發(fā)布時(shí)間：2018-06-07 18:22

  本文選題：多重PCR + 手部　； 參考：《遵義醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:構(gòu)建能同時(shí)檢測鳥、海、偶然、堪薩斯及潰瘍分枝桿菌等五種感染手部常見NTM的多重PCR方法,為手部NTM感染的早期診療提供實(shí)驗(yàn)方法。方法:根據(jù)前期研究成果,1.通過GeneBank數(shù)據(jù)庫和文獻(xiàn)查找鳥、海、偶然、堪薩斯及潰瘍分枝桿菌等五種感染手部常見NTM的特有基因序列,用MFEprimer-5.0及Olige6.0軟件設(shè)計(jì)引物,并用DNAMAN軟件對(duì)其進(jìn)行評(píng)估,篩選出特異性較高的引物構(gòu)建PCR。2.單一PCR擴(kuò)增標(biāo)準(zhǔn)菌DNA后進(jìn)行測序,以驗(yàn)證本研究所用的標(biāo)準(zhǔn)菌是否符合要求。3.利用其引物間無互補(bǔ)或互補(bǔ)性較小的特點(diǎn),構(gòu)建能同時(shí)檢測上述五種NTM的多重PCR,通過優(yōu)化多重PCR的退火溫度,評(píng)估其特異性及敏感性。4.用該多重PCR檢測并鑒定12份臨床考慮手部NTM感染的標(biāo)本。結(jié)果:1.成功為上述五種分枝桿菌設(shè)計(jì)了5對(duì)特異性引物;2.單一PCR檢測本研究所用的標(biāo)準(zhǔn)菌符合要求;3.成功構(gòu)建了能同步檢測鳥、海、偶然、堪薩斯及潰瘍分枝桿菌五種常見手部NTM感染的五重PCR方法;4.該五重PCR的最佳退火溫度為57.5℃,具有較高的特異性及敏感性;5.該五重PCR檢測12份臨床標(biāo)本,10份標(biāo)本擴(kuò)增出陽性條帶,檢出率為83%;6、利用該五重PCR檢測NTM可將時(shí)間縮短至4小時(shí)左右。結(jié)論:本研究建立的m PCR可同步檢測鳥、海、偶然、堪薩斯及潰瘍分枝桿菌等五種手部常見的NTM感染,具有較高的特異性及敏感性。該方法可迅速將NTM鑒定至種屬及亞種水平,為臨床鑒定NTM感染并制定最佳診療方案提供新的檢驗(yàn)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，

本文編號(hào)：1992263