细菌回复突变试验标准菌株分子质控方法的研究
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摘要
目的建立细菌回复突变试验标准菌株的分子生物学质量控制(质控)方法。方法使用分子生物学方法(16s rDNA、种属特异性鉴定PCR、组氨酸操纵子关键突变位点序列分析、凝胶电泳和多位点序列分析方法)鉴定细菌回复突变试验标准菌株,并与传统生物学特性的质控方法进行比对。结果细菌回复突变试验标准菌株的生物学特性结果符合标准要求;16S rDNA序列比对为沙门菌,且种属特异性扩增结果为鼠伤寒沙门菌;标准菌株均具有组氨酸操纵子区原始构建的突变位点;脉冲场凝胶电泳带型均不一致,最低相似度为82%;多位点序列分析可分为ST19和ST3438两个ST型。结论建立了细菌回复突变试验标准菌株的分子生物学质控方法,该方法具有操作性强和重复性好等特点,与传统生物学特征方法具有很好的一致性,特别是针对相应his基因突变位点的检测手段,确保了细菌回复突变试验标准菌株检测的遗传稳定性和准确性。
Objective To set up a method for molecular quality control of the standard strain for bacterial reverse mutation test(Ames test).Methods The identification of standard strain was conducted by using 5 different molecular biology methods including 16S rDNA,PCR,species-specific identification of key histidine operon mutation sequence analysis,pulsed field gel electrophoresis and sequence analysis in identification of the standard strain in Ames test,and compared detected related characteristics with the traditional quality control methods.Results The biological characteristics of the standard strain for Ames test met the standard requirements.The tested 16S rDNA sequence alignment was Salmonella,and the species-specific amplification was Salmonella typhimurium.The standard strain had all mutated sites derived from original construction by the histidine operon region.The band types were inconsistent by pulsed field gel electrophoresis,the minimum similarity was 82%.It showed two types of ST19 and ST3438 by multilocus sequence analysis.Conclusion The molecular biology quality control method of the standard bacterial strain was set up for Ames test,which had a well consistency with the conventional methods,espacially,in detection of corresponding mutations in his gene,with a stable genetic stability and accuracy.In addtion,it had adventagies in operation and repeatibility.
Objective To set up a method for molecular quality control of the standard strain for bacterial reverse mutation test(Ames test).Methods The identification of standard strain was conducted by using 5 different molecular biology methods including 16S rDNA,PCR,species-specific identification of key histidine operon mutation sequence analysis,pulsed field gel electrophoresis and sequence analysis in identification of the standard strain in Ames test,and compared detected related characteristics with the traditional quality control methods.Results The biological characteristics of the standard strain for Ames test met the standard requirements.The tested 16S rDNA sequence alignment was Salmonella,and the species-specific amplification was Salmonella typhimurium.The standard strain had all mutated sites derived from original construction by the histidine operon region.The band types were inconsistent by pulsed field gel electrophoresis,the minimum similarity was 82%.It showed two types of ST19 and ST3438 by multilocus sequence analysis.Conclusion The molecular biology quality control method of the standard bacterial strain was set up for Ames test,which had a well consistency with the conventional methods,espacially,in detection of corresponding mutations in his gene,with a stable genetic stability and accuracy.In addtion,it had adventagies in operation and repeatibility.
引文
[1]MARON D M,AMES B N.Revised methods for the Salmonella mutagenicity test[J].Mutat Res,1983,113(3/4):173-215.
[2]SIERRA,MARIA L,GAIVAO,et al.Genotoxicity and DNA Repair[M]//ARACELI PILLCO,EDUARDO DE LA PENA.A mes Test(Bacterial Reverse Mutation Test):Why,When,and How to Use.New York:Humana Press,2014:3-22.
[3]US.Food and Drug Administration.Guidance for Industry and Other Stakeholders Toxicological Principles for the Safety Assessment of Food Ingredients[M]//Redbook 2000:IV.C.1.a Bacterial Reverse Mutation Test.2007.
[4]OECD.GUIDELINE FOR TESTING OF CHEMICALS.Test No.471:Bacterial Reverse Mutation Test[S].1997.
[5]中国国家标准化管理委员会.化学品细菌回复突变试验方法:GB/T 21786—2008[S].北京:中国标准出版社,2008.
[6]国家食品药品监督管理总局.化妆品安全技术规范:2015年版[M].2015.
[7]中国国家标准化管理委员会.食品安全国家标准细菌回复突变试验:GB 15193.4—2014[S].北京:中国标准出版社,2014.
[8]MONTALVO-KATZ S,HUANG H,APPEL M D,et al.Association with soil bacteria enhances p38-dependent infection resistance in Caenorhabditis elegans[J].Infect Immun,2013,81(2):514-520.
[9]RAHN K,DE GRANDIS S A,CLARKE R C,et al.Amplification of an inv A gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J].Mol Cell Probes,1992,6(4):271-279.
[10]LIN J S,TSEN H Y.Development and use of polymerase chain reaction for the specific detection of Salmonella typhimurium in stool and food samples[J].J Food Prot,1999,62(10):1103-1110.
[11]AMES B N,LEE F D,DURSTON W E.An improved bacterial test system for the detection and classification of mutagens and carcinogens[J].Proc Natl Acad Sci,1973,70(3):782-786.
[12]MORTELMANS K,ZEIGER E.The Ames Salmonella/microsome mutagenicity assay[J].Mutat Res,2000,455(1/2):29-60.
[13]HARTMAN P E,AMES B N,ROTH J R,et al.Target sequences for mutagenesis in Salmonella histidine-requiring mutants[J].Environ Mutagen,1986,8(4):631-641.
[14]LEVIN D E,YAMASAKI E,AMES B N.A new Salmonella tester strain,TA97,for the detection of frameshift mutagens.A run of cytosines as a mutational hot-spot[J].Mutat Res,1982,94(2):315-330.
[15]HOLLAENDER A.Chemical Mutagens[M]//AMES BN.The Detection of Chemical Mutagens with Enteric Bacteria.Boston:Springer,1971:267-282.
[16]KUMAR Y.Salmonella-A Diversified Superbug[M]//KARADAYI M,BARI爦AND,GLLCE M.Salmonella as a unique tool for Genetic Toxicology.Rijeka:In Tech,2012:255-278.
[2]SIERRA,MARIA L,GAIVAO,et al.Genotoxicity and DNA Repair[M]//ARACELI PILLCO,EDUARDO DE LA PENA.A mes Test(Bacterial Reverse Mutation Test):Why,When,and How to Use.New York:Humana Press,2014:3-22.
[3]US.Food and Drug Administration.Guidance for Industry and Other Stakeholders Toxicological Principles for the Safety Assessment of Food Ingredients[M]//Redbook 2000:IV.C.1.a Bacterial Reverse Mutation Test.2007.
[4]OECD.GUIDELINE FOR TESTING OF CHEMICALS.Test No.471:Bacterial Reverse Mutation Test[S].1997.
[5]中国国家标准化管理委员会.化学品细菌回复突变试验方法:GB/T 21786—2008[S].北京:中国标准出版社,2008.
[6]国家食品药品监督管理总局.化妆品安全技术规范:2015年版[M].2015.
[7]中国国家标准化管理委员会.食品安全国家标准细菌回复突变试验:GB 15193.4—2014[S].北京:中国标准出版社,2014.
[8]MONTALVO-KATZ S,HUANG H,APPEL M D,et al.Association with soil bacteria enhances p38-dependent infection resistance in Caenorhabditis elegans[J].Infect Immun,2013,81(2):514-520.
[9]RAHN K,DE GRANDIS S A,CLARKE R C,et al.Amplification of an inv A gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J].Mol Cell Probes,1992,6(4):271-279.
[10]LIN J S,TSEN H Y.Development and use of polymerase chain reaction for the specific detection of Salmonella typhimurium in stool and food samples[J].J Food Prot,1999,62(10):1103-1110.
[11]AMES B N,LEE F D,DURSTON W E.An improved bacterial test system for the detection and classification of mutagens and carcinogens[J].Proc Natl Acad Sci,1973,70(3):782-786.
[12]MORTELMANS K,ZEIGER E.The Ames Salmonella/microsome mutagenicity assay[J].Mutat Res,2000,455(1/2):29-60.
[13]HARTMAN P E,AMES B N,ROTH J R,et al.Target sequences for mutagenesis in Salmonella histidine-requiring mutants[J].Environ Mutagen,1986,8(4):631-641.
[14]LEVIN D E,YAMASAKI E,AMES B N.A new Salmonella tester strain,TA97,for the detection of frameshift mutagens.A run of cytosines as a mutational hot-spot[J].Mutat Res,1982,94(2):315-330.
[15]HOLLAENDER A.Chemical Mutagens[M]//AMES BN.The Detection of Chemical Mutagens with Enteric Bacteria.Boston:Springer,1971:267-282.
[16]KUMAR Y.Salmonella-A Diversified Superbug[M]//KARADAYI M,BARI爦AND,GLLCE M.Salmonella as a unique tool for Genetic Toxicology.Rijeka:In Tech,2012:255-278.