本文關(guān)鍵詞：嗜尸性蠅類的分子標記種屬鑒別與法醫(yī)學應用研究　出處：《重慶醫(yī)科大學》2013年博士論文　論文類型：學位論文

  更多相關(guān)文章： 法醫(yī)昆蟲學 種屬鑒定 mtDNA COI 法醫(yī)昆蟲學 大頭金蠅 PMI 發(fā)育歷期 法醫(yī)昆蟲學 SPME GC/MS 氯氮平

【摘要】：第一部分：線粒體DNA中COI基因序列在常見嗜尸性蠅類種屬鑒別中的應用 目的利用線粒體DNA（mtDNA）細胞色素氧化酶輔酶I（COI）中的498bp和841bp基因序列，對常見嗜尸性蠅類進行種屬鑒別，解決依據(jù)形態(tài)學方法難以鑒別現(xiàn)場樣本種屬的難題，為法醫(yī)現(xiàn)場檢案提供幫助。 方法收集不同地區(qū)的2科4屬6個種的嗜尸性蠅類樣本，經(jīng)專業(yè)人員對樣本進行形態(tài)學檢驗，分別提取樣本mtDNA，利用COI基因序列中的兩對引物進行不同序列PCR擴增，以瓊脂糖電泳檢測擴增產(chǎn)物，PCR膠回收試劑盒純化，ABI377測序儀測序，將所得序列以DNAMAN6.0分析軟件分別截取498bp和841bp長度的序列，分別輸入GenBank進行BLAST檢索同源相似性比對，以MEGA5.1軟件進行序列分析并構(gòu)建系統(tǒng)發(fā)育樹，分析不同地區(qū)及不同種屬的樣本鑒別效果。 結(jié)果5個地區(qū)6種嗜尸性蠅類30個樣本，mtDNA的COI基因序列I（498bp）BLAST種屬同源性正確比對28個，正確比對率達到93.33%，種內(nèi)進化分歧均數(shù)在0.1%~1.6%之間，種間進化分歧均數(shù)在2.2%~11.2%之間，除絲光綠蠅與銅綠蠅2個近緣種屬難以區(qū)分外， 其余4個種屬通過系統(tǒng)發(fā)育樹可明確區(qū)分。3個地區(qū)6種嗜尸性蠅類36個樣本，mtDNA的COI基因序列II（841bp）BLAST種屬同源性正確比對35個，正確比對率達到97.22%，種內(nèi)進化分歧均數(shù)在 0.2%~1.5%之間，種間進化分歧均數(shù)在2.7%~10.6%之間，6個種屬通過系統(tǒng)發(fā)育樹均可明確區(qū)分。 結(jié)論COI基因序列同源性比對與系統(tǒng)發(fā)育樹構(gòu)建的種屬鑒別方法，是嗜尸性蠅類形態(tài)學種屬檢驗的重要補充手段，對嗜尸性蠅類樣本種屬的快速、準確鑒定有重要作用。 第二部分：不同食物成分對大頭金蠅生長發(fā)育的影響 目的觀察不同臟器及不同脂肪含量的肌肉組織對大頭金蠅生長發(fā)育的影響，為昆蟲學相關(guān)研究及死后間隔時間（Post mortem interval,PMI）的法醫(yī)學推斷提供理論依據(jù)。 方法取豬的腦、心臟、肝臟及腎臟制作4種臟器培養(yǎng)基，同時取肌肉和皮下脂肪組織，按照脂肪含量0%，10%，30%，50%和80%的質(zhì)量比重，分別制作5種混合食物培養(yǎng)基；在25℃恒溫條件下，以不同培養(yǎng)基飼養(yǎng)大頭金蠅初孵幼蟲，幼蟲孵化16h開始每12h取樣測量幼蟲體長和體重，化蛹后取樣測蛹長及蛹重，每次取樣10只，同時觀察并記錄不同階段的樣本數(shù)量，計算發(fā)育歷期，羽化完畢后計算各組幼蟲及蛹的死亡率和成蟲性別比率，實驗重復5次統(tǒng)計各組之 間的差異。 結(jié)果不同臟器組織對大頭金蠅幼蟲、蛹及成蟲的體長、體重產(chǎn)生明顯影響（P0.01），與其他3組比較取食肝組織的幼蟲生長緩慢 （P0.01），達到最大體長和體重的時間延遲36h，其2齡、3齡幼蟲期及總發(fā)育歷期明顯大于其他3組（P0.01）；食物脂肪含量對大頭金蠅幼蟲、蛹及成蟲的體長、體重產(chǎn)生明顯影響（P0.01），高脂肪組織食物早期可促進幼蟲生長，孵化40h后則抑制其生長并使發(fā)育歷期縮短（P0.01），，幼蟲及蛹的死亡率增高（P0.01）；不同組織源食物對成蟲的性別比率無明顯影響（P＞0.03）。 結(jié)論食物成分的不同會對大頭金蠅個體大小、生長發(fā)育歷期及死亡率可產(chǎn)生明顯影響，在昆蟲學相關(guān)研究及法醫(yī)學檢案的PMI推斷時應引起足夠重視。 第三部分：固相微萃取氣相色譜質(zhì)譜法測定大頭金蠅樣本內(nèi)的氯氮平 目的建立嗜尸性蠅類樣本內(nèi)氯氮平（clozapine, CLP）的有效定量檢測方法，為此類藥物中毒死亡案件的法醫(yī)學檢驗分析提供幫助。 方法采用固相微萃取結(jié)合氣相色譜質(zhì)譜聯(lián)用（SPME-GC-MS）的方法，以100m聚二甲基硅氧烷萃取頭萃取，洛沙平為內(nèi)標，氣相色譜質(zhì)譜選擇離子方式檢測，建立標準曲線，定量檢測大頭金蠅幼蟲及蛹樣本內(nèi)的CLP；分析不同發(fā)育時期，取食含有不同濃度CLP的肌肉組織的樣本內(nèi)CLP含量，比較食物與樣本體內(nèi)CLP濃度間的關(guān)系。 結(jié)果實驗建立的SPME-GC-MS法能準確定量檢測昆蟲樣本內(nèi)的氯氮平，CLP的檢出限為0.1ng/mL，在5～5000ng/mL范圍內(nèi)線性良好，不同濃度的回收率在93%～104%間，日內(nèi)及日間精密度（RSD）均小于8%；食物與樣本體內(nèi)CLP濃度間存在明顯相關(guān)性。 結(jié)論SPME-GC-MS法可用于大頭金蠅樣本體內(nèi)CLP的檢測，該方法簡便快捷，精密度好，結(jié)果可靠，可用于此類中毒案件中的法醫(yī)學分析。
[Abstract]:The first part: the application of the COI gene sequence in the mitochondrial DNA in the identification of common cadaver species
Objective to identify species of DNA and 841bp genes in mitochondrial DNA (mtDNA) cytochrome oxidase coenzyme I (COI), and to solve the problem of identifying the species and species in the field based on morphological methods, so as to provide help for forensic field investigation.
Methods collected from different areas of the 2 families, 4 genera and 6 species of sarcosaphagous flies samples, the samples of morphological examination by the professionals, were extracted from the samples mtDNA, different PCR sequences were amplified by using two pairs of primers of COI gene sequences of the PCR products by agarose gel electrophoresis, PCR Gel Extraction Kit, ABI377 sequencing, the sequence with DNAMAN6.0 sequence analysis software 498bp and 841bp were cut in length, respectively, into GenBank BLAST to retrieve the homology comparison with MEGA5.1 software for sequence analysis and phylogenetic tree analysis of different regions and different species of sample identification results.
Results 5 of 6 necrophagous flies 30 samples, COI gene sequence of mtDNA I (498bp) BLAST species homology comparison of 28 correct, correct matching rate reached 93.33%, a mean evolutionary divergence between 0.1%~1.6%, interspecific divergences were between 2.2%~11.2%, in addition to Lucilia sericata and l.cuprina 2 related species are difficult to distinguish,
The other 4 species were able to clearly distinguish 36 samples of 6 kinds of autopsy flies in.3 area by phylogenetic tree. The sequence of mtDNA COI gene sequence II (841bp) BLAST was 35, and the correct rate was 97.22%.
Between 0.2%~1.5%, the evolutionary divergence of interspecies is between 2.7%~10.6%, and the 6 species can be clearly distinguished by phylogenetic tree.
Conclusion the identification method of COI gene homology and phylogenetic tree is an important supplementary method for morphological examination of the body flies, and plays an important role in the rapid and accurate identification of the species of the body flies.
The second part: the effect of different food ingredients on the growth and development of the big head golden fly
Objective To observe the effects of different viscera and fat content on the growth and development of golden fly, and provide theoretical evidence for forensic inference of Post mortem interval (PMI).
Methods the porcine brain, heart, liver and kidney of 4 organ culture medium, at the same time, muscle and subcutaneous adipose tissue, in accordance with the fat content of 0%, 10%, 30%, 50% and 80% of the proportion of quality, are making 5 kinds of mixed food medium; at 25 DEG C under the condition of constant temperature, in different medium feeding head megacephala larva, larva of 16h 12h larvae begin each sampling to measure the body length and body weight, after pupation sampling for pupal length and pupal weight, while only 10 per sample, observe and record the number of samples in different stages, calculate the developmental period, after eclosion and adult sex ratio was calculated the mortality rate of larvae and pupae the experiment was repeated 5 times, each of the statistics
Differences between each other.
Results different organs and organs had significant effects on body length and body weight of the larvae, pupae and adults. (P0.01), compared with the other 3 groups, larvae fed on liver tissue grew slowly.
(P0.01), the maximum body length and body weight of the time delay of 36h, the age of 2, 3 instar larvae and total growth period was significantly higher than the other 3 groups (P0.01); the fat content of food of c.megacephala larva, pupa and adult body length, affect the body weight (P0.01), high fat group the fabric can promote food early larval growth after hatching, 40H inhibited their growth and development duration was shortened (P0.01), increased the mortality of larvae and pupae (P0.01); no significant effect of sex ratio of different tissue derived food for adults (P > 0.03).
Conclusion the difference of food composition can significantly affect the individual size, growth duration, and mortality of golden fly. We should pay enough attention to PMI inference in entomology related research and forensic examination.
The third part: the determination of clozapine in the sample of the fly by solid phase microextraction gas chromatography-mass spectrometry
Objective to establish an effective quantitative detection method for clozapine (clozapine, CLP) in specimens of the corpse fly, so as to provide help for forensic analysis of death cases.
Using the method of solid-phase microextraction combined with gas chromatography-mass spectrometry (SPME-GC-MS) method, 100m two polydimethylsiloxane SPME extraction, loxapine as internal standard, mass spectrometry detection of gas chromatography, establish the standard curve, quantitative detection of c.megacephala larvae and pupae samples in different developmental stages of CLP analysis; the content of CLP, feeding with different concentrations of CLP muscle tissue samples, relationship between food and the concentration of CLP in the sample room.
The results of SPME-GC-MS were established by accurate quantitative detection of clozapine in insect samples, the detection limit of CLP was 0.1ng/mL, 5 ~ 5000ng/mL in the linear range, the recovery rate in different concentrations of 93% to 104%, the intra day and inter day (RSD) is less than 8%; there was a significant correlation between food samples and in vivo CLP concentration.
Conclusion the SPME-GC-MS method can be used for the detection of CLP in the samples of the golden fly. The method is simple, quick, accurate and reliable. It can be used for forensic analysis of such poisoning cases.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：D919.1

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