耐甲氧西林金黄色葡萄球菌的核酸检测方法研究

发布时间：2017-12-27 12:17

本文关键词：耐甲氧西林金黄色葡萄球菌的核酸检测方法研究　出处：《中国科学院研究生院(武汉病毒研究所)》2016年博士论文　论文类型：学位论文

【摘要】：病原细菌在抗生素治疗中对其产生耐药性,导致抗生素失效和相关细菌感染难以治愈,一直是一个难以解决的全球化问题。因此快速准确地从复杂的样本中检测出耐药病原菌,以便制定有效的处置方案,成为了微生物学一个重要的研究方向。本课题针对目前耐甲氧西林金黄色葡萄球菌(Methicillin-Resistant Staphylococcus aureus,MRSA)核酸分子检测方法所面临的细胞壁厚难释放核酸、混合菌样本假阳性等问题,进行了一些新方法的研究。首先研究了破碎金黄色葡萄球菌细胞壁以便提取核酸的方法,该菌细胞壁由致密的肽聚糖构成,导致此类细菌的破壁比较困难。因此我们重点研究了新型噬菌体裂解酶取代现有商业化肽聚糖水解酶(如溶葡萄球菌素和无色肽酶)用于金黄色葡萄球菌核酸提取的可行性,实验证明噬菌体裂解酶用于提取金黄色葡萄球菌(包括MRSA)的核酸具有非常高的效率,明显优于现有的商业化水解酶。然后,我们进一步研究了磁珠标记抗体富集金黄色葡萄球菌(包括MRSA)和噬菌体裂解酶联用从复杂样本中检测MRSA的可行性,实验结果表明检测限达到~10 2CFU/ml。最后,我们研究了数字PCR(Droplet Digital PCR,dd PCR)在MRSA检测方面的应用,重点对混合样本进行了双重dd PCR分析。重点评估了不同的金黄色葡萄球菌核酸提取方法和不同PCR策略用于检测MRSA的特异性和敏感度,尤其是评估了这些策略是否能在MRSA检测中排除实际样本中的耐甲氧西林凝固酶阴性葡萄球菌(Methicillin-Resistant Coagulase-Negative Staphylocococci,MR-Co NS)对检测准确性和特异性的干扰。发现采用dd PCR双重扩增mec A和SCCmec-orf X junction既可以直接从混合葡萄球菌样本中准确检出MRSA,也可以对样本中MRSA的数量进行直接的定量分析。
[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.
【学位授予单位】：中国科学院研究生院(武汉病毒研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378.11;R440

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