黏菌素耐药基因mcr-1 TaqMan-MGB荧光定量PCR检测方法的建立  

Establishment of TaqMan-MGB fluorescent quantitative PCR for detection of colistin resistance gene mcr-1

作　　者：施开创[1] 尹彦文[1] 温丽霞[1] 屈素洁[1] 王海清[1] 胡杰[1] SHI Kai-chuang;YIN Yan-wen;WEN Li-xia;QU Su-jie;WANG Hai-qing;HU Jie(Guangxi Center for Animal Disease Control and Prevention,Nanning 530001,China)

机构地区：[1]广西动物疫病预防控制中心,南宁530001

出　　处：《南方农业学报》2018年第7期1447-1452,共6页Journal of Southern Agriculture

基　　金：广西科技重大专项项目(桂科AA17204057);广西水产畜牧兽医局科技计划项目(桂渔牧科1304559)

摘　　要：【目的】建立针对黏菌素耐药基因mcr-1的Taq Man-MGB荧光定量PCR检测方法,为监控mcr-1基因携带细菌提供技术支持。【方法】针对mcr-1基因保守序列设计特异性引物和MGB探针,构建重组质粒作为阳性标准品,优化引物、探针浓度及Rox参比染料用量、退火温度等反应条件,建立针对mcr-1基因的Taq Man-MGB荧光定量PCR检测方法,并通过特异性、敏感性、重复性试验及临床应用验证其实用性。【结果】Taq Man-MGB荧光定量PCR最佳反应体系20.00μL:Premix Ex Taq^(TM)10.00μL,Rox参比染料(50×)0.25μL,引物MCR-1F/MCR-1R(10μmol/L)各0.50μL,MCR-1-P探针(10μmol/L)0.50μL,重组质粒pMCR-1(模板)2.00μL,灭菌双蒸水6.25μL。扩增程序:95℃预变性20 s;95℃10 s,60℃20 s,进行40个循环。该检测方法能特异性扩增出mcr-1基因,其检测敏感性为2.85拷贝/μL,是常规PCR的100倍,组内和组间重复性试验的变异系数为0.37%~1.18%,均小于1.20%。采用建立的Taq Man-MGB荧光定量PCR对82株广西鸡源致病性沙门氏菌分离株进行检测,结果未发现有携带mcr-1基因的菌株。【结论】针对mcr-1基因建立的Taq Man-MGB荧光定量PCR检测方法具有特异性强、敏感性高、重复性好的特点,可用于临床筛查mcr-1基因,为监控mcr-1基因携带细菌提供技术支持。【Objective】TaqMan-MGB fluorescent quantitative PCR detection method targeting colistin resistant gene mcr-1was established in order to supply an effective technique for surveillance of bacteria harboring gene mcr-1.【Method】Specific primers and MGB probe were designed basbased on mcr-1 gene conserved sequence,and recombinant plasmid was constructed as positive standard.After optimizing the reaction conditions including primer concentration,probe concentration,Rox reference dyeamount and annealing temperature,a TaqMan-MGB fluorescent quantitative PCR for detection of mcr-1 gene was established.The assay was further verified by specificity test,sensitivity test,repeatability test and clinical application.【Result】The optimal 20.00μL reaction system of TaqMan-MGB fluorescent quantitative PCR included:Premix Ex TaqTM 10.00μL,Rox reference dye(50×)0.25μL,MCR-1F/MCR-1R primers(10μmol/L)0.50μL each,MCR-1-P probe(10μmol/L)0.50μL,recombinant plasmid pMCR-1(template)2.00μL and distilled water 6.25μL.The amplification procedure consisted of an initial predenaturation step for 20 s at 95℃,followed by 40 cycles of 10 s at 95℃and 20 s at 60℃.The established assay could specifically amplify gene mcr-1.The detection limit was 2.85 copy/μL,which was as 100 times as that of conventional PCR.The coefficient of variation for intra-and inter-assay was from 0.37%to 1.18%,which was less than 1.20%.Eighty-two pathogenic chicken Salmonella isolates from Guangxi were detected for mcr-1 gene by the assay and none was positive.【Conclusion】The established TaqMan-MGB fluorescent quantitative PCR detection method was highly specific,sensitive and reproducible,and it can be used for screening mcr-1 gene in clinical practice and provide technical support for surveillance of bacteria harboring mcr-1 gene.

关 键 词：黏菌素 mcr-1基因 TAQMAN-MGB探针 荧光定量PCR 

分 类 号：S854.44[农业科学—临床兽医学]