative results emerged during tests of 9 target and 23 non-target bacteria strains, and the pathogens could be determined by melting temperature on melting curves. DNA sequencing of LAMP products showed the sequences as deduced, confirming the correct LAMP amplification. The detection limits for each pathogen were both 10 fg/μL. The tests result of 232 samples showed that 2 pieces of raw pork were contaminated by Staphylococcus aureus. This method showed high specificity, and could be used to detect foodborne pathogens rapidly.
Publication Date
4-28-2015
First Page
87
Last Page
92
DOI
10.13652/j.issn.1003-5788.2015.02.018
Recommended Citation
Kan, JIANG; Hui, ZHANG; Xin, WANG; Pengpeng, LIU; Jianfeng, HUANG; and Zhinan, ZHOU
(2015)
"Development of multiplex LAMP combined with melting curves analysis for detection of two kinds of food borne pathogens,"
Food and Machinery: Vol. 31:
Iss.
2, Article 18.
DOI: 10.13652/j.issn.1003-5788.2015.02.018
Available at:
https://www.ifoodmm.cn/journal/vol31/iss2/18
References
[1] 卫生部. GB 4789.4—2010 食品安全国家标准 食品微生物学检验 沙门氏菌检验[S]. 北京:中国标准出版社, 2010.
[2] 卫生部. GB 4789.10—2010 食品安全国家标准 食品微生物学检验 金黄色葡萄球菌检验[S]. 北京:中国标准出版社, 2010.
[3] Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12): E63.
[4] 易海华, 赵金伟, 房超, 等. 志贺菌环介导等温扩增检测方法建立[J]. 中国公共卫生, 2011, 27(3): 380~382
[5] 董鑫悦, 满朝新, 卢雁, 等. 环介导等温扩增法快速检测乳中阪崎肠杆菌[J]. 食品工业科技, 2013, 34(5): 318~320.
[6] 王晶钰, 董睿, 王利勤, 等. 市售鲜鸡蛋中沙门氏菌的分离鉴定及毒力岛基因检测[J]. 食品科学, 2012, 33(16): 154~158.
[7] 邵美丽, 许岩, 刘思国, 等. TaqMan探针实时定量PCR检测肉中金黄色葡萄球菌的研究[J]. 食品工业, 2013, 34(3):109~112.
[8] 质检总局. SN/T 1869—2007 食品中多种致病菌快速检测方法 PCR法[S]. 北京:中国标准出版社, 2007.
[9] 刘园园, 肖性龙, 余以刚. 一种能同时富集沙门氏菌、金黄色葡萄球菌和单增李斯特菌的培养基[J]. 微生物学报, 2009, 49(10): 1 389~1 396.
[10] Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers[J]. Molecular and Cellular Probes, 2002, 16(3): 223~229.