四重荧光定量PCR法鉴定肠炎、鼠伤寒以及伤寒沙门氏菌
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摘要
目的建立四重荧光定量PCR体系鉴定肠炎沙门氏菌、鼠伤寒沙门氏菌以及伤寒沙门氏菌。方法针对沙门氏菌属特异性ompC基因、肠炎沙门氏菌sdf基因、鼠伤寒沙门氏菌STM4495和伤寒沙门氏菌STY2021序列设计引物和TaqMan探针,建立多重荧光定量PCR体系,进行特异性与敏感性研究。结果 28株不同血清型的沙门氏菌均扩增出ompC基因,其他13株非沙门氏菌均未出现ompC的非特异性扩增。sdf、STM4495、STY2021的探针和引物分别特异性扩增出肠炎沙门氏菌、鼠伤寒沙门氏菌以及伤寒沙门氏菌,而25株其他血清型沙门氏菌以及13株非沙门氏菌均未见扩增曲线。敏感性试验显示,该体系的最低检测限分别为48 pg/mL(ompC)、560 pg/mL(sdf)、530 pg/mL(STM4495)、35 pg/mL(STY2021)。结论该方法特异好、灵敏高、能够快速检测沙门氏菌并鉴定肠炎沙门氏菌、鼠伤寒沙门氏菌以及伤寒沙门氏菌。
Objective To establish quadruple fluorescence quantitative PCR for identification of Salmonella enteritidis, Salmonella typhimurium and Salmonella typhi. Methods Primers and TaqMan probes were designed for Salmonella species-specific ompC gene, Salmonella enteritis sdf gene, Salmonella thyphimurium STM4495 and Salmonella typhi STY2021 sequences, and the multiplex fluorescence quantitative PCR system was established to investigate the specificity and sensitivity. Results The ompC gene was amplified from 28 different serotypes of Salmonella, and the other 13 non-Salmonella did not show non-specific amplification of ompC. The probes and primers of sdf, STM4495 and STY2021 were used to specifically amplify Salmonella enteritis, Salmonella typhi and Salmonella typhi respectively, while no amplification curve was observed for 25 other serotype Salmonella and 13 non-Salmonella strains. The limits of detection of this method were 48 pg/mL(ompC), 560 pg/mL(sdf), 530 pg/mL(STM449), and 35 pg/mL(STY2021). Conclusion The method is specific, sensitive, and can quickly detect Salmonella and identify Salmonella enteritidis, Salmonella typhimurium and Salmonella typhi.
Objective To establish quadruple fluorescence quantitative PCR for identification of Salmonella enteritidis, Salmonella typhimurium and Salmonella typhi. Methods Primers and TaqMan probes were designed for Salmonella species-specific ompC gene, Salmonella enteritis sdf gene, Salmonella thyphimurium STM4495 and Salmonella typhi STY2021 sequences, and the multiplex fluorescence quantitative PCR system was established to investigate the specificity and sensitivity. Results The ompC gene was amplified from 28 different serotypes of Salmonella, and the other 13 non-Salmonella did not show non-specific amplification of ompC. The probes and primers of sdf, STM4495 and STY2021 were used to specifically amplify Salmonella enteritis, Salmonella typhi and Salmonella typhi respectively, while no amplification curve was observed for 25 other serotype Salmonella and 13 non-Salmonella strains. The limits of detection of this method were 48 pg/mL(ompC), 560 pg/mL(sdf), 530 pg/mL(STM449), and 35 pg/mL(STY2021). Conclusion The method is specific, sensitive, and can quickly detect Salmonella and identify Salmonella enteritidis, Salmonella typhimurium and Salmonella typhi.
引文
[1]刘秀梅,陈艳,王晓英,等.1992~2001年食源性疾病暴发资料分析一国家食源性疾病监测网[J].卫生研究,2004,(6):725-727.Liu XM,Chen Y,Wang XY,et al.Food-borne disease outbreaks in China from 1992 to 2001 national foodborne disease surveillance system[J].JHyg Res,2004,(6):725-727.
[2]Weill FX,Guesnier F,Guibert V,et al.Multidrug resistance in Salmonella enterica serotype Typhimurium from humans in France(1993 to 2003)[J].J Clin Microbiol,2006,44(3):700-708.
[3]Shi C,Singh P,Ranieri ML,et al.Molecular methods for serovar determination of Salmonella[J].Crit Rev Microbiol,2015,41(3):309-325.
[4]Deng SX,Cheng AC,Wang MS,et a1.Study on thegastrointestinal tract distribution of Salmonel&enteritidis in orally infected mice with a species-specific fluorescent quantitative polymerase chain reaction[J].World J Gastroenterol,2007,(13):6568-6574.
[5]陈玲,张菊梅,杨小鹃,等.沙门氏菌分型研究进展[J].微生物学通报,2016,43(3):648-665.Chen L,Zhang JM,Yang XJ,et al.Advances in Salmonella subtyping-a review[J].Microbiol China,2016,43(3):648-665.
[6]李可,方莹,张晓峰,等.沙门氏菌的血清分型及分子鉴定研究进展[J].食品安全质量检测学报,2016,7(10):3947-3951.Li K,Fang Y,Zhang XF,et al.Research progress of Salmonella serotyping and molecular identification methods[J].J Food Saf Qual,2016,7(10):3947-3951.
[7]郑秋月,战晓微,徐杨,等.食源性致病菌沙门氏菌MALDI-TOFMS溯源分析[J].食品科技,2013,38(12):315-320.Zheng QY,Zhan XW,Xu Y,et al.Traceability analysis of food-borne pathogen Salmonella by MALDI-TOF MS[J].Food Sci Technol,2013,38(12):315-320.
[8]刘慧玲,万志刚,洪小柳,等.进出口食品中不同血清型沙门氏菌PFGE和MLST分型比较研究[J].食品安全质量检测学报,2014,5(11):3454-3461.Liu HL,Wan ZG,Hong XL,et al.Comparison of pulsed-field gel electrophoresis and multilocus sequence typing of Salmonella in import and export foods[J].J Food Saf Qual,2014,5(11):3454-3461.
[9]邵彪,黄伟东,周鸣镝,等.多重荧光定量PCR检测食品污染菌[J].中国食品学报,2012,12(1):176-181.Shao B,Huang WD,Zhou MD,et al.Multiplex fluorescence quantitative PCR detection of food contaminative bacteria[J].J Chin Inst Food Sci Technol,2012,12(1):176-181.
[10]Luk JM,Kongmuang U,Reeves PR,et al.Selective amplification of abequose and paratose synthase genes(rfb)by polymerase chain reaction for identification of Salmonella major serogroups(A,B,C2,and D)[J].JClin Microbiol,1993,31(8):2118-2123.
[11]Chen J,Zhang L,Paoli GC,et al.A real-time PCR method 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.
[12]方婷子,史贤明,施春雷.沙门氏菌血清型快速PCR鉴定方法的建立[J].中国食品学报,2017,l7(2):212-219.Fang TZ,Shi XM,Shi CL.Development of rapid PCR determination method of Salmonella serovars[J].J Chin Inst Food Sci Technol,2017,l7(2):212-219.
[13]张童利,王曼宇,曹俊,等.鸡白痢沙门氏菌与其它致病性沙门氏菌的血清型特异性PCR鉴别检测方法的建立[J].中国预防兽医学报,2017,39(3):215-219.Zhang TL,Wang MY,Cao J,et al.Specific differential PCR method for detection of Salmonella pullorum from other pathogenic serotypes[J].Chin J Prev Vet Med,2017,39(3):215-219.
[14]杨林,娄亚坤,宿春虎,等.多重PCR方法快速鉴别肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌[J].畜牧兽医学报,2014,45(2):268-273.Yang L,Lou YK,Su CH,et al.Development of a multiplex PCR for rapid identification of Salmonella enteritidis,Salmonella typhimurium,Salmonella pullorumand Salmonella gallinarum[J].Chin J Anim Vet Sci,2014,45(2):268-273.
[15]袁慕云,刘二龙,谢力,等.基于TaqMan探针三重荧光PCR检测沙门菌、肠炎沙门菌和鼠伤寒沙门菌[J].中国预防医学杂志,2015,16(10):774-780.Yuan MY,Liu EL,Xie L,et al.Detection of salmonella,salmonella enteritidis and salmonella typhimurium by TaqMan real-time PCR[J].Chin Prev Med,2015,16(10):774-780.
[16]许龙岩,袁慕云,孙薇,等.基于TaqMan探针四重荧光PCR检测甲型、乙型、丙型副伤寒和伤寒沙门菌[J].卫生研究,2017,46(2):298-302.Xu LY,Yuan MY,Sun W,et al.TaqMan probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi[J].J Hyg Res,2017,46(2):298-302.
[2]Weill FX,Guesnier F,Guibert V,et al.Multidrug resistance in Salmonella enterica serotype Typhimurium from humans in France(1993 to 2003)[J].J Clin Microbiol,2006,44(3):700-708.
[3]Shi C,Singh P,Ranieri ML,et al.Molecular methods for serovar determination of Salmonella[J].Crit Rev Microbiol,2015,41(3):309-325.
[4]Deng SX,Cheng AC,Wang MS,et a1.Study on thegastrointestinal tract distribution of Salmonel&enteritidis in orally infected mice with a species-specific fluorescent quantitative polymerase chain reaction[J].World J Gastroenterol,2007,(13):6568-6574.
[5]陈玲,张菊梅,杨小鹃,等.沙门氏菌分型研究进展[J].微生物学通报,2016,43(3):648-665.Chen L,Zhang JM,Yang XJ,et al.Advances in Salmonella subtyping-a review[J].Microbiol China,2016,43(3):648-665.
[6]李可,方莹,张晓峰,等.沙门氏菌的血清分型及分子鉴定研究进展[J].食品安全质量检测学报,2016,7(10):3947-3951.Li K,Fang Y,Zhang XF,et al.Research progress of Salmonella serotyping and molecular identification methods[J].J Food Saf Qual,2016,7(10):3947-3951.
[7]郑秋月,战晓微,徐杨,等.食源性致病菌沙门氏菌MALDI-TOFMS溯源分析[J].食品科技,2013,38(12):315-320.Zheng QY,Zhan XW,Xu Y,et al.Traceability analysis of food-borne pathogen Salmonella by MALDI-TOF MS[J].Food Sci Technol,2013,38(12):315-320.
[8]刘慧玲,万志刚,洪小柳,等.进出口食品中不同血清型沙门氏菌PFGE和MLST分型比较研究[J].食品安全质量检测学报,2014,5(11):3454-3461.Liu HL,Wan ZG,Hong XL,et al.Comparison of pulsed-field gel electrophoresis and multilocus sequence typing of Salmonella in import and export foods[J].J Food Saf Qual,2014,5(11):3454-3461.
[9]邵彪,黄伟东,周鸣镝,等.多重荧光定量PCR检测食品污染菌[J].中国食品学报,2012,12(1):176-181.Shao B,Huang WD,Zhou MD,et al.Multiplex fluorescence quantitative PCR detection of food contaminative bacteria[J].J Chin Inst Food Sci Technol,2012,12(1):176-181.
[10]Luk JM,Kongmuang U,Reeves PR,et al.Selective amplification of abequose and paratose synthase genes(rfb)by polymerase chain reaction for identification of Salmonella major serogroups(A,B,C2,and D)[J].JClin Microbiol,1993,31(8):2118-2123.
[11]Chen J,Zhang L,Paoli GC,et al.A real-time PCR method 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.
[12]方婷子,史贤明,施春雷.沙门氏菌血清型快速PCR鉴定方法的建立[J].中国食品学报,2017,l7(2):212-219.Fang TZ,Shi XM,Shi CL.Development of rapid PCR determination method of Salmonella serovars[J].J Chin Inst Food Sci Technol,2017,l7(2):212-219.
[13]张童利,王曼宇,曹俊,等.鸡白痢沙门氏菌与其它致病性沙门氏菌的血清型特异性PCR鉴别检测方法的建立[J].中国预防兽医学报,2017,39(3):215-219.Zhang TL,Wang MY,Cao J,et al.Specific differential PCR method for detection of Salmonella pullorum from other pathogenic serotypes[J].Chin J Prev Vet Med,2017,39(3):215-219.
[14]杨林,娄亚坤,宿春虎,等.多重PCR方法快速鉴别肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌[J].畜牧兽医学报,2014,45(2):268-273.Yang L,Lou YK,Su CH,et al.Development of a multiplex PCR for rapid identification of Salmonella enteritidis,Salmonella typhimurium,Salmonella pullorumand Salmonella gallinarum[J].Chin J Anim Vet Sci,2014,45(2):268-273.
[15]袁慕云,刘二龙,谢力,等.基于TaqMan探针三重荧光PCR检测沙门菌、肠炎沙门菌和鼠伤寒沙门菌[J].中国预防医学杂志,2015,16(10):774-780.Yuan MY,Liu EL,Xie L,et al.Detection of salmonella,salmonella enteritidis and salmonella typhimurium by TaqMan real-time PCR[J].Chin Prev Med,2015,16(10):774-780.
[16]许龙岩,袁慕云,孙薇,等.基于TaqMan探针四重荧光PCR检测甲型、乙型、丙型副伤寒和伤寒沙门菌[J].卫生研究,2017,46(2):298-302.Xu LY,Yuan MY,Sun W,et al.TaqMan probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi[J].J Hyg Res,2017,46(2):298-302.