本文選題：種屬鑒定 + 線粒體DNA　； 參考：《山西醫(yī)科大學》2013年碩士論文

【摘要】：目的：種屬鑒定是法醫(yī)物證檢驗的重要步驟,對現(xiàn)場提取的各種生物檢材進行種屬鑒定是進行其他鑒定的首要及關鍵環(huán)節(jié)。利用線粒體的特點,構建一種特異性好,靈敏度高,穩(wěn)定性好,操作簡便適用于法醫(yī)學實踐的擴增體系,以期解決當前法醫(yī)種屬鑒定所存在的問題及不足。 方法：根據(jù)本課題的研究目的,探索復合擴增-瓊脂糖凝膠電泳進行種屬鑒定方法。參閱之前發(fā)表的種屬鑒定方面的文章,確定篩選人類特異性基因和內參對照基因的原則,依照該原則并結合基因庫中人及各種哺乳動物mtDNA序列,研究mt DNA的37個基因,并篩選出選出符合條件的編碼基因,然后仔細比對,尋找異同,進而鎖定目標基因序列；根據(jù)引物設計的原則,利用primer5及primer3(online)等多款引物設計軟件,設計引物并優(yōu)化；收集人和恒河猴,豬牛羊兔5種動物的肌肉或者血液樣本,提取DNA,復合擴增線粒體12SrRNA,16SrRNA,COX1基因片段,摸索優(yōu)化擴增條件,尋找特異性好、靈敏度高、結果穩(wěn)定的擴增條件；擴增產物用瓊脂糖凝膠電泳分離檢測、254nm紫外燈下觀察結果。 結果：內參基因選擇12SrRNA和16SrRNA,選擇COX-1基因作為檢測現(xiàn)代智人的特異性標志。設計四對引物12S1,12S2,16S和COX-1擴增相關區(qū)域。12S1和12S2引物擴增片段大小相同。電泳檢測結果顯示若16SrRNA、12SrRNA、 COX-1三個區(qū)域全部擴增出,顯示三條帶時,且大小分別為395bp、231bp、202bp判定樣品來自人。對于非人類標本,包括猴在內的其它動物僅有16SrRNA、12SrRNA區(qū)的擴增條帶。對于12SrRNA基因,我們在同一個擴增區(qū)域設計兩對引物12S1和12S2,其中12S2擴增區(qū),豬牛樣品顯示陽性,而12S1區(qū)域,除豬牛樣品外,所有哺乳動物樣品均可被擴增出。該擴增體系敏感度為2.5pg,特異性基因COX-1的靈敏度為10-8pg,因此適合對法醫(yī)學上極微量檢材的適用。 結論：初步建立了可運用于法醫(yī)學人類樣品認定的復合擴增體系。該方法穩(wěn)定性好、靈敏度高、特異性強、檢測過程方便,適合法醫(yī)學實踐中的極微量檢材的人類樣品認定,為今后法醫(yī)學種屬鑒定提供了更為方便而實用的技術方法基礎。
[Abstract]:Objective: species identification is an important step of forensic physical evidence examination, and the identification of species of various biological samples extracted in the field is the most important and key link of other identification.Based on the characteristics of mitochondria, an amplification system with good specificity, high sensitivity, good stability and easy operation was constructed to solve the problems and shortcomings in forensic identification.Methods: according to the purpose of this study, the method of multiplex amplification-agarose gel electrophoresis for species identification was explored.Referring to the previously published articles on species identification, the principles of screening human specific genes and internal reference control genes were determined. According to this principle, 37 genes of Mt DNA were studied in combination with the mtDNA sequences of human and various mammals in the gene bank.According to the principle of primer design, using several primer design software, such as primer5 and primer3online, the primers are designed and optimized.The muscle or blood samples of human, rhesus monkey, pig, sheep and rabbit were collected, and the DNA was extracted, the mitochondrial 12SrRNA-16SrRNA-COX1 gene fragment was amplified, and the optimized amplification conditions were explored to find the amplification conditions with good specificity, high sensitivity and stable results.The amplified products were separated by agarose gel electrophoresis and detected by UV lamp at 254 nm.Results: 12SrRNA and 16s rRNA were selected as the specific markers for the detection of modern Homo sapiens.Four pairs of primers, 12S1, 12S216s and COX-1, were designed to amplify the relevant region. 12S1 and 12S2 primers were the same size.The electrophoretic analysis showed that if the 16s rRNAs were 12s rRNAs, all the three regions of COX-1 were amplified, and the size of the three bands was 395bpn231bp20bp, respectively.For non-human specimens, there are only 16s rRNA 12s rRNA bands in other animals, including monkeys.For 12SrRNA gene, we designed two pairs of primers 12S1 and 12S2 in the same amplification region, in which 12S2 amplification region, porcine and bovine samples showed positive, and 12S1 region, all mammalian samples except porcine cattle samples could be amplified.The sensitivity of the amplification system was 2.5 PG and the sensitivity of specific gene COX-1 was 10-8 PG.Conclusion: a multiplex amplification system which can be applied to the identification of forensic human samples was established.This method has the advantages of good stability, high sensitivity, high specificity and convenient detection process. It is suitable for the identification of human samples in forensic practice and provides a more convenient and practical technical basis for the identification of forensic medicine species in the future.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：D919.4

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