应用原位PCR技术检测石蜡切片MN血型基因型的研究

发布时间：2018-06-12 12:36

本文选题：MN基因型 + 原位PCR　；参考：《中国医科大学》2004年硕士论文

【摘要】： 前言个人识别是以个人为研究对象，包括活体，尸体及来源于人体的生物学性检材，应用法医学、人类学的理论和技术检测分析，确定其个体的身源。遗传标记检测是个人识别最可靠、最确切的手段。DNA遗传标记多态性检测技术在法医学领域中的应用，特别是具有灵敏度高、特异性强、适用于降解DNA检材、检测方法操作简单、节省时间等特点的PCR技术的应用和普及，使法医物证检测由细胞水平发展到分子水平，由宏观水平发展到微观水平，由只能否定发展到可以肯定的水平，极大地扩展了法医物证的应用范围，提高了法医物证鉴定的质量。法医实践中某些案件却经常要求我们对送检的特殊检材—福尔马林固定后的人体组织(石蜡包埋块或病理切片)进行同一认定和个人识别。例如：罪犯保外就医提供的是否本人的病理组织和切片，或者是保险理赔时提供的他人的恶性肿瘤病理切片，或是病理室工作中偶尔遇到标本的标记错误，发现与临床资料不匹配时等案件都需要对这种特殊检材的组织来源进行鉴定，石蜡包埋组织或者切片的DNA分型鉴定在实践中已经愈发重要。本研究应用灵敏度高、特异性好的原位PCR技术，测定福尔马林固定不同时间的石蜡切片组织MN血型的基因型，并对蛋白酶消化时间等主要影响因素进行了初步的研究，以建立一种稳定、实用的检测石蜡组织切片DNA遗传标记的方法，为法医物证鉴定和个人识别提供一种新的检测手段。材料与方法样本来源和处理：样本来源：样本血液2ml、肌肉组织200mg各12例，分别取自本校法医病理教研室12例解剖尸体(死亡后48小时内，未腐败尸体)。12例血液样品制悬液备检。同时酚/氯仿法常规提取DNA。每例肌肉组织分别按照10％福尔马林固定24h、48h、72h、3个月分组。石蜡包埋，切5卜m厚切片数张。运用直接凝集法检测红细胞MN表型。运用常规 PCR检测样本血液MN基因型。石蜡切片进行原位PCR检，预处理分别滴加loom岁血的蛋白酶K20闪消化，以原位扩增显色后阳性细胞占总细胞的比值75%为标准，确定最适消化时间〔‘，〕，研究不同固定时间与蛋白酶消化的相互影响和关系，同时检测石蜡切片的MN基因型。结果血清学检测12例样本MN表型结果、PCR检测结果与原位PCR检测 MN基因型结果完全相同:12例被检样本，1、6、7、n号4个样品基因型为’ MM型;2、4、8、9、12号5个样本基因型为NN型;3、5、10号3个样本基因型为MN型。固定时间为24h、48h、72h、3个月组所需最适宜的蛋白酶消化时间分别为30.41土6.55而n、46.67土6.38min、56.25士8.49而n、58.33土6. 38而n。统计学分析表明不同固定时间分组所需蛋白酶最适消化时间存在显著差异 (P0 .01)。讨论在法医物证实践中，经过化学处理的特殊检材，例如福尔马林固定后的石蜡包埋组织块和切片的个人识别和来源鉴别，，因为福尔马林介导的甲基化直接影响DNA提取的效率，其中固定时间是一个重要的变异因素。在常规方法提取DNA过程中，由于包埋组织中DNA发生降解，同时DNA提取过程中，机械和化学的作用，也最容易导致模板DNA污染和造成DNA破坏。本研究根据文献合成了两条MN序列特异性引物，分别与一条共同引物构成两个可以分别检测M、N基因的PCR反应体系。运用原位PCR技术来对组织切片进行DNA分型，避免了提取过程中的破坏和污染，在切片上直接进行PcR反应进行组织切片的MN基因型检侧。12例样品基因分型结果与血清型分型结果完全一致。表明本研究合成的引物质量良好，PCR 扩增条件选择适宜〔‘，1。本研究采用的是直接原位PCR检测方法，即在PCR反应液中，按一定比例用Bi。一n一dUTP代替dT]叩，在PCR反应中，Bio一n一dUTP掺人到扩增的目的片段中，然后用亲和素化的酶显色系统显色观察。本研究结合临床病理诊断实际和法医物证实践，对10%福尔马林固定时间分别为24h，48h，72h和3个月的肌肉组织分组进行蛋白酶消化时间的梯度研究，通过蛋白酶消化时间的筛选实验，来研究固定时间和蛋白酶消化时间的相互影响和关系，成功地对固定时间从24h到3个月的组织石蜡包埋切片进行了MN血型的检测，从而为法医物证实践提供原位PCR检测组织切片的实验条件。研究表明:PCR信号强弱与福尔马林固定时间和蛋白酶消化程度密切相关，而最适宜的蛋白酶消化时间决定于福尔马林固定时间，蛋白酶消化时间随着固定时间的增长而递增，这种强相关性使得充分的蛋白酶消化在原位PCR检测中非常重要，蛋白酶消化不充分是原位PcR失败的最常见的原因。组织标本经蛋白酶消化后可增加通透性，使反应试剂进人细胞内，并暴露靶核酸序列。消化不足可导致细胞通透性差，蛋白质与核酸之间广泛交联，出现假阴性结果，而过度消化则可能造成细胞形态结构的破坏，同时导致扩增产物弥散，造成假阳性结果。我们对比了几种不同消化方式，确定以 100协扩mL的蛋白酶K溶液、针对不同固定时间的组织进行消化，可达到较佳结果。将原位PCR技术运用于法医血清学的个人识别和同一认定，目前国内外还?
[Abstract]:Foreword

Personal identification is an individual research object , including living body , body and biological sample derived from human body , applying forensic science , anthropology theory and technology detection analysis , to determine the body source of its individual .

Genetic marker detection is the most reliable and accurate method for personal identification . The application and popularization of DNA genetic marker polymorphism detection technology in forensic medicine field , especially the application and popularization of PCR technique with high sensitivity and specificity , is suitable for degrading DNA sample , detection method has simple operation , time saving and so on .

Some cases in the practice of forensic medicine often ask us to conduct the same identification and personal identification of the special sample - formalin - fixed human tissue ( paraffin embedded block or pathological section ) after the examination . For example , it is important to identify the tissue source of the special test material , and the paraffin embedding tissue or the DNA typing of the slice has become more and more important in practice .

In order to establish a stable and practical method for the detection of DNA genetic markers in paraffin sections , a new method for the identification of forensic evidence and personal identification was provided .

Materials and Methods

Sample source and treatment : sample source : sample blood 2ml , muscle tissue 200 mg each 12 cases , taken from 12 autopsy bodies ( within 48 hours after death , uncorrupted body ) from the department of forensic pathology of our school . 12 cases of blood sample suspension preparation . Meanwhile , the DNA was routinely extracted by phenol / chloroform method . Each muscle tissue was fixed for 24h , 48h , 72h , and 3 months according to 10 % formalin , respectively . Paraffin bag

The expression of erythrocyte MN phenotype was detected by direct agglutination test , and routine test was used to detect the phenotype of erythrocyte MN .

The blood MN genotype of sample was detected by PCR . The paraffin sections were subjected to in - situ PCR and pre - treatment respectively .

The protease K20 was digested with proteinase K20 , and the positive cells in situ were used to make up the total cells .

The ratio of 75 % was the standard , the optimal digestion time was determined &bra; ' , &ket; , the different fixing time and protease were studied . The mutual influence and the relationship between digestion and the MN genotype of paraffin sections were also detected . Results Serologic test of 12 samples of MN phenotype results , PCR detection results and in situ PCR detection The results of MN genotype were identical : 12 samples were tested , 1 , 6 , 7 , and 4 samples were of the same genotype .

Type MM ; 2 , 4 , 8 , 9 , 12 5 samples genotype NN ; 3 , 5 , 10 3 sample genotypes

is of the mn type .

the time of fixation is 24 h , 48 h , 72 h , the most suitable protease digestion time required for the 3 month group is

For 30.41 soil 6.55 , n , 46.67 soil 6.38min , 56.25 + 8.49 and n , 58.33 soil 6.38 and n .

The statistical analysis indicated that there was a significant difference in the optimal digestion time of protease required for different fixation time groups .

(P0 .01).

discuss

in that practice of forensic evidence , a special sample of chemical treatment , such as formalin fix ,

Individual identification and source identification of paraffin - embedded tissue blocks and slices , as formalin - mediated methyl

The efficiency of DNA extraction is directly affected , and the fixed time is an important variation factor .

in that proces of extracting DNA by the method , the DNA in the embedding tissue is degraded , and the DNA extraction

During the process , mechanical and chemical effects are most likely to lead to DNA contamination of the template and DNA damage

Bad .

Two specific primers of MN sequence were synthesized according to the literature .

The PCR reaction system can be used for detecting M and N genes respectively .

DNA typing of the tissue slices is carried out , the damage and the pollution in the extraction process are avoided ,

The genotype of MN genotype of tissue sections was examined by direct PCR reaction . 12 samples were genotyped .

Results The results were completely consistent with the results of serotyping . The results showed that the quality of the synthesized primers was good , PCR was carried out .
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2004
【分类号】：D919

【参考文献】