变性梯度凝胶电泳技术在食源性沙门氏菌分子分型中的应用研究
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摘要
目的建立基于变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术的沙门氏菌分型方法。方法以5株代表性沙门氏菌为供试菌,用引物对GC-rpoB1/ropB3R扩增获得目的片段后,经变性凝胶电泳分离获得谱图,量化分析谱带多样性及均一性,依据菌株谱图量化相似性构建聚类分析图,分析该技术用于沙门氏菌分型的可行性。结果 5个供试菌的PCR-DGGE谱图差异大,其中以标准菌株肠炎沙门氏菌(ATCC9184)的多样性及丰富度最高,标准菌株鼠伤寒沙门氏菌的最低;就谱带相似性而言,分离自鸭腿样品的2株沙门氏菌的相似性最高,戴斯系数高达85.7%,而分属于不同血清型的两个标准菌株则相似度指数为0%;基于谱带相似性建立的聚类树可将供试菌株很好地分为不同簇。结论本研究所建立的基于PCR-DGGE技术的沙门氏菌分型方法准确度和分辨力均较理想,具有一定的理论及实际意义。
Objective To establish a typing method for Salmonella based on polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE). Methods With 5 representative Salmonella isolates as sample, the target fragment was amplified by using primer pair GC-ropB1/ropB3 R. The spectrum was obtained by denatured gel electrophoresis, and the diversity and evenness of the spectrum were quantitatively analyzed. The cluster analysis chart was constructed according to the similarity of the bacterial strain, and the feasibility of using this technology for the classification of Salmonella was analyzed. Results The PCR-DGGE spectra of the 5 tested strains were significantly different, with the highest diversity and richness of the standard strain Salmonella Enteritidis(ATCC 9184) and the lowest of the standard strain Salmonella Typhimurium. In terms of spectral band similarity, the 2 Salmonella strains isolated from duck leg samples had the highest similarity, with the Deiss coefficient as high as 85.7%. The similarity index of 2 standard strains belonging to different serotypes was 0%. The cluster tree based on spectral band similarity could divide the tested strains well into different clusters. Conclusion The PCR-DGGE based Salmonella typing method established in this study is ideal in accuracy and resolving power, which is of certain theoretical and practical significance.
Objective To establish a typing method for Salmonella based on polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE). Methods With 5 representative Salmonella isolates as sample, the target fragment was amplified by using primer pair GC-ropB1/ropB3 R. The spectrum was obtained by denatured gel electrophoresis, and the diversity and evenness of the spectrum were quantitatively analyzed. The cluster analysis chart was constructed according to the similarity of the bacterial strain, and the feasibility of using this technology for the classification of Salmonella was analyzed. Results The PCR-DGGE spectra of the 5 tested strains were significantly different, with the highest diversity and richness of the standard strain Salmonella Enteritidis(ATCC 9184) and the lowest of the standard strain Salmonella Typhimurium. In terms of spectral band similarity, the 2 Salmonella strains isolated from duck leg samples had the highest similarity, with the Deiss coefficient as high as 85.7%. The similarity index of 2 standard strains belonging to different serotypes was 0%. The cluster tree based on spectral band similarity could divide the tested strains well into different clusters. Conclusion The PCR-DGGE based Salmonella typing method established in this study is ideal in accuracy and resolving power, which is of certain theoretical and practical significance.
引文
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[13]廖超,张昭寰,姚鑫,等.GC夹对三种食源性致病菌的rpoB-PCR-DGGE图谱的影响[J].生物技术通报,2016,32(5):212-219.Liao C,Zhang ZH,Yao X,et al.Effects of GC clamps on RpoB-PCR-DGGE fingerprint of three types of food borne pathogens[J].Biotechnol bull,2016,32(5):212-219.
[2]Scallan E,Hoekstra RM,Angulo FJ,et al.Foodborne illness acquired in the United States-major pathogens[J].Emerg Infect Dis,2011,17(1):7-15.
[3]EFSA.The European Union summary report on trends and sources of zoonoses,zoonotic agents and food-borne outbreaks in 2016[J].EFSA J,2017,15(12):77,228.
[4]Wang SJ,Duan HL,Zhang W,et al.Analysis of bacterial foodborne disease outbreaks in China between 1994 and 2005[J].FEMS Immunol Med Microbiol,2007,51:8-13.
[5]陈玲,张菊梅,杨小娟,等.沙门氏菌分型研究进展[J].微生物学通报,2016,43(3):648-654.Chen L,Zhang JM,Yang XJ,et al.Advances in Salmonella subtyping-a review[J].Microbiology,2016,43(3):648-654.
[6]王开,裴志花,陈中秋.常见食源性致病菌及其分子分型技术的研究进展[J].中国兽医杂志,2013,12(42):51-53.Wang K,Pei ZH,Chen ZQ.Research progress of common foodborne pathogens and their molecular typing techniques[J].Chin J Veter Med,2013,12(42):51-53.
[7]候美如,尹珺伊,王岩,等.PCR-DGGE技术及其在猪肠道微生物菌群变化的研究进展[J].猪病防控,2018,4:54-56.Hou MR,Yin JY,Wang Y,et al.Advance in PCR-DGGE and its application in swine gut microbiome variation research[J].Swine Dis Prev Control,2018,4:54-56.
[8]张振华,鲍王波,余冉,等.鹌鹑肠道微生物PCR-DGGE方法的优化[J].生物技术通报,2015,31(1):73-78.Zhang ZH,Bao WB,Yu R,et al.Optimization method for the extraction of total DNA from the intestinal microbes in quails[J].Biotechnol bull,2015,31(1):73-78.
[9]Lan WQ,Xie J.Characterization of the dynamic changes of microorganisms in cutlassfish(Trichius haumela)under the cold storage with composit natural preservatives based on culture-dependent and 16Sr RNA-DGGE technology[J].Adv Materials Res,2012,554:1498-1506.
[10]Wen CQ,Lai XT,Xue M,et al.Molecular typing and identification of Bdellovibrio-and-like organisms isolated from seawater shrimp ponds and adjacent coastal waters[J].J Appl Microbiol,2009,106(4):1154-1162.
[11]Probst M,Walde J,Pumpel T,et al.Lactic acid fermentation within a cascading approach for biowaste treatment[J].Appl Microbiol Biot,2015,99:2029-2040.
[12]兰阿峰,杨曼,郭素芬,等.免培养法对大鲵肠道微生物多样性的研究[J].微生物学通报,2014,41(7):1342-1349.Lan AF,Yang M,Guo SF,et al.Culture-independent analysis of intestinal microbial diversity from Chinese giant salamander[J].Microbiology,2014,41(7):1342-1349.
[13]廖超,张昭寰,姚鑫,等.GC夹对三种食源性致病菌的rpoB-PCR-DGGE图谱的影响[J].生物技术通报,2016,32(5):212-219.Liao C,Zhang ZH,Yao X,et al.Effects of GC clamps on RpoB-PCR-DGGE fingerprint of three types of food borne pathogens[J].Biotechnol bull,2016,32(5):212-219.