摘要
根据GenBank中公布的猪线粒体色素细胞b基因序列设计6条特异性环介导等温扩增(loop-mediated isothermalamplification,LAMP)引物,通过简化线粒体DNA提取步骤,优化反应体系,以常见的牛、羊、鸡、鸭、马、驴肉为阴性对照验证该方法的特异性,掺假率梯度稀释以验证该LAMP法的最低检测限,建立常见肉制品中猪肉成分的可视化LAMP检测方法。结果表明:该方法提取目的基因操作简单、稳定,该可视化LAMP检测法以4-(2-吡啶偶氮)-间苯二酚钠盐为指示剂,能准确检测出常见肉类中猪肉是否掺杂,检测结果肉眼可见,灵敏度为10 pg/μL。
Six primers specific to the mitochondrial cytochrome b gene of swine were designed for use in loop-mediated isothermal amplification(LAMP). A simplified procedure was used for mitochondrial DNA extraction, and the reaction system was optimized. The specificity was tested with mitochondrial DNA samples from beef, mutton, chicken, duck meat,horse meat, and donkey meat as the negative controls, and the limit of detection(LOD) was determined with gradient dilution of adulterated meat with pork. A visual LAMP method for the detection of pork components in common meat products was established. The results showed that the modified mitochondrial DNA extraction method was simple and repeatable, and the visual LAMP method with 4-(2-pyridylazo)-resorcinol sodium salt as the indicator could accurately detect pork adulteration in common meat products with an LOD of 10 pg/μL.
引文
[1]陶然,王昕,苏志伟.食品中大肠杆菌快速检测方法的研究综述[J].食品安全导刊,2017(24):72.DOI:10.3969/j.issn.1674-0270.2017.24.075.
[2]MCMULLAN R,MENON V,BEUKERS A G,et al.Cronobacter sakazakii infection from expressed breast milk,Australia[J].Emerging Infectious Diseases,2018,24(2):393-394.DOI:10.3201/eid2402.171411.
[3]龙诚.一起副溶血性弧菌食物中毒的调查与分析[J].食品与发酵科技,2010,46(1):97-99.DOI:10.3969/j.issn.1674-506X.2010.01-025.
[4]冯学慧,黄素珍.肉类产品兽药残留的危害与对策[J].肉类研究,2010,24(5):3-7.DOI:10.3969/j.issn.1001-8123.2010.05.003.
[5]杨杰,高洁,苗虹.论食品欺诈和食品掺假[J].食品与发酵工业,2015,41(12):235-240.DOI:10.13995/j.cnki.11-1802/ts.201512045.
[6]任君安.羊肉及其制品中掺假动物源性成分数字PCR技术精准定量研究[D].北京:中国农业大学,2017.
[7]冯永巍,王琴.肉类掺假检验技术研究进展[J].食品与机械,2013,29(4):237-240.DOI:10.3969/j.issn.1003-5788.2013.04.058.
[8]李欣南,关一夫.掺假肉检验技术发展现状[J].食品研究与开发,2016,37(5):189-193.DOI:10.3969/j.issn.1005-6521.2016.05.044.
[9]NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-mediated isothermalamplification of DNA[J].Nucleic Acids Research,2000,28(12):e63.DOI:10.1093/nar/28.12.e63.
[10]ZAHRADNIK C,MARTZY R,MACH R L,et al.Loop-mediated isothermal amplification(LAMP)for the detection of horse meat in meat and processed meat products[J].Food Analytical Methods,2015,8(6):1576-1581.DOI:10.1007/s12161-014-0072-8.
[11]DEB R,SENGAR G S,SINGH U,et al.Application of a loopmediated isothermal amplification assay for rapid detection of cow components adulterated in buffalo milk/meat[J].Molecular Biotechnology,2016,58(12):850-860.DOI:10.1007/s12033-016-9984-4.
[12]MA B,DAI M Y,FANG J H,et al.Visual loop-mediated isothermal amplification(LAMP)method for identification bovine and ovine gene in animal foodstuff[J].American Journal of Food Technology,2016,11:193-203.DOI:10.3923/ajft.2016.193.203.
[13]杨丽霞,邱华丽,蒋成,等.可视化环介导等温扩增技术检测鸡鸭源性成分[J].食品安全质量检测学报,2017,8(2):624-628.DOI:10.3969/j.issn.2095-0381.2017.02.043.
[14]徐淑菲,孔繁德,苗丽,等.牛源性成分L A M P检测方法的建立[J].中国动物检疫,2016,33(12):94-99.DOI:10.3969/j.issn.1005-944X.2016.12.025.
[15]刘少宁,陈智,张志民,等.鉴别绵羊肉中狐狸源性成分的环介导等温扩增检测方法的建立[J].中国食品卫生杂志,2016,28(1):75-78.DOI:10.13590/j.cjfh.2016.01.017.
[16]王爱彦.中国猪肉产业的现状及未来[J].北方牧业,2017(11):13-14.
[17]LEE S Y,KIM M J,HONG Y,et al.Development of a rapid on-site detection method for pork in processed meat products using real-time loop-mediated isothermal amplification[J].Food Control,2016,66:53-61.DOI:10.1016/j.foodcont.2016.01.041.
[18]ROY S,RAHMAN I A,AHMED M U.based rapid detection of pork and chicken using LAMP-magnetic bead aggregates[J].Analytical Methods,2016,8(11):2391-2399.DOI:10.1039/C6AY00274A.
[19]RAN G Y,REN L Z,HAN X L,et al.Development of a rapid method for the visible detection of pork DNA in halal products by loopmediated isothermal amplification[J].Food Analytical Methods,2016,9(3):565-570.DOI:10.1007/s12161-015-0246-z.
[20]杨丽霞,淑君,彭新凯.环介导等温扩增法检测牛羊肉中的猪肉成分[J].食品与机械,2013,29(5):63-65.DOI:10.3969/j.issn.1003-5788.2013.05.017.
[21]李向丽,刘垚,谭贵良,等.基于LAMP法快速检测羊肉及其制品中的猪、鸭和羊源性成分[J].中国食品卫生杂志,2015,27(3):247-252.DOI:10.13590/j.cjfh.2015.03.006.
[22]陈文秀,姜旋,马晓燕,等.实时荧光环介导等温扩增技术检测乳粉中的肺炎克雷伯氏菌[J].食品科学,2014,35(20):192-197.DOI:10.7506/spkx1002-6630-201420038.
[23]贺庆华,邱翔.一种低危害的基因组D N A和总R N A的提取方法[J].西南民族大学学报(自然科学版),2017,43(6):562-566.DOI:10.11920/xnmdzk.2017.06.003.
[24]吴琼,宋安东.食用植物油中DNA提取和基因检测研究进展[J].粮油食品科技,2017(6):50-55.DOI:10.3969/j.issn.1007-7561.2017.06.011.
[25]龙绛雪,曹福祥,王志文.油菜线粒体DNA提取纯化方法研究[J].中南林业科技大学学报,2009,29(3):106-110.DOI:10.3969/j.issn.1673-923X.2009.03.017.
[26]刘昊,黄文胜,邓婷婷,等.LAMP法检测食品中开心果过敏原成分[J].食品科学,2013,34(22):128-132.DOI:10.7506/spkx1002-6630-201322026.
[27]唐毓祎.水产品弧菌的等温核酸扩增可视化检测方法的建立与应用[D].上海:上海海洋大学,2017.
[28]刘威.现场传染病诊断关键技术的研究及应用[D].北京:中国人民解放军军事医学科学院,2014.
[29]罗敏红.建立环介导等温扩增方法检测腹泻患者中的小肠结肠炎耶尔森菌[D].广州:南方医科大学,2013.
[30]SHI Y,FENG Y,XU C P,et al.Loop-mediated isothermal amplification assays for the rapid identification of duck-derived ingredients in adulterated meat[J].Food Analytical Methods,2017,10(7):2325-2331.DOI:10.1007/s12161-016-0767-0.
[31]何玮玲,胡序建,程欣,等.含有扩增内标的食品中猪肉和鸡肉成分Taqman探针实时荧光PCR检测方法的建立[J].中国农业科学,2013,46(21):4578-4585.DOI:10.3864/j.issn.0578-1752.2013.21.021.
[32]宋丽萍,薛晨玉,路勇,等.应用实时荧光P C R技术定量检测羊肉中的猪肉成分[J].食品科技,2014,39(10):319-322;326.DOI:10.13684/j.cnki.spkj.2014.10.069.
[33]李津,刘巍,李俊超,等.实时荧光LAMP法检测转基因猪的标记基因hCD3[J].畜牧兽医学报,2017,48(3):538-543.DOI:10.11843/j.issn.0366-6964.2017.03.018.
[34]姚贵哲,王伟利,夏明,等.沙门氏菌现场快速检测方法的建立[J].黑龙江畜牧兽医,2018(3):172-175;260.DOI:10.13881/j.cnki.hljxmsy.2017.06.0493.
[35]马骉,吴莹莹,戴明雁,等.副溶血性弧菌PMA-LAMP方法的建立[J].现代食品科技,2016,32(7):205-213.DOI:10.13982/j.mfst.1673-9078.2016.7.032.
