沙门菌LAMP检测方法的建立
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摘要
为动物产品沙门菌快速检测提供技术支持,针对沙门菌属特异性的侵袭蛋白(Invasion protein,invA)基因设计2对引物,建立了一种环介导等温扩增(Loop-mediated iso?thermal amplification,LAMP)方法。对优化后的LAMP方法进行了特异性检测、灵敏度检测及沙门菌人工污染鸡蛋内容物检测。结果表明,该LAMP法可快速、特异地检测出沙门菌。LAMP法对细菌培养物检出限为8.7×100cfu/mL,对人工污染鸡蛋内容物中沙门菌的检出限为9.5×100cfu/mL。该方法有望用于鸡蛋中沙门菌的快速检测。
To provide technical support for rapid detection of Salmonella in animal products, after designing 2 pairs of primers against Salmonella specific invasion protein gene, a loop-mediated isothermal amplification(LAMP)method was established. On the basis of optimizing LAMP reaction system and reaction conditions, specific detection and sensitivity test of the method were carried out. The content of eggs contaminated by Salmonella was detected by this method. The results showed that the LAMP method can detect Salmonella rapidly and specifically. The detection limit of this LAMP method for bacterial culture was 8.7×100 cfu/mL and for the contents of artificially contaminated eggs was 9.5×100 cfu/mL. This method was expected to be used for rapid detection of Salmonella in eggs.
To provide technical support for rapid detection of Salmonella in animal products, after designing 2 pairs of primers against Salmonella specific invasion protein gene, a loop-mediated isothermal amplification(LAMP)method was established. On the basis of optimizing LAMP reaction system and reaction conditions, specific detection and sensitivity test of the method were carried out. The content of eggs contaminated by Salmonella was detected by this method. The results showed that the LAMP method can detect Salmonella rapidly and specifically. The detection limit of this LAMP method for bacterial culture was 8.7×100 cfu/mL and for the contents of artificially contaminated eggs was 9.5×100 cfu/mL. This method was expected to be used for rapid detection of Salmonella in eggs.
引文
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[15]欧新华,张如胜,宋克云,等.环介导等温扩增(LAMP)技术检测沙门菌属方法的建立[J].实用预防医学,2008,15(6):1945-1947.
[16]袁发浒,尹欢,李琦,等.肠炎沙门氏菌的LAMP检测方法的建立[J].中国酿造,2013,32(4):146-149.
[2]MARLOW M A,LUNA-GIERKE R E,GRIFFIN P M,et al.Foodborne disease outbreaks in correctional institutionsUnited States,1998~2014[J].Am J Public Health,2017,107(7):1150-1156.
[3]王霄晔,任婧寰,王哲,等.2017年全国食物中毒事件流行特征分析[J].疾病监测,2018,33(5):359-364.
[4]WAGENAAR J A,HENDRIKSEN R S,CARRIQUE-MASJ.Practical considerations of surveillance of Salmonella serovars other than Enteritidis and Typhimurium[J].Rev Sci Techn 2013,32(2):509-519.
[5]陆承平.兽医微生物学[M].5版.北京:中国农业出版社,2013:102-108.
[6]RIEMANN H,HIMATHONGKHAM S,WILLOUGHBY D,et al.A survey for Salmonella by drag swabbing manure piles in California egg ranches[J].Avian Dis,1998,42(1):67-71.
[7]CRUMP J A,GRIFFIN P M,ANGULO F J.Bacterial contamination of animal feed and its relationship to human foodborne illness[J].Clin Infect Dis,2002,35(7):859-865.
[8]陈飞,成大荣,田志高,等.鸡蛋中沙门氏菌的快速检测[J].江苏农业科学,2009(5)5:284-285.
[9]CHEN J,ZHANG L,PAOLI G C,et al.A real-time PCRmethod for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis[J].Int J Food Microbiol,2010,137(2/3):168-174.
[10]NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loopmediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[11]王明俊.兽医生物制品学[M].北京:中国农业出版社,1997:296.
[12]陈金顶,索青利,廖明,等.沙门氏菌的invA基因序列分析与分子检测[J].中国人兽共患病杂志,2004,20(10):868-871.
[13]DARWIN K H,MILLER V L.Molecular basis of the interaction of Salmonella with the intestinal mucosa[J].Clin Microbiol Rev,1999,12(3):405-428.
[14]刘艳环,苗利光,刘晓颖,等.沙门氏菌入侵因子基因保守序列的克隆与分析[J].特产研究,2005,27(4):1-3.
[15]欧新华,张如胜,宋克云,等.环介导等温扩增(LAMP)技术检测沙门菌属方法的建立[J].实用预防医学,2008,15(6):1945-1947.
[16]袁发浒,尹欢,李琦,等.肠炎沙门氏菌的LAMP检测方法的建立[J].中国酿造,2013,32(4):146-149.