利用AFLP方法研究嗜肺巴斯德杆菌的遗传多态性
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摘要
目的针对嗜肺巴斯德杆菌分类变更及其在实验动物中感染率高的问题,建立该菌的快速基因分型方法,为其检测方法的修订及有效控制提供支撑。方法利用扩增片段长度多态性方法(AFLP),对实验动物中分离的314株嗜肺巴斯德杆菌及2株标准菌株进行遗传多态性及分子流行病学分析。结果 AFLP方法将受试分离株共分为了11个基因簇、190个基因型,多态性条带主要有31～36条,辛普森多样性指数0. 992。基因簇AC7为北京地区的主要流行群。结论本研究表明北京地区实验动物中的嗜肺巴斯德杆菌基因型丰富,2株标准菌株所在的基因簇在多态性上有明显区别。某些不同来源动物存在交叉污染、污染源多,以及长期污染的情况。
Objective To establish a rapid genotyping method for detection of Pasteurella pneumotropica as a standard revision and effective control,in view of problems with classification of P. pneumotropica and its high infection rate in laboratory animals. Methods Genetic polymorphism and molecular epidemiological analyses of 314 isolates and two standard strains of P. pneumotropica from laboratory animals were performed using an amplified fragment length polymorphism( AFLP) method. Results The AFLP method divided tested isolates into 11 gene clusters and 190 genotypes; gene cluster AC7 was the main epidemic group. Polymorphic bands mainly occurred between 31 ～ 36,and the Simpson diversity index was 0. 992. Conclusions This study shows that P. pneumotropica genotypes are abundant in laboratory animals in Beijing area,and different strains represented by the two standard strains have significantly different polymorphisms. Our results indicate the characteristics of cross contamination, abundant sources, and long-term contamination in some laboratory animals of different sources.
Objective To establish a rapid genotyping method for detection of Pasteurella pneumotropica as a standard revision and effective control,in view of problems with classification of P. pneumotropica and its high infection rate in laboratory animals. Methods Genetic polymorphism and molecular epidemiological analyses of 314 isolates and two standard strains of P. pneumotropica from laboratory animals were performed using an amplified fragment length polymorphism( AFLP) method. Results The AFLP method divided tested isolates into 11 gene clusters and 190 genotypes; gene cluster AC7 was the main epidemic group. Polymorphic bands mainly occurred between 31 ～ 36,and the Simpson diversity index was 0. 992. Conclusions This study shows that P. pneumotropica genotypes are abundant in laboratory animals in Beijing area,and different strains represented by the two standard strains have significantly different polymorphisms. Our results indicate the characteristics of cross contamination, abundant sources, and long-term contamination in some laboratory animals of different sources.
引文
[1]田克恭,贺争鸣,刘群等.实验动物疫病学[M].北京:中国农业出版社,2015:621-623.
[2] GB 14922. 2-2011实验动物微生物学等级及监测[S].北京:中国标准出版社,2002:2.
[3] GB/T 14926. 12-2001实验动物嗜肺巴斯德杆菌检测方法[S].北京:中国标准出版社,2011.
[4] Adhikary S,Nicklas W,Bisgaard M,et al. Rodentibacter gen.nov. including Rodentibacter pneumotropicus comb. nov.,Rodentibacter heylii sp. nov.,Rodentibacter myodis sp. nov.,Rodentibacter ratti sp. nov.,Rodentibacter heidelbergensis sp.nov.,Rodentibacter trehalosifermentans sp. nov.,Rodentibacter rarus sp. nov.,Rodentibacter mrazii and two genomospecies[J].Int J Syst Evol Microbiol,2017,67(6):1793-1806.
[5]邢进,冯育芳,岳秉飞等.北京地区实验动物中嗜肺巴斯德杆菌的表型分析[J].中国比较医学杂志,2014,24(6):54-57.
[6]邢进,冯育芳,岳秉飞等.嗜肺巴斯德杆菌研究进展[J].中国实验动物学报,2014,22(2):90-94.
[7] Vos P,Hogers R,Bleeker M,et al. AFLP:a new technique for DNA fingerprinting[J]. Nucleic Acids Res,1995,23(21):4407-4414.
[8] Sthitmatee N,Kataoka Y,Sawada T. Molecular epidemiology of Japanese avian Pasteurella multocida strains by the singleenzyme amplified fragment length polymorphism and pulsed-field gel electrophoresis[J]. J Vet Med Sci,2010,72(11):1465-1470.
[9] Sasaki H,Kawamoto E,Okiyama E,et al. Molecular typing of Pasteurella pneumotropica isolated from rodents by amplified 16S ribosomal DNA restriction analysis and pulsed-field gel electrophoresis[J]. Microbiol Immunol,2006,50(4):265-272.
[10] In silico AFLP-PCR amplification[DB/OL],http://insilico.ehu.es/AFLP/index.php? mo=Pasteurella,2015-5-18.
[11] Protocol:AFLP Microbial Fingerprinting(English)[DB/OL],https://www. thermofisher. com/search/results? query=AFLP%20Microbial%20Fingerprinting&focusarea,2010-7-23/2015-5-18.
[12] Hazen SP,Leroy P,Ward R. AFLP in Triticum aestivum L.:patterns of genetic diversity and genome distribution[J].Euphytica,2002,125(1):89-102.
[13] Hunter PR,Gaston MA. Numerical index of the discriminatory ability of typing systems:An application of simpson’s index of diversity[J]. J Clin Microbiol,1988,26(11):2465-2466.
[14] Boot R. A Pasteurella pneumotropica strain of mouse origin colonizes rats but is out competed by a P. pneumotropica strain of rat origin[J]. Scand J Lab Anim Sci,2010,37(4):261-265.
[15] Hayashimoto N,Takakura A,Itoh T. Genetic diversity on 16S r DNA sequence and phylogenic tree analysis in Pasteurella pneumotropica strains isolated from laboratory animals[J]. Curr Microbiol,2005,51(4):239-243.
[16] Kawamoto E,Sasaki H,Okiyama E,et al. Pathogenicity of Pasteurella pneumotropica in immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1(ICR)mice[J]. Exp Anim,2011,60(5):463-470.
[17] Hayashimoto N, Yasuda M, Ueno M, et al. Experimental infection studies of Pasteurella pneumotropica and V-factor dependent Pasteurellaceae for F344-rnu rats[J]. Exp Anim,2008,57(1):57-63.
[2] GB 14922. 2-2011实验动物微生物学等级及监测[S].北京:中国标准出版社,2002:2.
[3] GB/T 14926. 12-2001实验动物嗜肺巴斯德杆菌检测方法[S].北京:中国标准出版社,2011.
[4] Adhikary S,Nicklas W,Bisgaard M,et al. Rodentibacter gen.nov. including Rodentibacter pneumotropicus comb. nov.,Rodentibacter heylii sp. nov.,Rodentibacter myodis sp. nov.,Rodentibacter ratti sp. nov.,Rodentibacter heidelbergensis sp.nov.,Rodentibacter trehalosifermentans sp. nov.,Rodentibacter rarus sp. nov.,Rodentibacter mrazii and two genomospecies[J].Int J Syst Evol Microbiol,2017,67(6):1793-1806.
[5]邢进,冯育芳,岳秉飞等.北京地区实验动物中嗜肺巴斯德杆菌的表型分析[J].中国比较医学杂志,2014,24(6):54-57.
[6]邢进,冯育芳,岳秉飞等.嗜肺巴斯德杆菌研究进展[J].中国实验动物学报,2014,22(2):90-94.
[7] Vos P,Hogers R,Bleeker M,et al. AFLP:a new technique for DNA fingerprinting[J]. Nucleic Acids Res,1995,23(21):4407-4414.
[8] Sthitmatee N,Kataoka Y,Sawada T. Molecular epidemiology of Japanese avian Pasteurella multocida strains by the singleenzyme amplified fragment length polymorphism and pulsed-field gel electrophoresis[J]. J Vet Med Sci,2010,72(11):1465-1470.
[9] Sasaki H,Kawamoto E,Okiyama E,et al. Molecular typing of Pasteurella pneumotropica isolated from rodents by amplified 16S ribosomal DNA restriction analysis and pulsed-field gel electrophoresis[J]. Microbiol Immunol,2006,50(4):265-272.
[10] In silico AFLP-PCR amplification[DB/OL],http://insilico.ehu.es/AFLP/index.php? mo=Pasteurella,2015-5-18.
[11] Protocol:AFLP Microbial Fingerprinting(English)[DB/OL],https://www. thermofisher. com/search/results? query=AFLP%20Microbial%20Fingerprinting&focusarea,2010-7-23/2015-5-18.
[12] Hazen SP,Leroy P,Ward R. AFLP in Triticum aestivum L.:patterns of genetic diversity and genome distribution[J].Euphytica,2002,125(1):89-102.
[13] Hunter PR,Gaston MA. Numerical index of the discriminatory ability of typing systems:An application of simpson’s index of diversity[J]. J Clin Microbiol,1988,26(11):2465-2466.
[14] Boot R. A Pasteurella pneumotropica strain of mouse origin colonizes rats but is out competed by a P. pneumotropica strain of rat origin[J]. Scand J Lab Anim Sci,2010,37(4):261-265.
[15] Hayashimoto N,Takakura A,Itoh T. Genetic diversity on 16S r DNA sequence and phylogenic tree analysis in Pasteurella pneumotropica strains isolated from laboratory animals[J]. Curr Microbiol,2005,51(4):239-243.
[16] Kawamoto E,Sasaki H,Okiyama E,et al. Pathogenicity of Pasteurella pneumotropica in immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1(ICR)mice[J]. Exp Anim,2011,60(5):463-470.
[17] Hayashimoto N, Yasuda M, Ueno M, et al. Experimental infection studies of Pasteurella pneumotropica and V-factor dependent Pasteurellaceae for F344-rnu rats[J]. Exp Anim,2008,57(1):57-63.