多重PCR法检测产志贺毒素性大肠杆菌的4个血清型
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摘要
目的建立多重PCR法检测产志贺毒素性大肠杆菌(shiga toxin-producing Escherichia coli, STEC)主要血清型O26、O145、O45和O121的分析方法。方法根据GenBank登录序列,设计扩增O抗原翻转酶(wzx)基因的引物,建立PCR方法,并以STECO26、O145、O45和O121基因组为模板,检验多重PCR的灵敏度和特异性。使用建立的PCR,检测牛胴体表面拭子,阳性扩增条带送测序,以验证PCR扩增的可靠性。同时将阳性扩增样品,涂布显色平板,分离靶标血清型细菌。结果本研究成功建立STEC O26、O145、O45和O121的多重PCR方法, PCR循环参数中退火温度为60℃,扩增片段分别为249、353、890和587 bp。多重PCR直接检测O26、O145、O45和O121时,最低检测限介于10~3～10~4 CFU/mL,而增菌后再检测,最低检测限均为1CFU/m L。多重PCR用于其他血清型STEC,和非大肠杆菌扩增时,均未扩增出目的条带,只有O26、O145、O45和O121能够扩增出相应条带。当使用多重PCR直接检测胴体擦拭子时,阳性率为5.45%(3/55),主要为O26、O145血清型;增菌后检测阳性率为7.27%(4/55),主要为O26、O145和O121血清型。阳性PCR扩增样品,成功分离到O26两株、O145和O121各一株。分离菌株具有典型大肠杆菌的生化特性,携带STEC代表性毒力因子志贺毒素和紧密素,且具有多重耐药性。结论以STECO26、O145、O45和O121的wzx基因为检测靶标,成功建立多重PCR方法,灵敏度和特异性良好,与细菌分离联合使用,可减少工作量,精准分离目的病原菌。
Objective To establish a multiplex PCR method for determination of 4 predominant serotype O26, O145, O45 and O121 of shiga toxin-producing E.coli(STEC). Methods According to the GenBank accession sequence, the primers for amplifying the O antigen flipping enzyme(wzx) gene were designed, and the PCR method was established. The sensitivity and specificity of multiplex PCR were tested using the STEC O26, O145, O45 and O121 genomes as templates. Using the established PCR, the swabs on the surface of the bovine corpus callosum were detected, and the positive amplified bands were sent for sequencing to verify the reliability of the PCR amplification. At the same time, the sample was positively amplified, coated with a chromogenic plate, and the target serotype bacteria were isolated. Results This study successfully established STEC O26, O145, O45 and O121 multiplex PCR method and PCR cycle parameters in the annealing temperature was 60 ℃, fragments amplified 249, 353, 890 and 353 bp, respectively. When O26, O145, O45 and O121 were directly detected by multiple PCR, the minimum limit of detection was between 10~3 and 10~4 CFU/mL, while the minimum limit of detection was 1 CFU/mL after bacteria enrichment. When multiple PCR was applied to other serotypes of STEC and non-Escherichia coli, the target bands were not amplified, only O26, O145, O45 and O121 could amplify the corresponding bands. When the carcass swabs were directly detected by multiple PCR, the positive rate was 5.45%(3/55), mainly O26 and O145 serotypes. The positive rate after bacteria enrichment was 7.27%(4/55), mainly O26, O145 and O121 serotypes. Two strains of O26, one strain of O145 and one strain of O121 were isolated by positive PCR amplification. The isolated strain had the biochemical characteristics of typical Escherichia coli, carried STEC representative virulence factors shiga toxin and ghrelin, and had multi-drug resistance. Conclusion Using the wzx gene of STEC O26, O145, O45 and O121 as the detection target, the multiplex PCR method is successfully established, and the sensitivity and specificity are good. Combined with the separation of bacteria, the workload can be reduced and the pathogenic bacteria can be accurately separated.
Objective To establish a multiplex PCR method for determination of 4 predominant serotype O26, O145, O45 and O121 of shiga toxin-producing E.coli(STEC). Methods According to the GenBank accession sequence, the primers for amplifying the O antigen flipping enzyme(wzx) gene were designed, and the PCR method was established. The sensitivity and specificity of multiplex PCR were tested using the STEC O26, O145, O45 and O121 genomes as templates. Using the established PCR, the swabs on the surface of the bovine corpus callosum were detected, and the positive amplified bands were sent for sequencing to verify the reliability of the PCR amplification. At the same time, the sample was positively amplified, coated with a chromogenic plate, and the target serotype bacteria were isolated. Results This study successfully established STEC O26, O145, O45 and O121 multiplex PCR method and PCR cycle parameters in the annealing temperature was 60 ℃, fragments amplified 249, 353, 890 and 353 bp, respectively. When O26, O145, O45 and O121 were directly detected by multiple PCR, the minimum limit of detection was between 10~3 and 10~4 CFU/mL, while the minimum limit of detection was 1 CFU/mL after bacteria enrichment. When multiple PCR was applied to other serotypes of STEC and non-Escherichia coli, the target bands were not amplified, only O26, O145, O45 and O121 could amplify the corresponding bands. When the carcass swabs were directly detected by multiple PCR, the positive rate was 5.45%(3/55), mainly O26 and O145 serotypes. The positive rate after bacteria enrichment was 7.27%(4/55), mainly O26, O145 and O121 serotypes. Two strains of O26, one strain of O145 and one strain of O121 were isolated by positive PCR amplification. The isolated strain had the biochemical characteristics of typical Escherichia coli, carried STEC representative virulence factors shiga toxin and ghrelin, and had multi-drug resistance. Conclusion Using the wzx gene of STEC O26, O145, O45 and O121 as the detection target, the multiplex PCR method is successfully established, and the sensitivity and specificity are good. Combined with the separation of bacteria, the workload can be reduced and the pathogenic bacteria can be accurately separated.
引文
[1]Rangel JM,Sparling PH,Crowe C,et al.Epidemiology of Escherichia coli O157:H7 outbreaks,United States,1982-2002[J].Emerg Infect Dis,2005,11(4):603-609.
[2]Pradel N,Bertin Y,Martin C,et al.Molecular analysis of shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients and dairy samples in France[J].Appl Environ Microbiol,2008,74(7):2118-2128.
[3]Brooks JT,Sowers EG,Wells JG,et al.Non-O157 shiga toxin-producing Escherichia coli infections in the United States,1983-2002[J].J Infect Dis,2005,192(8):1422-1429.
[4]Bettelheim KA.The non-O157 shiga-toxigenic(verocytotoxigenic)Escherichia coli:Under-rated pathogens[J].Crit Rev Microbiol,2007,33(1):67-87.
[5]Byrne L,Jenkins C,Launders N,et al.The epidemiology,microbiology and clinical impact of Shiga toxin-producing Escherichia coli in England,2009-2012[J].Epidemiol Infect,2015,143(16):3475-3487.
[6]Ekiri AB,Landblom D,Doetkott D,et al.Isolation and characterization of shiga toxin-producing Escherichia coli serogroups O26,O45,O103,O111,O113,O121,O145,and O157 shed from range and feedlot cattle from postweaning to slaughter[J].J Food Prot,2014,77(7):1052-1061.
[7]Brooks JT,Sowers EG,Wells JG,et al.Non-O157 Shiga toxin-producing Escherichia coli infections in the United States,1983-2002[J].J Infect Dis,2005,192(8):1422-1429.
[8]Karmali MA,Gannon V,Sargeant JM.Verocytotoxin-producing Escherichia coli(VTEC)[J].Vet Microbiol,2010,140(3):360-370.
[9]Bosilevac JM,Koohmaraie M.Prevalence and characterization of non-O157 shiga toxin producing Escherichia coli isolated from commercial ground beef in the United States[J].Appl Environ Microbiol,2011,77(6):2103-2112.
[10]Arthur TM,Barkocy-Gallagher GA,Rivera-Betancourt M,et al.Prevalence and characterization of non-O157 Shiga toxin-producing Escherichia coli on carcasses in commercial beef cattle processing plants[J].Appl Environ Microbiol,2002,68(10):4847-4852.
[11]Arthur TM,Nou X,Kalchayanand N,et al.Survival of Escherichia coli O157:H7 on cattle hides[J].Appl Environ Microbiol,2011,77(9):3002-3008.
[12]GB 29921-2013食品安全国家标准食品中致病菌限量[S].GB 29921-2013 National food safety standard-Limit of pathogenic bacteria in food[S].
[13]Bai J,Paddock ZD,Shi X,et al.Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces[J].Foodborne Pathog Dis,2012,9(6):541-548.
[14]张雪寒,汪伟,何孔旺,等.通用引物检测志贺毒素和紧密素多个基因亚型[J].中国人兽共患病学报,2014,30(6):607-611.Zhang XH,Wang W,He KW,et al.Universal primers detection of shiga toxin and in timin variants[J].Chin J Zoon,2014,30(6):607-611.
[15]李丽.大肠杆菌O157:H7多重PCR检测和溶血素蛋白的表达及其免疫原性分析[D].南京:南京农业大学,2009.Li L.Detection of Escherichia coli O157:H7 by multiplex polymerase chain reaction and expression and immunogenicity analysis of hemolysin protein[D].Nanjing:Nanjing Agricultural University,2009.
[16]Hughes JM,Wilson ME,Johnson KE,et al.The emerging clinical importance of non-O157 shiga toxin-producing Escherichia coli[J].Clin Infect Dis,2006,43(12):1587-1595.
[17]Valilis E,Ramsey A,Sidiq S,et al.Non-O157 Shiga toxin-producing Escherichia coli-A poorly appreciated enteric pathogen:Systematic review[J].Int J Infect Dis,2018,(76):82-87.
[18]De-Rauw K,Jacobs S,Piérard D.Twenty-seven years of screening for shiga toxin-producing Escherichia coli in a university hospital.Brussels,Belgium,1987-2014[J].PLoS One,2018,13(7):e0199968.
[19]Scallan E,Griffin PM,Angulo FJ,et al.Foodborne illness acquired in the United States unspecified agents[J].Emerg Infect Dis,2011,17(1):16-22.
[20]Stromberg ZR,Redweik G,Mellata M.Detection,prevalence,and Pathogenicity of non-o157 shiga toxin-producing Escherichia coli from cattle hides and carcasses[J].Foodborne Pathog Dis,2018,15(3):119-131.
[21]Paddock Z,Shi X,Bai J,et al.Applicability of a multiplex PCR to detect O26,O45,O103,O111,O121,O145,and O157 serogroups of Escherichia coli in cattle feces[J].Vet Microbiol,2012,156(3-4):381-388.
[22]Bai J,Shi X,Nagaraja TG.A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7[J].J Microbiol Methods,2010,82(1):85-89.
[23]European Food Safety Authority.The European Union summary report on trends and sources of zoonoses,zoonotic agents and food-borne outbreaks in 2015[J].EFSA J,2016,(14):e04634.
[24]Sanchez S,Llorente MT,Echeita MA,et al.Development of three multiplex PCR assays targeting the 21 most clinically relevant serogroups associated with Shiga toxin-producing E.coli infection in humans[J].PLoS One,2015,(10):e0117660.
[25]European Committee for Standardization.Real-time polymerase chain reaction(PCR)-based method for the detection of food-borne pathogens-horizontal method for the detection of shiga toxin-producing Escherichia coli(STEC)and the determination of O157,O111,O26,O103and O145 serogroups(ISO/TS 13136:2012)[Z].
[26]Dewsbury DM,Renter DG,Shridhar PB,et al.Summer and winter prevalence of shiga toxin-producing Escherichia coli(STEC)O26,O45,O103,O111,O121,O145,and O157 in feces of feedlot cattle[J].Foodborne Pathog Dis,2015,12(8):726-732.
[2]Pradel N,Bertin Y,Martin C,et al.Molecular analysis of shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients and dairy samples in France[J].Appl Environ Microbiol,2008,74(7):2118-2128.
[3]Brooks JT,Sowers EG,Wells JG,et al.Non-O157 shiga toxin-producing Escherichia coli infections in the United States,1983-2002[J].J Infect Dis,2005,192(8):1422-1429.
[4]Bettelheim KA.The non-O157 shiga-toxigenic(verocytotoxigenic)Escherichia coli:Under-rated pathogens[J].Crit Rev Microbiol,2007,33(1):67-87.
[5]Byrne L,Jenkins C,Launders N,et al.The epidemiology,microbiology and clinical impact of Shiga toxin-producing Escherichia coli in England,2009-2012[J].Epidemiol Infect,2015,143(16):3475-3487.
[6]Ekiri AB,Landblom D,Doetkott D,et al.Isolation and characterization of shiga toxin-producing Escherichia coli serogroups O26,O45,O103,O111,O113,O121,O145,and O157 shed from range and feedlot cattle from postweaning to slaughter[J].J Food Prot,2014,77(7):1052-1061.
[7]Brooks JT,Sowers EG,Wells JG,et al.Non-O157 Shiga toxin-producing Escherichia coli infections in the United States,1983-2002[J].J Infect Dis,2005,192(8):1422-1429.
[8]Karmali MA,Gannon V,Sargeant JM.Verocytotoxin-producing Escherichia coli(VTEC)[J].Vet Microbiol,2010,140(3):360-370.
[9]Bosilevac JM,Koohmaraie M.Prevalence and characterization of non-O157 shiga toxin producing Escherichia coli isolated from commercial ground beef in the United States[J].Appl Environ Microbiol,2011,77(6):2103-2112.
[10]Arthur TM,Barkocy-Gallagher GA,Rivera-Betancourt M,et al.Prevalence and characterization of non-O157 Shiga toxin-producing Escherichia coli on carcasses in commercial beef cattle processing plants[J].Appl Environ Microbiol,2002,68(10):4847-4852.
[11]Arthur TM,Nou X,Kalchayanand N,et al.Survival of Escherichia coli O157:H7 on cattle hides[J].Appl Environ Microbiol,2011,77(9):3002-3008.
[12]GB 29921-2013食品安全国家标准食品中致病菌限量[S].GB 29921-2013 National food safety standard-Limit of pathogenic bacteria in food[S].
[13]Bai J,Paddock ZD,Shi X,et al.Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces[J].Foodborne Pathog Dis,2012,9(6):541-548.
[14]张雪寒,汪伟,何孔旺,等.通用引物检测志贺毒素和紧密素多个基因亚型[J].中国人兽共患病学报,2014,30(6):607-611.Zhang XH,Wang W,He KW,et al.Universal primers detection of shiga toxin and in timin variants[J].Chin J Zoon,2014,30(6):607-611.
[15]李丽.大肠杆菌O157:H7多重PCR检测和溶血素蛋白的表达及其免疫原性分析[D].南京:南京农业大学,2009.Li L.Detection of Escherichia coli O157:H7 by multiplex polymerase chain reaction and expression and immunogenicity analysis of hemolysin protein[D].Nanjing:Nanjing Agricultural University,2009.
[16]Hughes JM,Wilson ME,Johnson KE,et al.The emerging clinical importance of non-O157 shiga toxin-producing Escherichia coli[J].Clin Infect Dis,2006,43(12):1587-1595.
[17]Valilis E,Ramsey A,Sidiq S,et al.Non-O157 Shiga toxin-producing Escherichia coli-A poorly appreciated enteric pathogen:Systematic review[J].Int J Infect Dis,2018,(76):82-87.
[18]De-Rauw K,Jacobs S,Piérard D.Twenty-seven years of screening for shiga toxin-producing Escherichia coli in a university hospital.Brussels,Belgium,1987-2014[J].PLoS One,2018,13(7):e0199968.
[19]Scallan E,Griffin PM,Angulo FJ,et al.Foodborne illness acquired in the United States unspecified agents[J].Emerg Infect Dis,2011,17(1):16-22.
[20]Stromberg ZR,Redweik G,Mellata M.Detection,prevalence,and Pathogenicity of non-o157 shiga toxin-producing Escherichia coli from cattle hides and carcasses[J].Foodborne Pathog Dis,2018,15(3):119-131.
[21]Paddock Z,Shi X,Bai J,et al.Applicability of a multiplex PCR to detect O26,O45,O103,O111,O121,O145,and O157 serogroups of Escherichia coli in cattle feces[J].Vet Microbiol,2012,156(3-4):381-388.
[22]Bai J,Shi X,Nagaraja TG.A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7[J].J Microbiol Methods,2010,82(1):85-89.
[23]European Food Safety Authority.The European Union summary report on trends and sources of zoonoses,zoonotic agents and food-borne outbreaks in 2015[J].EFSA J,2016,(14):e04634.
[24]Sanchez S,Llorente MT,Echeita MA,et al.Development of three multiplex PCR assays targeting the 21 most clinically relevant serogroups associated with Shiga toxin-producing E.coli infection in humans[J].PLoS One,2015,(10):e0117660.
[25]European Committee for Standardization.Real-time polymerase chain reaction(PCR)-based method for the detection of food-borne pathogens-horizontal method for the detection of shiga toxin-producing Escherichia coli(STEC)and the determination of O157,O111,O26,O103and O145 serogroups(ISO/TS 13136:2012)[Z].
[26]Dewsbury DM,Renter DG,Shridhar PB,et al.Summer and winter prevalence of shiga toxin-producing Escherichia coli(STEC)O26,O45,O103,O111,O121,O145,and O157 in feces of feedlot cattle[J].Foodborne Pathog Dis,2015,12(8):726-732.