Diagnosis of Sudden death by in-situ-PCR using tissues from Autopsy

使用尸检组织通过原位 PCR 诊断猝死

• 批准号: 11670415
• 负责人: HONDA Katsuya
• 金额: $ 1.6万
• 依托单位: University of Tsukuba (2001)Osaka University (1999-2000)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670415/
• 关键词: in-situ-PCR; Direct method; hypoxia; Pneumonia; mitochondorial DNA; in-situ-PCR; 感染症診断; 内因性急死; 窒息; ウイルス検出; 法医解剖; ウイルス感染; 急死

This research did the, detection which used peculiar primer of the bacteria to fix the experimental conditions of in-situ-PCR. As a result, the best experimental conditions could be fixed, and applied for practical work. In the present study, I tried to detect the presence of bacterial infections in lung tissue samples, using a DIG Oligonucleotide 3'-End Labeling Kit (Boehringer Mannheim). I was able to confirm a stage of pneumonia to differ in time course, and high sensitivity of these methods was proved by these procedure.After that, it fixed experimental conditions detectable for virus, especially, the herpes group, too. Also, it detected the RNA which is manifested in the hypoxia condition by the in-situ-RT ・ PCR. I detected some kind of cytokines, for example, erythropoietin, interleukin-1a, in addition to the vascular endothelium cell growth factor.However, these manifestations were not too strong in an autopsy sample to diagnosis, and found the difficulty for practical use. Particularly, sudden death from suffocation, there is a extremely little manifestation of these markers.Therefore, to do practicing application, I expanded a search range for mitochondria DNA having relevance to be close to cellular respiration, and found necessity to analyze synthetically for the next step.

本研究利用细菌特异性引物，确定了原位PCR的实验条件，进行了检测。确定了最佳实验条件，并应用于实际工作。在本研究中，我尝试使用DIG寡核苷酸3 '末端标记试剂盒（Boehringer曼海姆）检测肺组织样本中细菌感染的存在。我能够确认肺炎的一个阶段在时间过程中的不同，并通过这些程序证明了这些方法的高灵敏度。之后，它确定了可检测病毒的实验条件，特别是疱疹组。同时，通过原位RT · PCR检测了缺氧条件下的RNA表达。我检测到了一些细胞因子，例如促红细胞生成素、白细胞介素-1a，此外还有血管内皮细胞生长因子，但这些表现在尸检标本中并不太强，难以诊断，难以发现实际应用。特别是窒息猝死，这些标志物的表现非常少，因此，为了进行实践应用，我将与细胞呼吸相关的线粒体DNA的搜索范围扩大到接近细胞呼吸，并发现有必要为下一步进行综合分析。

项目成果

Honda K,Tun Z,Young DR: "Examination of Y-STR mutations in sex chromosomal abnormality in forensic cases"Furensic Science International. Special issue(in press). (2001)

Honda K、Tun Z、Young DR：“法医案件中性染色体异常的 Y-STR 突变检查”Furensic Sc​​ience International。

Nakamura M.: "Application of in-situ-PCR to diagnose pneumonia in medico-legal autopsy Cases"Legal Med.. 3. 127-133 (2001)

Nakamura M.：“原位PCR在法医尸检病例中诊断肺炎中的应用”Legal Med. 3. 127-133 (2001)

瀧北祥子,本田克也,中村正巳: "生体由来試料のDNA保存性に関する定量的研究"DNA多型. 8. 264-267 (2000)

Shoko Takikita、Katsuya Honda、Masami Nakamura：“生物样品 DNA 保存的定量研究”DNA 多态性。

Nakamura M: "Application of insitu-PCR to diagnose pneumonia in medico-legal autopsy cases."Legal Med.. 3. 127-133 (2001)

Nakamura M：“应用原位PCR诊断法医尸检病例中的肺炎。”Legal Med.. 3. 127-133 (2001)

Tun Z,Hondak,Harihara S: "Cluster analysis of Hsian population by mitochondria DNA pdynerphisin"DNA polymorphism. 8. 175-178 (2000)

Tun Z，Hondak，Harihara S：“通过线粒体 DNA pdynerphisin 对西安人群进行聚类分析”DNA 多态性。