多重耐药基因lsa(E)实时荧光定量PCR检测方法的创新  

Innovation of qRT-PCR assay for detection of multiple drug resistance gene lsa(E)

作　　者：张博 陈峥[1] 李琼[1] 刘伟成 杜聪阳 杜向党[1] 许春燕 ZHANG Bo;CHEN Zheng;LI Qiong;LIU Weicheng;DU Congyang;DU Xiangdang;XU Chunyan(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046

出　　处：《河南农业大学学报》2023年第4期639-645,共7页Journal of Henan Agricultural University

基　　金：国家自然科学基金项目(U1704108);河南省高校科技创新团队支持计划项目(18IRTSTHN020);中国博士后科学基金项目(2021M690924)。

摘　　要：【目的】创新多重耐药基因lsa(E)实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)的检测方法,为lsa(E)基因流行和传播的监测提供参考。【方法】根据GenBank中lsa(E)基因的参考序列设计特异性引物,通过纯化、连接和转化等步骤,提取含有lsa(E)基因片段的重组质粒DNA为标准品进行检测,绘制标准曲线,验证其特异性和重复性,从而对多重耐药基因lsa(E)qRT-PCR检测方法进行创新;使用该检测方法对牛场粪便、土壤、淤泥和卧料等环境样品中lsa(E)基因的拷贝数量和相对丰度进行检测。【结果】该多重耐药基因lsa(E)qRT-PCR检测方法熔解曲线均为平滑单峰,特异性强;扩增效率良好(101.56%);标准曲线呈现良好的线性关系(r^(2)≥0.997);检测拷贝数量范围广(1.71×10^(2)~1.71×10^(8) copies·μL^(-1))且灵敏度高,组内组间变异系数范围为0.45%~1.66%,重复性强;使用该方法检测牛场环境样品,lsa(E)基因拷贝数量范围为2.54×10^(4)~1.52×10^(8) copies·μL^(-1),相对丰度范围为9.39×10^(-4)~1.20×10^(3),而普通PCR方法仅能在拷贝数量大于1×10^(6) copies·μL^(-1)的环境样品中检测到lsa(E)基因。【结论】本研究创新了多重耐药基因lsa(E)的qRT-PCR检测方法,特异性强,灵敏度高,检测范围广,且能应用于不同养殖环境样品(粪便、土壤和淤泥等)中lsa(E)基因的定量检测,为lsa(E)基因流行和传播的监测提供技术支持和理论指导。【Objective】To provide a reference for monitoring the prevalence and spread of lsa(E)gene by innovating a real-time quantitative PCR(qRT-PCR)method for the detection of multi-drug resistance gene lsa(E).【Method】Specific primers were designed according to the reference sequence of the lsa(E)gene in GenBank.The recombinant plasmid DNA containing lsa(E)gene was extracted following purification,ligation and transformation and used as the standard substance for detection.The standard curve was drawn and its specificity and repeatability were verified to establish the innovative method of a qRT-PCR assay for multidrug-resistant gene lsa(E).Then,the method was applied to detect the copy numbers and relative abundance of lsa(E)gene in feces,soil,sludge and padding collected from cattle farms.【Result】The results showed that the melting curves of this method were smooth and unimodal,and exhibited a strong specificity;the amplification efficiency was 101.56%;the value of r^(2) was≧0.997,indicating a good linearity relationship of the standard curve;the detection range(number of gene copies)of the assay for lsa(E)was wide with 1.71×10^(2) to 1.71×10^(8) copies·μL^(-1) and its sensitivity was high,and both the variable coefficients of intra and inter batch repeated tests were within 0.45%to 1.66%,with a good repeatability.For environmental samples from cattle farm,the copy number of the lsa(E)gene was from 2.54×10^(4) to 1.52×10^(8) copies·μL^(-1) and the relative abundance was from 9.39×10^(-4) to 1.20×10-3 detected by qRT-PCR.However,the conventional PCR method could only detect lsa(E)in environment samples with more than 1×10^(6) copies·μL^(-1) of the copy number of gene.【Conclusion】This study innovated a qRT-PCR assay for the detection of multidrug-resistant gene lsa(E)with high specificity,sensitivity,and wide range of application.The innovative method could be used in different environment samples(including feces,soil and sludge)collected from cattle farms,and provided a technical and theor

关 键 词：lsa(E)基因 多重耐药 实时荧光定量PCR 拷贝数量 相对丰度 

分 类 号：S852.61[农业科学—基础兽医学]