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南京医科大学学报(自然科学版) 第44卷第1期
· 24 · Journal of Nanjing Medical University(Natural Sciences) 2024年1月
·基础研究·
重组酶聚合酶扩增结合侧向流动试纸条技术快速可视化检测
嗜麦芽窄食单胞菌方法的建立与应用
季 拓 ,高玉芝 ,王 彦 ,高绪柱 1,2*
1,2
1,2
1,2
江苏大学附属连云港市第二人民医院中心实验室,江苏 连云港 222000;南京医科大学康达学院附属连云港市第二人民
1 2
医院中心实验室,江苏 连云港 222000
[摘 要] 目的:建立一种基于重组酶聚合酶扩增(recombinant polymerase amplification,RPA)和侧向流动试纸条(lateral flow
strips,LFS)的嗜麦芽窄食单胞菌快速可视化检测方法。方法:以嗜麦芽窄食单胞菌特异性序列(NC_010943.1)为模板,设计
RPA 引物。通过基础型 RPA 反应和琼脂糖凝胶电泳,根据候选引物对的扩增性能及引物二聚体的形成情况筛选最佳引物
对。根据最佳引物对,设计探针和修饰引物。在引物和探针中引入碱基错配以消除假阳性信号,建立RPA⁃LFS反应体系。根
据检测线的显色情况,优化RPA⁃LFS的最佳反应条件。使用临床常见的12种致病菌和12个临床来源的嗜麦芽窄食单胞菌检
测该方法的特异性。以10倍梯度稀释的嗜麦芽窄食单胞菌基因组为模板,检测该方法的灵敏度。收集108例临床样本,将该
方法与qPCR检测法和生化培养法对比,对RPA⁃LFS检测法进行kappa一致性检验及临床应用评价。结果:RPA⁃LFS检测法在
37 ℃恒温条件下8 min即可完成扩增反应,1 min内可在LFS上观察到结果。该方法灵敏度高,最低检出限为1.107 CFU,并且
与其他病原菌无交叉反应,特异性强。应用于临床样本检测时,该方法与qPCR相比,检测结果准确性一致。与生化培养方法
的符合率为99.07%,kappa指数值为0.972,具有良好的一致性。结论:本研究建立了一种不依赖于精密仪器和专业技术人员
的RPA⁃LFS检测方法,能够短时间内精准鉴定嗜麦芽窄食单胞菌。该方法的建立可为及时制定合理的抗菌治疗方案提供信
息,具有较大的临床应用潜力。
[关键词] 嗜麦芽窄食单胞菌;重组酶聚合酶扩增;侧向流动试纸条
[中图分类号] R446.5 [文献标志码] A [文章编号] 1007⁃4368(2024)01⁃024⁃08
doi:10.7655/NYDXBNSN230558
Establishment and application of a rapid and visual detection method of Stenotrophomonas
maltophilia by recombinant polymerase amplification combined with lateral flow strip
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JI Tuo ,GAO Yuzhi ,WANG Yan ,GAO Xuzhu 1,2*
Department of Central Laboratory,Lianyungang Hospital Affiliated to Jiangsu University,Lianyungang 222000;
1
2 Department of Central Laboratory,Lianyungang Hospital Affiliated to Kangda College of Nanjing Medical
University,Lianyungang 222000,China
[Abstract] Objective:To develop a rapid and visual detection method of Stenotrophomonas maltophilia(S.maltophilia) by
recombinant polymerase amplification(RPA)combined with lateral flow strip(LFS). Methods:RPA primers were designed based on
specific sequences(NC_010943.1)of S. maltophilia. Through basic RPA reaction and agarose gel electrophoresis,the best primer pair
was selected according to the amplification performance and the formation of cross dimers. According to the best primer pair,the probe
and modified⁃primer were designed. Base mismatches were introduced into the primer and probe to eliminate false positive signals,and
then the RPA⁃LFS reaction system was established. The optimal reaction conditions of RPA⁃LFS were optimized based on the color of
the test line. The specificity of the method was identified by detecting 12 common clinically pathogens and 12 S. maltophilia of clinical
origin. The sensitivity of this method was tested by diluting the genome template. Kappa analysis and clinical application evaluation of
[基金项目] 连云港市卫生科技项目(202217);连云港市第六期“521工程”科研资助立项项目(LYG06521202157);连云港市
科技局重点研发计划(社会发展)(SF2224)
∗
通信作者(Corresponding author),E⁃mail:alexgwan@163.com