猪源大肠杆菌氟苯尼考耐药基因SYBR GreenⅠ荧光定量PCR检测试剂盒的研制
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摘要
根据Gen Bank中猪源大肠杆菌氟苯尼考耐药基因floR的序列设计1对特异性引物,扩增出floR基因片段,克隆到p MD-18T载体上,构建p MD-18T-floR阳性标准质粒。通过SYBR GreenⅠ荧光定量PCR反应条件的优化、荧光定量PCR标准曲线的建立、熔解曲线的分析及敏感性、特异性、重复性试验,建立针对猪源大肠杆菌氟苯尼考耐药基因floR的荧光定量PCR检测方法,并研制出检测试剂盒,同时对从养殖场分离的156株大肠杆菌进行耐药表型和耐药基因检测。结果显示,研制的试剂盒灵敏度可达1.0×101拷贝,重复性好,特异性强,对大肠杆菌磺胺类耐药菌株、β-内酰胺类耐药菌株、大环内酯类耐药菌株检测均为阴性,熔解曲线分析,扩增产物的熔解温度为88.8~89.2℃,没有引物二聚体和非特异性扩增峰出现,耐药表型与耐药基因检测符合率为96.7%。表明研制的试剂盒适合于临床样品猪源大肠杆菌氟苯尼考耐药基因的检测。
According to the florfenicol resistance gene floR of Escherichia coli in Gen Bank,one pair of specific primers was designed for amplifying the specific fragments of floR gene. Then floR gene was amplified by PCR and cloned into p MD-18 T vector,which was used as positive standard plasmid. Through optimization of the reaction conditions,establishment of standard curve,analysis of melting curve and test of sensitivity,specificity and repeatability,a SYBR Green Ⅰ real-time quantitative PCR was established and diagnostic kit was developed. 156 strains of Escherichia coli which were isolated from swine farms were studied on drug resistance phenotype and drug resistance gene. The PCR kit was highly sensitive( 1. 0 ×101 copies DNA),and had a good reproducibility,specificity. The kit test result was negative for sulfonamides resistant strains,beta lactam resistant strains and macrolide resistant strains of Escherichia coli isolated from swine. The melting curve analysis showed that the dissolution temperature of the amplified product was between 88. 8 ℃ and 89. 2 ℃,and there were no primer dimers and non-specific amplification peaks. The coincidence rate of drug resistance phenotype and resistance gene was 96. 7%. The results showed that the SYBR GreenⅠreal-time quantitative PCR kit was suitable for the detection of florfenicol resistance gene floR of Escherichia coli in clinical samples.
According to the florfenicol resistance gene floR of Escherichia coli in Gen Bank,one pair of specific primers was designed for amplifying the specific fragments of floR gene. Then floR gene was amplified by PCR and cloned into p MD-18 T vector,which was used as positive standard plasmid. Through optimization of the reaction conditions,establishment of standard curve,analysis of melting curve and test of sensitivity,specificity and repeatability,a SYBR Green Ⅰ real-time quantitative PCR was established and diagnostic kit was developed. 156 strains of Escherichia coli which were isolated from swine farms were studied on drug resistance phenotype and drug resistance gene. The PCR kit was highly sensitive( 1. 0 ×101 copies DNA),and had a good reproducibility,specificity. The kit test result was negative for sulfonamides resistant strains,beta lactam resistant strains and macrolide resistant strains of Escherichia coli isolated from swine. The melting curve analysis showed that the dissolution temperature of the amplified product was between 88. 8 ℃ and 89. 2 ℃,and there were no primer dimers and non-specific amplification peaks. The coincidence rate of drug resistance phenotype and resistance gene was 96. 7%. The results showed that the SYBR GreenⅠreal-time quantitative PCR kit was suitable for the detection of florfenicol resistance gene floR of Escherichia coli in clinical samples.
引文
[1]马增军,芮萍,逯春香,等.猪源肠外致病性大肠杆菌的分离与鉴定[J].中国人兽共患病学报,2015,31(2):130-134.
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[3]李红丽,詹丽娥,王彩先,等.山西省猪致病性大肠杆菌血清型调查及耐药性监测[J].山西农业科学,2012,40(11):1226-1330.
[4]赵凤菊,曹东,李井春,等.猪源大肠埃希菌超广谱β-内酰胺酶的检测及其耐药性分析[J].河南农业科学,2017,46(2):111-115.
[5]孙硕,吕世明,谭艾娟,等.适应度代价对大肠杆菌药物敏感性恢复的作用[J].天津农业科学,2016,22(4):33-35.
[6]俱雄,党亚锋,刘祥,等.大肠杆菌外膜蛋白Omp C的生物信息学分析及表位多肽疫苗的重组预测[J].河南农业大学学报,2017,51(1):94-100.
[7]李进福,丁海峰,李小申,等.河南地区猪呼吸道隐性感染大肠杆菌耐药基因检测及分析[J].河南农业科学,2017,46(9):144-148.
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[9]刘艳红,吕淑霞,李颖,等.大肠杆菌耐药性及氟苯尼考耐药基因floR的研究[J].中国畜牧杂志,2017,53(1):115-118.
[10]崔耀明,林莉,唐桂芬,等.复方氟苯尼考注射液的临床应用试验[J].黑龙江畜牧兽医,2016(14):176-178.
[11]文丽,吴泽华,李仕超.氟苯尼考手性特性及在临床上的应用[J].猪业科学,2016,33(8):70-71.
[12]冯世文,李军,曾芸,等.大肠杆菌O157∶H7对氟苯尼考耐药机制的初步研究[J].中国人兽共患病学报,2016,32(3):271-275.
[13]王娟.革兰阳性细菌氟苯尼考耐药机制的研究进展[J].中国兽医杂志,2011,47(11):60-62.
[14]吴蓓蓓,杜向党.氟苯尼考耐药菌的构建及大肠杆菌中floR蛋白定位[J].畜牧兽医学报,2008,39(10):1438-1441.
[15]Blickwede M,Schwarz S.Molecular analysis of florfenicol-resistant Escherichia coli isolates from pigs[J].Journal of Antimicrobial Chemotherapy,2004,53(1):58-64.
[16]冯世文,曾芸,李军,等.细菌对氟苯尼考的耐药机制研究[J].黑龙江畜牧兽医,2014(3):52-54.
[17]郑朝朝,刘聚祥,刘静.保定地区不同动物源大肠杆菌氟苯尼考耐药基因floR的同源性分析[J].中国兽医科学,2012,42(7):677-680.
[18]陈阳阳,薛慧亮,马红霞,等.氟苯尼考联合多西环素缩小猪源大肠杆菌耐药突变选择窗[J].中国兽医学报,2013,33(11):1720-1723.
[2]Martinezmedina M,Mora A,Blanco M,et al.Similarity and divergence among adherent-invasive Escherichia coli and extraintestinal pathogenic E.coli strains[J].Journal of Clinical Microbiology,2009,47(12):3968-3979.
[3]李红丽,詹丽娥,王彩先,等.山西省猪致病性大肠杆菌血清型调查及耐药性监测[J].山西农业科学,2012,40(11):1226-1330.
[4]赵凤菊,曹东,李井春,等.猪源大肠埃希菌超广谱β-内酰胺酶的检测及其耐药性分析[J].河南农业科学,2017,46(2):111-115.
[5]孙硕,吕世明,谭艾娟,等.适应度代价对大肠杆菌药物敏感性恢复的作用[J].天津农业科学,2016,22(4):33-35.
[6]俱雄,党亚锋,刘祥,等.大肠杆菌外膜蛋白Omp C的生物信息学分析及表位多肽疫苗的重组预测[J].河南农业大学学报,2017,51(1):94-100.
[7]李进福,丁海峰,李小申,等.河南地区猪呼吸道隐性感染大肠杆菌耐药基因检测及分析[J].河南农业科学,2017,46(9):144-148.
[8]颜友荣,方向红,王琳琳.苏中地区猪大肠杆菌的耐药性与耐药基因的试验[J].中国兽医杂志,2016,52(2):90-92.
[9]刘艳红,吕淑霞,李颖,等.大肠杆菌耐药性及氟苯尼考耐药基因floR的研究[J].中国畜牧杂志,2017,53(1):115-118.
[10]崔耀明,林莉,唐桂芬,等.复方氟苯尼考注射液的临床应用试验[J].黑龙江畜牧兽医,2016(14):176-178.
[11]文丽,吴泽华,李仕超.氟苯尼考手性特性及在临床上的应用[J].猪业科学,2016,33(8):70-71.
[12]冯世文,李军,曾芸,等.大肠杆菌O157∶H7对氟苯尼考耐药机制的初步研究[J].中国人兽共患病学报,2016,32(3):271-275.
[13]王娟.革兰阳性细菌氟苯尼考耐药机制的研究进展[J].中国兽医杂志,2011,47(11):60-62.
[14]吴蓓蓓,杜向党.氟苯尼考耐药菌的构建及大肠杆菌中floR蛋白定位[J].畜牧兽医学报,2008,39(10):1438-1441.
[15]Blickwede M,Schwarz S.Molecular analysis of florfenicol-resistant Escherichia coli isolates from pigs[J].Journal of Antimicrobial Chemotherapy,2004,53(1):58-64.
[16]冯世文,曾芸,李军,等.细菌对氟苯尼考的耐药机制研究[J].黑龙江畜牧兽医,2014(3):52-54.
[17]郑朝朝,刘聚祥,刘静.保定地区不同动物源大肠杆菌氟苯尼考耐药基因floR的同源性分析[J].中国兽医科学,2012,42(7):677-680.
[18]陈阳阳,薛慧亮,马红霞,等.氟苯尼考联合多西环素缩小猪源大肠杆菌耐药突变选择窗[J].中国兽医学报,2013,33(11):1720-1723.