本文關(guān)鍵詞：耐甲氧西林金黃色葡萄球菌的核酸檢測(cè)方法研究　出處：《中國(guó)科學(xué)院研究生院(武漢病毒研究所)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 耐甲氧西林金黃色葡萄球菌 噬菌體裂解酶 核酸提取 實(shí)時(shí)定量PCR 數(shù)字PCR

【摘要】：病原細(xì)菌在抗生素治療中對(duì)其產(chǎn)生耐藥性,導(dǎo)致抗生素失效和相關(guān)細(xì)菌感染難以治愈,一直是一個(gè)難以解決的全球化問(wèn)題。因此快速準(zhǔn)確地從復(fù)雜的樣本中檢測(cè)出耐藥病原菌,以便制定有效的處置方案,成為了微生物學(xué)一個(gè)重要的研究方向。本課題針對(duì)目前耐甲氧西林金黃色葡萄球菌(Methicillin-Resistant Staphylococcus aureus,MRSA)核酸分子檢測(cè)方法所面臨的細(xì)胞壁厚難釋放核酸、混合菌樣本假陽(yáng)性等問(wèn)題,進(jìn)行了一些新方法的研究。首先研究了破碎金黃色葡萄球菌細(xì)胞壁以便提取核酸的方法,該菌細(xì)胞壁由致密的肽聚糖構(gòu)成,導(dǎo)致此類(lèi)細(xì)菌的破壁比較困難。因此我們重點(diǎn)研究了新型噬菌體裂解酶取代現(xiàn)有商業(yè)化肽聚糖水解酶(如溶葡萄球菌素和無(wú)色肽酶)用于金黃色葡萄球菌核酸提取的可行性,實(shí)驗(yàn)證明噬菌體裂解酶用于提取金黃色葡萄球菌(包括MRSA)的核酸具有非常高的效率,明顯優(yōu)于現(xiàn)有的商業(yè)化水解酶。然后,我們進(jìn)一步研究了磁珠標(biāo)記抗體富集金黃色葡萄球菌(包括MRSA)和噬菌體裂解酶聯(lián)用從復(fù)雜樣本中檢測(cè)MRSA的可行性,實(shí)驗(yàn)結(jié)果表明檢測(cè)限達(dá)到~10 2CFU/ml。最后,我們研究了數(shù)字PCR(Droplet Digital PCR,dd PCR)在MRSA檢測(cè)方面的應(yīng)用,重點(diǎn)對(duì)混合樣本進(jìn)行了雙重dd PCR分析。重點(diǎn)評(píng)估了不同的金黃色葡萄球菌核酸提取方法和不同PCR策略用于檢測(cè)MRSA的特異性和敏感度,尤其是評(píng)估了這些策略是否能在MRSA檢測(cè)中排除實(shí)際樣本中的耐甲氧西林凝固酶陰性葡萄球菌(Methicillin-Resistant Coagulase-Negative Staphylocococci,MR-Co NS)對(duì)檢測(cè)準(zhǔn)確性和特異性的干擾。發(fā)現(xiàn)采用dd PCR雙重?cái)U(kuò)增mec A和SCCmec-orf X junction既可以直接從混合葡萄球菌樣本中準(zhǔn)確檢出MRSA,也可以對(duì)樣本中MRSA的數(shù)量進(jìn)行直接的定量分析。
[Abstract]:Pathogenic bacteria are resistant to antibiotics in the treatment of antibiotics, which leads to the failure of antibiotics and related bacterial infections. Therefore, it is an important research direction for microbiology to detect pathogenic bacteria quickly and accurately from complex samples, so as to develop effective disposal plan. Aiming at the problems of nucleic acid molecular detection methods for Methicillin-Resistant Staphylococcus aureus (MRSA), such as cell wall thickness, difficult to release nucleic acid and false positive of mixed bacteria, some new methods were studied. First, we studied the method of breaking the cell wall of Staphylococcus aureus to extract nucleic acid. The cell wall is composed of dense peptidoglycan, which leads to the difficulty of breaking the bacteria. We focus our study on the new type of phage lysin to replace the existing commercial peptidoglycan hydrolase (e.g. lysostaphin and achromopeptidase) for feasibility of extraction of Staphylococcus aureus DNA, experiments show that phage lyase for extraction of Staphylococcus aureus (including MRSA) nucleic acid with very high efficiency, superior to the existing commercial enzymes. Then we further studied the feasibility of magnetic beads labeled antibody enrichment of Staphylococcus aureus (including MRSA) and phage lyase in detecting MRSA from complex samples. The experimental results showed that the detection limit reached ~10 2CFU/ml. Finally, we studied the application of digital PCR (Droplet Digital PCR, DD PCR) in MRSA detection, focusing on the dual DD PCR analysis of the mixed samples. The evaluation focused on the different Staphylococcus aureus nucleic acid extraction methods and different PCR strategies for specific detection of MRSA and sensitivity, especially the assessment of whether these strategies can exclude the actual sample of methicillin resistant coagulase negative Staphylococcus aureus in detection of MRSA (Methicillin-Resistant Coagulase-Negative Staphylocococci, MR-Co NS) on the accuracy and interference specific detection. It is found that double amplification of MEC A and SCCmec-orf X junction by DD PCR can detect MRSA accurately from mixed staphylococcal samples, and the number of MRSA in samples can be directly quantified.
【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(武漢病毒研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R378.11;R440

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