禽大肠杆菌、肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌多重PCR方法的建立及应用
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摘要
【背景】大肠杆菌病和沙门菌病是最常见的家禽细菌性疾病,给养禽业造成严重经济损失。另外,禽大肠杆菌和沙门菌也是重要的人畜共患病原菌,可通过禽类及其产品传播给人类,对人类健康造成严重威胁。加强禽大肠杆菌和沙门菌的快速鉴别检测,对养禽业和公共卫生都具有重要意义。【目的】建立禽大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的多重PCR检测方法。【方法】通过比较分析确定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的特异靶标基因,设计5对特异性引物,通过条件优化建立多重PCR方法,分析该多重PCR方法的特异性、敏感性及可靠性。【结果】该方法能特异性地鉴定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌,每个PCR反应的最低检出限分别为10~3 CFU细菌和100 pg基因组DNA。临床分离菌株检测显示,多重PCR与传统血清学方法结果一致。【结论】建立的多重PCR方法能够快速鉴别禽致病性大肠杆菌和不同血清型沙门菌,对禽大肠杆菌病和沙门菌病的流行病学调查及临床检测具有重要意义。
[Background] Colibacillosis and salmonellosis are the most common bacterial diseases in poultry, causing economically devastating to poultry industries. Salmonella and avian pathogenic E. coli(APEC) are important zoonotic pathogens, also with potential threat to human health. Thus, it is necessary to strengthen the rapid detection and differential diagnosis of these bacterial diseases. [Objective] We developed multiplex PCR to efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium,Salmonella gallinarum and Salmonella pullorum. [Methods] The special genes of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum were screened and selected for primers designing. A multiplex PCR method was developed by optimization of conditions.Then, the sensitivity and usage for detection of clinical isolates were determined. [Results] A multiplex PCR was established for accurately and effectively detection of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum. The sensitivity of the multiplex PCR was 10~3 bacterial colony forming units(CFUs) and 100 pg genomic DNA. The results of multiplex PCR for detection of clinical isolates agreed well with those of traditional serological assays. [Conclusion] Multiplex PCR developed in this study could efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum.
[Background] Colibacillosis and salmonellosis are the most common bacterial diseases in poultry, causing economically devastating to poultry industries. Salmonella and avian pathogenic E. coli(APEC) are important zoonotic pathogens, also with potential threat to human health. Thus, it is necessary to strengthen the rapid detection and differential diagnosis of these bacterial diseases. [Objective] We developed multiplex PCR to efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium,Salmonella gallinarum and Salmonella pullorum. [Methods] The special genes of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum were screened and selected for primers designing. A multiplex PCR method was developed by optimization of conditions.Then, the sensitivity and usage for detection of clinical isolates were determined. [Results] A multiplex PCR was established for accurately and effectively detection of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum. The sensitivity of the multiplex PCR was 10~3 bacterial colony forming units(CFUs) and 100 pg genomic DNA. The results of multiplex PCR for detection of clinical isolates agreed well with those of traditional serological assays. [Conclusion] Multiplex PCR developed in this study could efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum.
引文
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[5]Foley SL,Nayak R,Hanning IB,et al.Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production[J].Applied and Environmental Microbiology,2011,77(13):4273-4279
[6]Martinez-Ballesteros I,Paglietti B,Rementeria A,et al.Intra-and inter-laboratory evaluation of an improved multiplex-PCRmethod for detection and typing of Salmonella[J].The Journal of Infection in Developing Countries,2012,6(5):443-451
[7]Shah DH,Park JH,Cho MR,et al.Allele-specific PCR method based on rfbS sequence for distinguishing Salmonella gallinarum from Salmonella pullorum:serotype-specific rfbS sequence polymorphism[J].Journal of Microbiological Methods,2005,60(2):169-177
[8]Hu QH,Tu J,Han XG,et al.Development of multiplex PCRassay for rapid detection of Riemerella anatipestifer,Escherichia coli,and Salmonella enterica simultaneously from ducks[J].Journal of Microbiological Methods,2011,87(1):64-69
[9]Zhang M,Han F,Shen WZ,et al.Establishment and application of multiplex PCR assay for detecting Pasteurella multocida,Haemophilus paragallinarum and Escherichia coli[J].Acta Agriculturae Boreali-Occidentalis Sinica,2018,27(2):163-168(in Chinese)张曼,韩飞,沈文正,等.禽多杀性巴氏杆菌、副鸡嗜血杆菌和大肠杆菌多重PCR检测方法的建立及应用[J].西北农业学报,2018,27(2):163-168
[10]Pai?o FG,Arisitides LGA,Murate LS,et al.Detection of Salmonella spp.,Salmonella enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay[J].Brazilian Journal of Microbiology,2013,44(1):37-42
[11]Pui CF,Wong WC,Chai LC,et al.Multiplex PCR for the concurrent detection and differentiation of Salmonella spp.,Salmonella Typhi and Salmonella Typhimurium[J].Tropical Medicine and Health,2011,39(1):9-15
[12]de Freitas CG,Santana?P,Da Silva PHC,et al.PCR multiplex for detection of Salmonella Enteritidis,Typhi and Typhimurium and occurrence in poultry meat[J].International Journal of Food Microbiology,2010,139(1/2):15-22
[13]Lee SH,Jung BY,Rayamahji N,et al.A multiplex real-time PCRfor differential detection and quantification of Salmonella spp.,Salmonella enterica serovar Typhimurium and Enteritidis in meats[J].Journal of Veterinary Science,2009,10(1):43-51
[14]Yang L,Lou YK,Su CH,et al.Development of a multiplex PCRfor rapid identification of Salmonella enteritidis,Salmonella typhimurium,Salmonella pollorum and Salmonella gallinarum[J].Acta Veterianria et Zootechnica Sinica,2014,45(2):268-273(in Chinese)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
[15]Gong JS,Zhuang LL,Lu GW,et al.Development and application of a multiplex PCR for detection of avian Salmonella[J].China Poultry,2016,38(4):14-18(in Chinese)龚建森,庄林林,陆光武,等.禽源沙门菌多重PCR检测方法的建立与应用[J].中国家禽,2016,38(4):14-18
[16]Geng SZ,Pan ZM,Jiang CH,et al.Development of allele-specific PCR assay for detecting Salmonella pullorum[J].Veterinary Science in China,2007,37(2):113-116(in Chinese)耿士忠,潘志明,蒋春红,等.鸡白痢沙门菌等位基因特异性PCR检测方法的建立[J].中国兽医科学,2007,37(2):113-116
[17]Kang MS,Kwon YK,Jung BY,et al.Differential identification of Salmonella enterica subsp.enterica serovar Gallinarum biovars Gallinarum and Pullorum based on polymorphic regions of glgCand speC genes[J].Veterinary Microbiology,2011,147(1/2):181-185
[18]Boyd EF,Porwollik S,Blackmer F,et al.Differences in gene content among Salmonella enterica serovar typhi isolates[J].Journal of Clinical Microbiology,2003,41(8):3823-3828
[2]Pang T,Bhutta ZA,Finlay BB,et al.Typhoid fever and other salmonellosis:a continuing challenge[J].Trends in Microbiology,1995,3(7):253-255
[3]Mora A,Viso S,López C,et al.Poultry as reservoir for extraintestinal pathogenic Escherichia coli O45:K1:H7-B2-ST95in humans[J].Veterinary Microbiology,2013,167(3/4):506-512
[4]Mellata M.Human and avian extraintestinal pathogenic Escherichia coli:infections,zoonotic risks,and antibiotic resistance trends[J].Foodborne Pathogens and Disease,2013,10(11):916-932
[5]Foley SL,Nayak R,Hanning IB,et al.Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production[J].Applied and Environmental Microbiology,2011,77(13):4273-4279
[6]Martinez-Ballesteros I,Paglietti B,Rementeria A,et al.Intra-and inter-laboratory evaluation of an improved multiplex-PCRmethod for detection and typing of Salmonella[J].The Journal of Infection in Developing Countries,2012,6(5):443-451
[7]Shah DH,Park JH,Cho MR,et al.Allele-specific PCR method based on rfbS sequence for distinguishing Salmonella gallinarum from Salmonella pullorum:serotype-specific rfbS sequence polymorphism[J].Journal of Microbiological Methods,2005,60(2):169-177
[8]Hu QH,Tu J,Han XG,et al.Development of multiplex PCRassay for rapid detection of Riemerella anatipestifer,Escherichia coli,and Salmonella enterica simultaneously from ducks[J].Journal of Microbiological Methods,2011,87(1):64-69
[9]Zhang M,Han F,Shen WZ,et al.Establishment and application of multiplex PCR assay for detecting Pasteurella multocida,Haemophilus paragallinarum and Escherichia coli[J].Acta Agriculturae Boreali-Occidentalis Sinica,2018,27(2):163-168(in Chinese)张曼,韩飞,沈文正,等.禽多杀性巴氏杆菌、副鸡嗜血杆菌和大肠杆菌多重PCR检测方法的建立及应用[J].西北农业学报,2018,27(2):163-168
[10]Pai?o FG,Arisitides LGA,Murate LS,et al.Detection of Salmonella spp.,Salmonella enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay[J].Brazilian Journal of Microbiology,2013,44(1):37-42
[11]Pui CF,Wong WC,Chai LC,et al.Multiplex PCR for the concurrent detection and differentiation of Salmonella spp.,Salmonella Typhi and Salmonella Typhimurium[J].Tropical Medicine and Health,2011,39(1):9-15
[12]de Freitas CG,Santana?P,Da Silva PHC,et al.PCR multiplex for detection of Salmonella Enteritidis,Typhi and Typhimurium and occurrence in poultry meat[J].International Journal of Food Microbiology,2010,139(1/2):15-22
[13]Lee SH,Jung BY,Rayamahji N,et al.A multiplex real-time PCRfor differential detection and quantification of Salmonella spp.,Salmonella enterica serovar Typhimurium and Enteritidis in meats[J].Journal of Veterinary Science,2009,10(1):43-51
[14]Yang L,Lou YK,Su CH,et al.Development of a multiplex PCRfor rapid identification of Salmonella enteritidis,Salmonella typhimurium,Salmonella pollorum and Salmonella gallinarum[J].Acta Veterianria et Zootechnica Sinica,2014,45(2):268-273(in Chinese)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
[15]Gong JS,Zhuang LL,Lu GW,et al.Development and application of a multiplex PCR for detection of avian Salmonella[J].China Poultry,2016,38(4):14-18(in Chinese)龚建森,庄林林,陆光武,等.禽源沙门菌多重PCR检测方法的建立与应用[J].中国家禽,2016,38(4):14-18
[16]Geng SZ,Pan ZM,Jiang CH,et al.Development of allele-specific PCR assay for detecting Salmonella pullorum[J].Veterinary Science in China,2007,37(2):113-116(in Chinese)耿士忠,潘志明,蒋春红,等.鸡白痢沙门菌等位基因特异性PCR检测方法的建立[J].中国兽医科学,2007,37(2):113-116
[17]Kang MS,Kwon YK,Jung BY,et al.Differential identification of Salmonella enterica subsp.enterica serovar Gallinarum biovars Gallinarum and Pullorum based on polymorphic regions of glgCand speC genes[J].Veterinary Microbiology,2011,147(1/2):181-185
[18]Boyd EF,Porwollik S,Blackmer F,et al.Differences in gene content among Salmonella enterica serovar typhi isolates[J].Journal of Clinical Microbiology,2003,41(8):3823-3828
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