应用基因芯片技术检测肉及肉制品中5种致病菌

摘要/Abstract

摘要： 建立一种运用多重聚合酶链式反应(PCR)结合基因芯片技术检测大肠埃希氏菌、沙门氏菌、金黄色葡萄球菌、志贺氏菌和单核细胞增生李斯特菌5种食源性致病菌的快速、准确、灵敏的方法。分别选取编码大肠埃希氏菌的slt基因、沙门氏菌invA基因、金黄色葡萄球菌nuc基因、志贺氏菌ipaH基因和单核细胞增生李斯特菌inlA基因，并以细菌16S rDNA基因作为阳性对照，设计引物和探针，进行多重PCR扩增，产物与含特异性探针的芯片杂交。结果表明：该基因芯片可同时特异性地检测5种致病菌，多重PCR检测灵敏度为20pg，而DNA芯片检测灵敏度可达2pg；用所制备的基因芯片检测实际肉及肉制品样品，准确率高于传统培养法。所建立的基因芯片检测方法特异性好、灵敏度高，可为食源性致病菌的检测提供理想手段。

关键词: 肉及肉制品, 多重PCR, 致病菌, 基因芯片

Abstract: A rapid, accurate and sensitive method for simultaneous detection of Escherichia coli, Salmonella spp, Staphylococcus aureus, Shigella dysenteriae and Listeria monocytogenes was developed by using DNA microarray coupled with multiplex PCR. Six pairs of primers and oligo-nucleotide probes were designed and synthesized by using the Slt gene of Escherichia coli, the invA gene of Salmonella spp, the nuc gene of Staphylococcus aureus, the ipaH gene of Shigella dysenteriae and the inlA gene of Listeria monocytogenes as target genes, and 16S rDNA of bacteria as positive control. The multiplex PCR products were hybridized with DNA microarray containing specific probes for five food-borne pathogenic microorganisms. DNA microarray could allow simultaneous detection of five specific pathogens with detection limit of 2 pg, which was more sensitive than multiplex PCR with detection limit of 20 pg. Similarly, the application of DNA microarrary in the detection of actual meat and meat products revealed higher accuracy than traditional culture method. Therefore, DNA microarray provides a specific, sensitive and promising approach for the detection of pathogenic microorganisms.

Key words: meat and meat products, multiplex PCR, pathogens, DNA chip

中图分类号:

TS207.4

祝儒刚1，李拖平1,*，宋立峰2. 应用基因芯片技术检测肉及肉制品中5种致病菌[J]. 食品科学, 2012, 33(14): 211-215.

参考文献

[1] 陈双雅，张永祥.基因芯片在食品微生物检测中的应用[J].食品工业科技，2008, 29(4): 314-320.
[2] 李巧云，蔡挺，常燕子.基因芯片技术在检测临床致病菌中的应用[J].现代实用医学，2010, 22(5): 487-500.
[3] 李林海，程颖，陈丽丹，等.基因芯片法鉴定血培养标本中致病菌的临床评价[J].南方医科大学学报，2009, 29(10): 2070-2072.
[4] 王洪敏，何冬梅，周辉，等.华南地区常见的8种食源性致病菌检测基因芯片研制[J].南方医科大学学报，2010, 30(11): 2472-2476.
[5] 金大智，文思远，王升启.基因芯片技术在检测肠道致病菌方面的应用[J].微生物学报，2006, 46(2): 500-505.
[6] 李禾，徐琦，吴忠华，等.基因芯片技术检测3种肠道病原微生物方法的建立[J].生物技术通讯，2007, 18(3): 441-445
[7] 陈昱，潘迎捷，赵勇，等.基因芯片技术检测3种食源性致病微生物方法的建立[J].微生物学通报，2009, 36(2): 285-291.
[8] 黎昊雁，章国祥.十二种食源性致病菌可见光基因芯片检测方法的建立[J].检验检疫学刊，2009, 19(3): 17-21.
[9] ABOLMAATY A, VU C, OLIVER J, et al. Development of a new lysis solution for releasing genomic DNA from bacterial cells for DNA amplification by polymerase chain reaction[J]. Microbios, 2000, 101(6): 181-189.
[10] JIN D Z, WEN S Y, CHEN S H, et al. Detection and identification of intestinal pathogensin clinical specimensusing DNA microarrays[J]. Molecular and Cellular Probes, 2006, 20(6): 337-347.
[11] 毛正果，郑浩轩，王新颖，等.基因芯片检测常见肠道致病菌感染的研究与评价[J].胃肠病学和肝病学杂志，2008, 17(10): 809-812.
[12] SPEEL E J M, HOPMAN A H N, KOMMINOTH P. Amplification methods to increase the sensitivity of in situ hybridization: play CARD(S)[J]. The Journal of Histochemistry and Cytochemistry, 1999, 47(3): 281-288.
[13] SPEEL E J M, RAMACEKERS F C S, HOPMAN A H N. Sensitivity multicolor fluorescence in situ hybridization using catalyzed reporter deposition (CARD) amplification[J]. The Journal of Histochemistry and Cytochemistry, 1997, 45(10): 1439-1446.
[14] KERSTENS H M, PODDIGHE P J, HANSELAAR A G. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine[J]. J Histochem Cytochem, 1995, 43(4): 347-352.
[15] 李玉锋，何洋，刘红露.应用基因芯片技术鉴定食源性金黄色葡萄球菌的研究[J].食品科学，2007, 28(12): 294-297.
[16] 王大勇，方振东，谢朝新，等.水体中致病菌快速检测的基因芯片技术研究[J].解放军医学杂志，2010, 35(9): 1117-1122.
[17] 高爽，谢明杰，金大智，等.运用基因芯片技术建立检测水产品中常见病原微生物方法的研究[J].生物技术通讯，2007, 18(1): 72-76.
[18] 任莉莉，丛彦丽.用于食源性疾病病原菌诊断的基因芯片研究及评价[J].食品科学，2011, 32(4): 208-211.
[19] 杨小鹃，吴清平，张菊梅，等.畜禽肉沙门氏菌和大肠杆菌O157多重PCR检测研究[J].微生物学通报，2008, 35(3): 470-474.
[20] 王大勇，方振东，谢朝新，等.食源性致病菌快速检测技术研究进展[J].微生物学杂志，2009, 29(5): 67-72.
[21] 贺晨，孙鸿燕，邵丽筠，等.多重PCR结合基因芯片技术检测11种致病菌方法的建立[J].中国实验诊断学，2011, 27(4): 587-591.
[22] Moreira A N, Conceicao F R, Conceicao R C, et al. Detection of Salmonella Typhimurium in Raw Meats using In-House Prepared Monoclonal Antibody Coated Magnetic Beads and PCR Assay of the fimA Gene[J]. J Immunoassay Immunochem, 2008, 29(1): 58-66.