氟苯尼考耐药基因floR LAMP检测方法的建立及应用
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摘要
为建立快速、灵敏且可定量检测氟苯尼考耐药基因floR的环介导等温扩增方法(LAMP),根据GenBank登录的革兰阴性菌floR基因保守序列,利用PrimerExploerV4设计特异性LAMP引物,成功建立了快速检测氟苯尼考耐药floR基因的LAMP方法。该LAMP检测方法反应温度为63℃,反应时间为60 min,具有可实时监测反应、定量检测出floR基因的拷贝数,以及操作简便的特点,灵敏度高,检测限为6.24×10~0拷贝,是普通PCR的100倍。用建立的方法检测对氟苯尼考不同敏感性的大肠埃希菌floR基因,结果显示,对氟苯尼考敏感、中介和耐药的菌株均检测到floR基因,其拷贝数与菌株MIC相关,提示floR基因不仅存在于氟苯尼考耐药菌中。所建立的floR基因LAMP检测方法可为floR基因的监测,以及氟苯尼考耐药性产生和传播机制的研究提供新的技术手段。
A loop-mediated isothermal amplification method was developed for the rapid detection of florfenicol resistance gene floR and a set of primers were designed by using Primer Exploer V4 based on the gene floR from Gram-negative bacteria in GenBank.The assay was optimized to amplify gene floR by incubating at 63 ℃ for 60 min in an instrument of real-time turbidity meter LA-320.The assay can quantitatively determine the gene copy number of gene floR,and it is easy and simple to handle.The sensitivity test proved that this assay had a high sensitivity,the detection limit was up to 6.24×10~0 copies,which was 100 times higher than the general PCR. The gene floR from E.coli strains which have different MIC to florfenicol were detected by this assay,the results showed that gene floR exist in the E.coli strains which were sensitive,intermediary,or resistant to florfenicol,but the gene copy number of floR is different,relative to the MIC.The results suggested that gene floR not only exist in the E.coli strains which resistant to florfenicol.The established method will provide a new technical for the monitoring of floR gene and the study of the resistance generation and transmission mechanism of florfenicol.
A loop-mediated isothermal amplification method was developed for the rapid detection of florfenicol resistance gene floR and a set of primers were designed by using Primer Exploer V4 based on the gene floR from Gram-negative bacteria in GenBank.The assay was optimized to amplify gene floR by incubating at 63 ℃ for 60 min in an instrument of real-time turbidity meter LA-320.The assay can quantitatively determine the gene copy number of gene floR,and it is easy and simple to handle.The sensitivity test proved that this assay had a high sensitivity,the detection limit was up to 6.24×10~0 copies,which was 100 times higher than the general PCR. The gene floR from E.coli strains which have different MIC to florfenicol were detected by this assay,the results showed that gene floR exist in the E.coli strains which were sensitive,intermediary,or resistant to florfenicol,but the gene copy number of floR is different,relative to the MIC.The results suggested that gene floR not only exist in the E.coli strains which resistant to florfenicol.The established method will provide a new technical for the monitoring of floR gene and the study of the resistance generation and transmission mechanism of florfenicol.
引文
[1] Graham R,Palmer D,Pratt B,et al.In vitro activity of florphenicol[J].Eur J Clin Microbiol Infect Dis,1988,7(5):691.
[2] 陈晓慧,刘明春,焦阳.氟苯尼考的研究应用进展[J].中国家禽,2006,28(14):50-52.
[3] 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.
[4] Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[5] National Committee for Clinical Laboratoray Standards.Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals approved standard[M].2nd.Wayne Pa:NCCLS,2009:47-55.
[6] 于学辉,李旭玲,杨发龙,等.鸭源致病性大肠杆菌耐氟苯尼考floR基因的克隆与序列分析[J].中国畜牧兽医,2010,37(3):84-86.
[7] 冯世文,李军,曾芸,等.猪源大肠杆菌O157:H7体外氟苯尼考耐药诱导及对抗菌药物敏感性变化研究[J].中国畜牧兽医,2015,42(12):127-134.
[8] 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.
[2] 陈晓慧,刘明春,焦阳.氟苯尼考的研究应用进展[J].中国家禽,2006,28(14):50-52.
[3] 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.
[4] Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[5] National Committee for Clinical Laboratoray Standards.Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals approved standard[M].2nd.Wayne Pa:NCCLS,2009:47-55.
[6] 于学辉,李旭玲,杨发龙,等.鸭源致病性大肠杆菌耐氟苯尼考floR基因的克隆与序列分析[J].中国畜牧兽医,2010,37(3):84-86.
[7] 冯世文,李军,曾芸,等.猪源大肠杆菌O157:H7体外氟苯尼考耐药诱导及对抗菌药物敏感性变化研究[J].中国畜牧兽医,2015,42(12):127-134.
[8] 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.