嗜尸性蝇类的分子标记种属鉴别与法医学应用研究

发布时间：2018-01-06 11:37

本文关键词：嗜尸性蝇类的分子标记种属鉴别与法医学应用研究　出处：《重庆医科大学》2013年博士论文　论文类型：学位论文

【摘要】：第一部分：线粒体DNA中COI基因序列在常见嗜尸性蝇类种属鉴别中的应用目的利用线粒体DNA（mtDNA）细胞色素氧化酶辅酶I（COI）中的498bp和841bp基因序列，对常见嗜尸性蝇类进行种属鉴别，解决依据形态学方法难以鉴别现场样本种属的难题，为法医现场检案提供帮助。方法收集不同地区的2科4属6个种的嗜尸性蝇类样本，经专业人员对样本进行形态学检验，分别提取样本mtDNA，利用COI基因序列中的两对引物进行不同序列PCR扩增，以琼脂糖电泳检测扩增产物，PCR胶回收试剂盒纯化，ABI377测序仪测序，将所得序列以DNAMAN6.0分析软件分别截取498bp和841bp长度的序列，分别输入GenBank进行BLAST检索同源相似性比对，以MEGA5.1软件进行序列分析并构建系统发育树，分析不同地区及不同种属的样本鉴别效果。结果5个地区6种嗜尸性蝇类30个样本，mtDNA的COI基因序列I（498bp）BLAST种属同源性正确比对28个，正确比对率达到93.33%，种内进化分歧均数在0.1%~1.6%之间，种间进化分歧均数在2.2%~11.2%之间，除丝光绿蝇与铜绿蝇2个近缘种属难以区分外，其余4个种属通过系统发育树可明确区分。3个地区6种嗜尸性蝇类36个样本，mtDNA的COI基因序列II（841bp）BLAST种属同源性正确比对35个，正确比对率达到97.22%，种内进化分歧均数在 0.2%~1.5%之间，种间进化分歧均数在2.7%~10.6%之间，6个种属通过系统发育树均可明确区分。结论COI基因序列同源性比对与系统发育树构建的种属鉴别方法，是嗜尸性蝇类形态学种属检验的重要补充手段，对嗜尸性蝇类样本种属的快速、准确鉴定有重要作用。第二部分：不同食物成分对大头金蝇生长发育的影响目的观察不同脏器及不同脂肪含量的肌肉组织对大头金蝇生长发育的影响，为昆虫学相关研究及死后间隔时间（Post mortem interval,PMI）的法医学推断提供理论依据。方法取猪的脑、心脏、肝脏及肾脏制作4种脏器培养基，同时取肌肉和皮下脂肪组织，按照脂肪含量0%，10%，30%，50%和80%的质量比重，分别制作5种混合食物培养基；在25℃恒温条件下，以不同培养基饲养大头金蝇初孵幼虫，幼虫孵化16h开始每12h取样测量幼虫体长和体重，化蛹后取样测蛹长及蛹重，每次取样10只，同时观察并记录不同阶段的样本数量，计算发育历期，羽化完毕后计算各组幼虫及蛹的死亡率和成虫性别比率，实验重复5次统计各组之间的差异。结果不同脏器组织对大头金蝇幼虫、蛹及成虫的体长、体重产生明显影响（P0.01），与其他3组比较取食肝组织的幼虫生长缓慢（P0.01），达到最大体长和体重的时间延迟36h，其2龄、3龄幼虫期及总发育历期明显大于其他3组（P0.01）；食物脂肪含量对大头金蝇幼虫、蛹及成虫的体长、体重产生明显影响（P0.01），高脂肪组织食物早期可促进幼虫生长，孵化40h后则抑制其生长并使发育历期缩短（P0.01），，幼虫及蛹的死亡率增高（P0.01）；不同组织源食物对成虫的性别比率无明显影响（P＞0.03）。结论食物成分的不同会对大头金蝇个体大小、生长发育历期及死亡率可产生明显影响，在昆虫学相关研究及法医学检案的PMI推断时应引起足够重视。第三部分：固相微萃取气相色谱质谱法测定大头金蝇样本内的氯氮平目的建立嗜尸性蝇类样本内氯氮平（clozapine, CLP）的有效定量检测方法，为此类药物中毒死亡案件的法医学检验分析提供帮助。方法采用固相微萃取结合气相色谱质谱联用（SPME-GC-MS）的方法，以100m聚二甲基硅氧烷萃取头萃取，洛沙平为内标，气相色谱质谱选择离子方式检测，建立标准曲线，定量检测大头金蝇幼虫及蛹样本内的CLP；分析不同发育时期，取食含有不同浓度CLP的肌肉组织的样本内CLP含量，比较食物与样本体内CLP浓度间的关系。结果实验建立的SPME-GC-MS法能准确定量检测昆虫样本内的氯氮平，CLP的检出限为0.1ng/mL，在5～5000ng/mL范围内线性良好，不同浓度的回收率在93%～104%间，日内及日间精密度（RSD）均小于8%；食物与样本体内CLP浓度间存在明显相关性。结论SPME-GC-MS法可用于大头金蝇样本体内CLP的检测，该方法简便快捷，精密度好，结果可靠，可用于此类中毒案件中的法医学分析。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：D919.1

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