应用液态悬浮芯片技术建立17种大肠杆菌耐药基因多重快速检测方法
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S851.3

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青岛市科技惠民示范引导专项(21-1-4-ny-11-nsh);国家重点研究开发项目(2018YFD0500505)


A Multiplex Rapid Assay for Detection of 17 Drug Resistance Genes of E. coli Established by Liquid Suspension Microarray
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    摘要:

    为建立一种快速、高通量的多重耐药基因检测方法,利用Luminex液态芯片平台,建立了可同时检测17种耐药基因的液态芯片检测方法。该方法对大肠杆菌常见的七大类抗菌药物所对应的17种耐药基因(blaSHV、blaCMY-1、Aph3-IIa-1、aac(6)-Ib-cr、aadA-1、cmlA-1、gyrA、mcr-1、NDM-1、parC、qnrS-1、sul-1、sul-2、sul-3、tetA、tetB、tetX)进行序列分析,随后依次对其设计多重PCR特异性引物,构建特异的阳性质粒作为阳性参比品进行液态芯片检测条件优化,从而进行多重耐药基因液态芯片检测方法的研发。结果显示:成功构建了17种耐药基因的阳性质粒,并建立了两套体系用于检测17种耐药基因。在特异性试验中,两套体系中的检测信号无干扰,具有较高的特异性数值;敏感性试验中,体系一的单一质粒最低检测量为102~104 copies/μL,混合质粒为103~105 copies/μL;体系二的单一质粒最低检测量为102~105 copies/μL,混合质粒为104~106 copies/μL;有效性试验中,液态芯片法与PCR检测结果的Kappa值多在0.60以上,具有高度一致性。结果表明,本研究建立的2套液态芯片检测体系具有高通量、高灵敏和高特异性的优点,可同时对17种耐药基因进行检测,能够达到快速检测的目的。

    Abstract:

    In order to establish a rapid and high throughput method for detecting multidrug resistance genes,a liquid microarray method was established for simultaneous detection of 17 drug resistance genes by using Luminex liquid microarray platform. The sequences of 17 genes(including blaSHV,blaCMY-1,Aph3-IIa-1,aac(6)-Ib-cr,aadA-1,cmlA-1,gyrA,mcr-1,NDM-1,parC,qnrS-1,sul-1,sul-2,sul-3,tetA,tetB and tetX)corresponding to seven classes of antibiotics against Escherichia coli were analyzed by the method,then specific primers for multiplex PCR were successively designed,and specific positive plasmids were constructed as positive reference to optimize the detection conditions of liquid microarray,so as to develop a liquid microarray detection method for multidrug resistance genes. The results showed that positive plasmids of the 17 genes and two systems for detecting the genes were successively established. In the specificity test,detection signals in the two systems did not interfere with each other,and with high value of specificity;in the sensitivity test,the minimum detection amounts of single and mixed plasmids were 102~104 and 103~105 copies/μL respectively in system 1;and 102~105 and 104~106 copies/μL respectively in system 2;in the validity test,Kappa values of the test results by liquid microarray and PCR were mostly above 0.60,which were highly consistent. It was concluded that the two systems established in the paper could be used to simultaneously detect 17 drug resistance genes to ensure rapid detection with the help of their advantages of high throughput,sensitivity and specificity.

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韩天飞,赵建梅,刘娜,张青青,王娟,张喜悦,刘俊辉,李月华,黄秀梅,王君玮,曲志娜,祁克宗.应用液态悬浮芯片技术建立17种大肠杆菌耐药基因多重快速检测方法[J].《中国动物检疫》编辑部,2021,38(10):120-126.

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  • 在线发布日期: 2021-10-08
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