多重PCR同步检测感染手部五种非结核分枝杆菌检验方法的建立

发布时间：2018-06-07 18:22

  本文选题：多重PCR + 手部　； 参考：《遵义医学院》2017年硕士论文

【摘要】：目的:构建能同时检测鸟、海、偶然、堪萨斯及溃疡分枝杆菌等五种感染手部常见NTM的多重PCR方法,为手部NTM感染的早期诊疗提供实验方法。方法:根据前期研究成果,1.通过GeneBank数据库和文献查找鸟、海、偶然、堪萨斯及溃疡分枝杆菌等五种感染手部常见NTM的特有基因序列,用MFEprimer-5.0及Olige6.0软件设计引物,并用DNAMAN软件对其进行评估,筛选出特异性较高的引物构建PCR。2.单一PCR扩增标准菌DNA后进行测序,以验证本研究所用的标准菌是否符合要求。3.利用其引物间无互补或互补性较小的特点,构建能同时检测上述五种NTM的多重PCR,通过优化多重PCR的退火温度,评估其特异性及敏感性。4.用该多重PCR检测并鉴定12份临床考虑手部NTM感染的标本。结果:1.成功为上述五种分枝杆菌设计了5对特异性引物;2.单一PCR检测本研究所用的标准菌符合要求;3.成功构建了能同步检测鸟、海、偶然、堪萨斯及溃疡分枝杆菌五种常见手部NTM感染的五重PCR方法;4.该五重PCR的最佳退火温度为57.5℃,具有较高的特异性及敏感性;5.该五重PCR检测12份临床标本,10份标本扩增出阳性条带,检出率为83%;6、利用该五重PCR检测NTM可将时间缩短至4小时左右。结论:本研究建立的m PCR可同步检测鸟、海、偶然、堪萨斯及溃疡分枝杆菌等五种手部常见的NTM感染,具有较高的特异性及敏感性。该方法可迅速将NTM鉴定至种属及亚种水平,为临床鉴定NTM感染并制定最佳诊疗方案提供新的检验方法。
[Abstract]:Objective: to construct a multiplex PCR method for simultaneous detection of NTM in the hands of birds, sea, accidental, Kansas and Mycobacterium ulcers, so as to provide an experimental method for the early diagnosis and treatment of NTM infection in the hands. Methods: according to the previous research results. By using the GeneBank database and literature, five common NTM genes of bird, sea, accidental, Kansas and Mycobacterium ulcerans were found. Primers were designed with MFEprimer-5.0 and Olige6.0 software and evaluated by DNAMAN software. High specific primers were screened to construct PCR.2. The standard bacteria DNA was amplified by single PCR and sequenced to verify whether the standard bacteria used in this study met the requirements. The multiplex PCRs which can simultaneously detect the above five kinds of NTM were constructed by using the characteristics of no complementation or little complementarity among the primers. The specificity and sensitivity of the PCR were evaluated by optimizing the annealing temperature of the multiplex PCR. The multiplex PCR was used to detect and identify 12 specimens of NTM infection in the hands. The result is 1: 1. Five pairs of specific primers were successfully designed for the above five mycobacteria. The standard bacteria used in this study were tested by single PCR. A five-fold PCR method for simultaneous detection of NTM infection in the hands of birds, sea, accidental, Kansas and Mycobacterium ulcers was successfully constructed. The optimum annealing temperature of the five-fold PCR is 57.5 鈩，

本文编号：1992263