激光显微捕获切割技术在脱落细胞类检材中的方法学建立及其法医学应用研究
发布时间：2018-11-17 06:50
【摘要】：目的 本课题旨在建立一套适于脱落细胞检测的激光显微捕获切割技术(Laser Capture Microdissection, LCM)的方法,并对该方法在脱落细胞类检材特别是降解的脱落细胞样本检验中的应用进行探索性研究。 方法 1以口腔上皮脱落细胞为模型,从染色方法、提取方法、循环参数等方面进行比较研究,建立LCM方法。 2以酸性饮料和口腔上皮细胞混合物为研究对象,对Chelex-100法、Chelex-100与Microcon100相结合法、DNAIQTM提取法等不同方法分别与LCM方法进行比较研究。 3以尿液中脱落细胞为研究对象,对目前法庭科学常用检测方法与LCM方法进行比较研究。结果 1利用本实验所建立的LCM方法捕获10个口腔上皮细胞,即可获得完整的DNA分型,重复性较好,为法医实际案件中微量生物检材的DNA检验提供了一种参考方法。 2酸性饮料和口腔上皮细胞混合物仅采用Chelex-100法或与Microcon100相结合,难以获得较高质量的DNA,从而难以获得较满意的检测结果；对于柠檬酸类混合物,可直接采用DNA IQTM提取法提取,并可以通过减小洗脱体积,提高DNA浓度,从而提高DNA的检出率；乳酸类富含蛋白质的混合物,可考虑采用LCM法,将靶细胞从复杂环境中分离,更利于获得较好的检测结果。 3新鲜尿液组：采用LCM技术提取的DNA在所有基因座上均获得较满意的结果,而采用Chelex-100提取法提取的DNA在各基因座上存在等位基因丢失、非特异性扩增、峰值低等问题；储存尿液组：4℃储存10天以内和室温储存4天以内的尿液采用LCM技术联合DNA IQTM提取法的均可明确判读12个以上基因座；而采用Chelex-100提取法未获得有效分型。结论 LCM法为脱落细胞类检材,特别是降解的脱落细胞的DNA检测提供了一种有效的方法,同时也为其他种类的微量生物检材的检验提供了一种参考方法。
[Abstract]:Objective to establish a method of laser microcapture and cutting (Laser Capture Microdissection, LCM) for the detection of exfoliated cells. The application of this method in the detection of exfoliated cell samples, especially the samples of degraded exfoliated cells, was studied. Methods 1 the oral epithelial exfoliated cells were used as the model, and the LCM method was established by comparing the staining methods, extraction methods and circulatory parameters. 2 the mixture of acid beverage and oral epithelial cells was used as the research object. The methods of Chelex-100, Chelex-100 combined with Microcon100, DNAIQTM extraction and LCM were compared with each other. (3) taking exfoliated cells in urine as the research object, the common methods of forensic science and LCM were compared. Results 1 10 oral epithelial cells were captured by LCM method, and the complete DNA typing was obtained, which provided a reference method for the DNA test of trace biological samples in forensic cases. (2) the mixture of acid beverage and oral epithelial cells was only by Chelex-100 method or combined with Microcon100, so it was difficult to obtain high quality DNA, and obtain satisfactory results. The citric acid mixture can be extracted directly by DNA IQTM extraction, and the detection rate of DNA can be improved by reducing the volume of elution and increasing the concentration of DNA. Since lactic acid is rich in protein, LCM method can be used to isolate target cells from complex environment. (3) fresh urine group: DNA extracted by LCM technique had satisfactory results at all loci, while DNA extracted by Chelex-100 method had some problems such as allele loss, non-specific amplification and low peak value. In the urine storage group, the urine stored at 4 鈩，
本文编号：2336833
[Abstract]:Objective to establish a method of laser microcapture and cutting (Laser Capture Microdissection, LCM) for the detection of exfoliated cells. The application of this method in the detection of exfoliated cell samples, especially the samples of degraded exfoliated cells, was studied. Methods 1 the oral epithelial exfoliated cells were used as the model, and the LCM method was established by comparing the staining methods, extraction methods and circulatory parameters. 2 the mixture of acid beverage and oral epithelial cells was used as the research object. The methods of Chelex-100, Chelex-100 combined with Microcon100, DNAIQTM extraction and LCM were compared with each other. (3) taking exfoliated cells in urine as the research object, the common methods of forensic science and LCM were compared. Results 1 10 oral epithelial cells were captured by LCM method, and the complete DNA typing was obtained, which provided a reference method for the DNA test of trace biological samples in forensic cases. (2) the mixture of acid beverage and oral epithelial cells was only by Chelex-100 method or combined with Microcon100, so it was difficult to obtain high quality DNA, and obtain satisfactory results. The citric acid mixture can be extracted directly by DNA IQTM extraction, and the detection rate of DNA can be improved by reducing the volume of elution and increasing the concentration of DNA. Since lactic acid is rich in protein, LCM method can be used to isolate target cells from complex environment. (3) fresh urine group: DNA extracted by LCM technique had satisfactory results at all loci, while DNA extracted by Chelex-100 method had some problems such as allele loss, non-specific amplification and low peak value. In the urine storage group, the urine stored at 4 鈩，
本文编号：2336833
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