动物源细菌对β-内酰胺类药物耐药基因三重PCR检测方法建立与应用
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摘要
β-内酰胺类抗生素包括青霉素、头孢菌素类、单环β-内酰胺类、碳青霉烯类、β-内酰胺酶抑制剂等,是兽医和人医临床应用最广的抗菌药物。随着分子生物学技术的发展,细菌对β-内酰胺类药物耐药基因陆续被鉴定。本研究采用多重PCR方法检测细菌β-内酰胺类耐药基因,建立快速、灵敏、简便的多重PCR检测方法,为β-内酰胺类抗生素耐药性的快速检测及耐药基因的分子流行病学调查提供新的手段。
本研究在我国19个省市共分离鉴定动物源细菌771株,用常规药敏实验(K-B)法检测了细菌对β-内酰胺类抗生素的耐药性。细菌对供试药物率耐药头孢西丁68.22%,头孢他啶58.88%,头孢曲松32.94%,头孢噻吩31.26%,头孢噻肟19.71%,头孢泊肟6.49%,头孢噻夫4.15%,氨曲南2.98%。
选择三个β-内酰胺类药物耐药基因(TEM,SHV,CTX-M)作为扩增目的基因,根据GenBank上提供的序列,分别设计3对特异性引物,并对反应体系和反应参数进行了优化,筛选最佳Mg~(2+)浓度、dNTPs浓度、Taq酶浓度和引物浓度,选定了最适的退火温度和循环次数,优化模板制备方法。优化后的反应体系和反应参数为:反应总体积为25μL,其中10×PCR buffer 2.5μL,MgCL_2(25 mmoL/L)2.5μL,dNTP(2.5mmoL/L)3μL TEM基因、SHV基因和CTX-M基因的特异性上下游引物(25μmoL/L)分别0.25μL、0.5μL、1.0μL,Taq DNA聚合酶(2.5 U/μL)0.35μL,模板5μL;反应参数:预变性94℃5 min,变性94℃50s,退火55℃55s,延伸72℃1min,32个循环,72℃终延伸5min。建立了多重PCR检测β-内酰胺类药物耐药基因的方法;该方法利用单重PCR技术筛选出β-内酰胺类药物耐药基因的阳性菌株hu112作为三重PCR阳性对照,大肠杆菌质控菌株ATCC25922作为阴性对照。
对建立的三重PCR方法方法进行了特异性、灵敏性、重复性检测试验,检测结果表明:本方法特异性强,只特异性的扩增耐药目的基因;本方法最低检测浓度为2×10~4cfu/mL,灵敏度高;方法批内和批间重复检测结果相同,稳定性好。
用建立的方法对我国19个省、市规模化养殖场771株分离菌进行了β-内酰胺类药物耐药基因(TEM、SHV、CTX-M)的检测,771分离菌中阳性菌的检出率为89.1%。各省的耐药基因检出率差异较大,三种基因的平均检出率分别为:42.5%、32.8%和24.7%。用本方法和常规药敏实验(K-B)法,分别对30株分离菌的耐药基因型和耐药表型同时进行检测比较,检测时间分别为7h、35-36h。同时比较771菌三重方法检测结果与常规药敏实验(K-B)法检测结果,两种方法检测结果的符合率为92.3%.
本研究建立的β-内酰胺类药物耐药基因(TEM、SHV、CTX-M)三重PCR检测方法,为β-内酰胺类抗生素耐药性的快速检测及耐药基因的分子流行病学调查提供了新的手段。
β-Lactamases are among the criticaLLy important antibiotics in human and veterinary medicine,which incLuding peniciLLin,CephaLosporins,Carbapenems,β-Lactams enzyme inhibitors,etc.Manyβ-Lactams resistance genes were found wordwide.The aim of this study was to estabLish a fast,sensitive and simple method for detecting threeβ-Lactams resistance genes based on muLtipLex PCR assay,which can be used as an usefuL method for moLecuLar epidemioLogy study ofβ-Lactams resistance genes.
A drug sensitivity test was carried out in 771 strains.The resuLts showed That the CephaLothin resistance rate was 68.22%,the Ceftazidime resistance rate was 58.88%,the Ceftriaxone resistance rate was 32.94%,the CefaLotin resistance rate was 31.26%,the Cefotaxime resistance rate was 19.71%,the Cefpodoxime resistance rate was 6.49%,the ceftiofur resistance rate was 4.15%.the Aztreonam resistance rate was 2.98%.
Three kinds ofβ-Lactamase resistance genes(TEM,SHV,CTX-M) were chosen as target genes in this study.Three pairs of specific primers were designed according to the correlative TEM,SHV and CTX-M gene sequences that pubLished in GenBank.A isoLate hu112 that positive for aLL the three resistance genes was seLected by PCR method.The muLti-PCR was estabLished on the basis of simpLe PCR technique.The reaction conditions were optimized,incLuding the concentration of Mg2+,dNTPs,Taq poLymerase and primers,the anneaLing temperature and the cycLe times.The best parameters of ampLification system as foLLows:10×PCR buffer 2.5μL,MgCL2(25 mmoL/L)2.5μL,dNTPs(2.5 mmoL/L)3μL,primer of TEM,SHV and CTX-M(25μmoL/L)were 0.25μL,0.5μL and 1.0μL respectiveLy,Taq DNA poLymerase(2.5 U/μL) 0.35μL,DNA tempLate 5μL,and ensure the totaL voLume was 25μL by adding ddH2O. The reaction was performed with 5 min initiaL denaturation at 94℃.The PCR program comprised 32 cycLes(50 s at 94℃,55 s at 55℃,1min at 72℃).A finaL elongation of 5min at 72℃foLLowed the Last cycle.ResuLt shows that,we have estabLished a muLti-PCR method to detectionβ-Lactamase resistance genes successfuLLy.Strain hu112 and standard Escherichia coLi strain(ATCC259922) were used as positive and negative strains respectiveLy.
ResuLts show that the muLtipLex PCR method was a good specificity and repeatabiLity method,which couLd detect the genes with the concentration of tempLate at 2.4×104CFU.
Phenotypic and genotypic characterization of 30 strains was tested by K-B method and muLtiplex PCR method respectiveLy,and we found that the muLtiplex PCR method was more specific,repetitive and than the K-B method.The comparation between muLtipLex PCR and drug-sensitive test ResuLt shows that the consistency rate was 92.3%, 771 strains of bacteria selected from intensive Lifestock farms of 19 provinces in China,were test by the muLtipLex PCR method.The resuLt showed that the positive strains detection rate is 89.1%.And the average detection rate of the three genes was 42.5%,32.8%and 24.7%.
In concLusion,it is the first time estabLished muLtipLex PCR detection of animaL originaL bacteriaβ-Lactamase resistance genes,it offered a rapid,convenient and accurate method to the detection ofβ-Lactamase resistance genes.
本研究在我国19个省市共分离鉴定动物源细菌771株,用常规药敏实验(K-B)法检测了细菌对β-内酰胺类抗生素的耐药性。细菌对供试药物率耐药头孢西丁68.22%,头孢他啶58.88%,头孢曲松32.94%,头孢噻吩31.26%,头孢噻肟19.71%,头孢泊肟6.49%,头孢噻夫4.15%,氨曲南2.98%。
选择三个β-内酰胺类药物耐药基因(TEM,SHV,CTX-M)作为扩增目的基因,根据GenBank上提供的序列,分别设计3对特异性引物,并对反应体系和反应参数进行了优化,筛选最佳Mg~(2+)浓度、dNTPs浓度、Taq酶浓度和引物浓度,选定了最适的退火温度和循环次数,优化模板制备方法。优化后的反应体系和反应参数为:反应总体积为25μL,其中10×PCR buffer 2.5μL,MgCL_2(25 mmoL/L)2.5μL,dNTP(2.5mmoL/L)3μL TEM基因、SHV基因和CTX-M基因的特异性上下游引物(25μmoL/L)分别0.25μL、0.5μL、1.0μL,Taq DNA聚合酶(2.5 U/μL)0.35μL,模板5μL;反应参数:预变性94℃5 min,变性94℃50s,退火55℃55s,延伸72℃1min,32个循环,72℃终延伸5min。建立了多重PCR检测β-内酰胺类药物耐药基因的方法;该方法利用单重PCR技术筛选出β-内酰胺类药物耐药基因的阳性菌株hu112作为三重PCR阳性对照,大肠杆菌质控菌株ATCC25922作为阴性对照。
对建立的三重PCR方法方法进行了特异性、灵敏性、重复性检测试验,检测结果表明:本方法特异性强,只特异性的扩增耐药目的基因;本方法最低检测浓度为2×10~4cfu/mL,灵敏度高;方法批内和批间重复检测结果相同,稳定性好。
用建立的方法对我国19个省、市规模化养殖场771株分离菌进行了β-内酰胺类药物耐药基因(TEM、SHV、CTX-M)的检测,771分离菌中阳性菌的检出率为89.1%。各省的耐药基因检出率差异较大,三种基因的平均检出率分别为:42.5%、32.8%和24.7%。用本方法和常规药敏实验(K-B)法,分别对30株分离菌的耐药基因型和耐药表型同时进行检测比较,检测时间分别为7h、35-36h。同时比较771菌三重方法检测结果与常规药敏实验(K-B)法检测结果,两种方法检测结果的符合率为92.3%.
本研究建立的β-内酰胺类药物耐药基因(TEM、SHV、CTX-M)三重PCR检测方法,为β-内酰胺类抗生素耐药性的快速检测及耐药基因的分子流行病学调查提供了新的手段。
β-Lactamases are among the criticaLLy important antibiotics in human and veterinary medicine,which incLuding peniciLLin,CephaLosporins,Carbapenems,β-Lactams enzyme inhibitors,etc.Manyβ-Lactams resistance genes were found wordwide.The aim of this study was to estabLish a fast,sensitive and simple method for detecting threeβ-Lactams resistance genes based on muLtipLex PCR assay,which can be used as an usefuL method for moLecuLar epidemioLogy study ofβ-Lactams resistance genes.
A drug sensitivity test was carried out in 771 strains.The resuLts showed That the CephaLothin resistance rate was 68.22%,the Ceftazidime resistance rate was 58.88%,the Ceftriaxone resistance rate was 32.94%,the CefaLotin resistance rate was 31.26%,the Cefotaxime resistance rate was 19.71%,the Cefpodoxime resistance rate was 6.49%,the ceftiofur resistance rate was 4.15%.the Aztreonam resistance rate was 2.98%.
Three kinds ofβ-Lactamase resistance genes(TEM,SHV,CTX-M) were chosen as target genes in this study.Three pairs of specific primers were designed according to the correlative TEM,SHV and CTX-M gene sequences that pubLished in GenBank.A isoLate hu112 that positive for aLL the three resistance genes was seLected by PCR method.The muLti-PCR was estabLished on the basis of simpLe PCR technique.The reaction conditions were optimized,incLuding the concentration of Mg2+,dNTPs,Taq poLymerase and primers,the anneaLing temperature and the cycLe times.The best parameters of ampLification system as foLLows:10×PCR buffer 2.5μL,MgCL2(25 mmoL/L)2.5μL,dNTPs(2.5 mmoL/L)3μL,primer of TEM,SHV and CTX-M(25μmoL/L)were 0.25μL,0.5μL and 1.0μL respectiveLy,Taq DNA poLymerase(2.5 U/μL) 0.35μL,DNA tempLate 5μL,and ensure the totaL voLume was 25μL by adding ddH2O. The reaction was performed with 5 min initiaL denaturation at 94℃.The PCR program comprised 32 cycLes(50 s at 94℃,55 s at 55℃,1min at 72℃).A finaL elongation of 5min at 72℃foLLowed the Last cycle.ResuLt shows that,we have estabLished a muLti-PCR method to detectionβ-Lactamase resistance genes successfuLLy.Strain hu112 and standard Escherichia coLi strain(ATCC259922) were used as positive and negative strains respectiveLy.
ResuLts show that the muLtipLex PCR method was a good specificity and repeatabiLity method,which couLd detect the genes with the concentration of tempLate at 2.4×104CFU.
Phenotypic and genotypic characterization of 30 strains was tested by K-B method and muLtiplex PCR method respectiveLy,and we found that the muLtiplex PCR method was more specific,repetitive and than the K-B method.The comparation between muLtipLex PCR and drug-sensitive test ResuLt shows that the consistency rate was 92.3%, 771 strains of bacteria selected from intensive Lifestock farms of 19 provinces in China,were test by the muLtipLex PCR method.The resuLt showed that the positive strains detection rate is 89.1%.And the average detection rate of the three genes was 42.5%,32.8%and 24.7%.
In concLusion,it is the first time estabLished muLtipLex PCR detection of animaL originaL bacteriaβ-Lactamase resistance genes,it offered a rapid,convenient and accurate method to the detection ofβ-Lactamase resistance genes.
引文
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