嗜尸性蝇类的分子标记种属鉴别与法医学应用研究
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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（P<0.01），与其他3组比较取食肝组织的幼虫生长缓慢
（P<0.01），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（P<0.01）；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（P<0.01），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（P<0.01），幼虫及蛹的死亡率增高（P<0.01）；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。
PART I: Application of mtDNA cytochrome oxidase subunit I gene inspecies identification between common sarcosaphagous flies
Objective To identify species of sarcosaphagous flies by analysisbased on498bp and841bp region of the gene for cytochrome oxidasesubunit I (COI) encoding region of mtDNA and provide assistance for theforensic science application.
Methods Sarcosaphagous fly samples in different regions belongingto2families,4genera and6species were collected for morphologicalindentify. MtDNA was extracted and the PCR amplification reaction wasoptimized by two pairs of primers in COI gene. The PCR products wereobtained and purified through agar gel electrophoresis and sequenced onABI Model377DNA sequencers. Sequences of498bp and841bp in COIgene were acquired by multiple-alignment DNAMAN (version6.0) andthe sequence alignments were disposed. BLAST search and homologyalignment was carried out in the GenBank database. Phylogenetic treeswere constructed by the MEGA5.1package.
Results28of30sarcosaphagous fly samples collected from5different regions were identified in correct alignment rate of93.33%by
sequence I (489bp) of COI gene. The divergence of sequence I within andbetween species was0.1%～1.6%and2.2%～11.2%respectively.Different species can be identified successfully except2samples of luciliacuprina (Wiedemann) and lucilia sericata (Meigen).35of36sarcosaphagous fly samples collected from3diffrent regions wereidentified in correct alignment rate of97.22%in sequence II (841bp) ofCOI gene. The divergence of sequence II within and between species was0.2%～1.5%and2.7%～10.6%respectively. All the different species canbe identified successfully by the phylogenetic trees.
Conclusion Methods of sequence homology alignment andphylogenetic tree of COI gene was an important supplementary means inthe species identification of sarcosaphagous flies, which realized the rapid,accurate and easy identification in forensic science application.
PART II: Effect of feeding on different tissues on larva developmentof Chrysomya megacephala (Diptera: Calliphoridae)
Objective To observe the effect of dietary factors on the developmentof Chrysomya megacephala larvae and provide assistance for the entomology research and estimating of post mortem interval (PMI) inforensic science application.
Methods The pig’s brain, heart, liver and kidney were collected tomake different substrates for the subsequent experiment. Fresh pure leanpork and subcutaneous fat was minced and mixed for5groups substratesand the proportion of fat in the mixture was0%,10%,30%,50%,80%byweight. The Chrysomya megacephala larvae were reared on the differentsubstrates at a constant temperature of25℃. Length and weight of larvaeand pupae were measured at12h interval16h after eclosion.10larvae orpupae were collected each time. The time of development, mortality, andsex ratio of adults were recorded for statistical analysis of the5replicatedexperiments.
Results Variation in the type of tissue that larvae feed on canproduce marked differences in developmental rate (P<0.01). The larvaefed on liver grew slowly and the time of reaching maximum length andweight was delayed for about36hours compared with other groups(P<0.01). Duration of second instar, third instar and total larvadevelopment was longer than that of other groups (P<0.01). More dietaryfat content was beneficial for development of larvae in the early stage butit was adverse in the later larval stages (P<0.01). Larvae fed on high-fatdiet reached the wandering stage earlier and the mortality of larvae andpupae was also hiher (P<0.01). There was no significant difference in the sex ratio between the four groups (P﹥0.05).
Conclusion The body size, development duration and mortality of
Chrysomya megacephala were different due to feed on the different tissuesor diets. This results should be considered in the entomology experimentsand forensic science application.
PART III: Determination of clozapine in samples of Chrysomyamegacephala by SPME-GC-MS method
Objective To explore and establish effective and reliably quantitativedetection methods of clozapine in samples of necrophagous flies so as tooffer a new detection methods and forensic medical analysis strategies forthis kind of poisoning cases.
Methods Method including standard curve of Solid-phasemicroextraction (SPME)-Gas Chromatography-Mass Spectrometry (GC-MS) was established to determine clozapine concentration in samples.Clozapine was extracted by100m polydimethylsiloxane (PDMS) fiber.Loxapine was used as internal standard and detected by GC-MS inselected ion monitoring (SIM). Samples of Chrysomya megacephala fedon different diet substrate which added a certain amount of clozapine wasdetected and statistical analyzed at different developmental stages including larvae and pupae.
Results The analytical results showed that SPME-GC-MS can beused in the samples detection and the detection limit of clozapine was0.1ng/mL. The standard curve was linear in the range of5～5000ng/mL. Theaverage recovery of clozapine in the range of93%～104%in differentconcentration. The precisions (RSD) of within-day and between-day wasless than8%. The concentration of CLP in samples was related with theconcentration of diet which Chrysomya megacephala fed on.
Conclusion This method was validated with respect to simplicity,accuracy and precision and provided a new potential effective detectionstrategies. It can be used for in necrophagous flies samples in this kind oftoxic poisoning cases.
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（P<0.01），与其他3组比较取食肝组织的幼虫生长缓慢
（P<0.01），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（P<0.01）；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（P<0.01），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（P<0.01），幼虫及蛹的死亡率增高（P<0.01）；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。
PART I: Application of mtDNA cytochrome oxidase subunit I gene inspecies identification between common sarcosaphagous flies
Objective To identify species of sarcosaphagous flies by analysisbased on498bp and841bp region of the gene for cytochrome oxidasesubunit I (COI) encoding region of mtDNA and provide assistance for theforensic science application.
Methods Sarcosaphagous fly samples in different regions belongingto2families,4genera and6species were collected for morphologicalindentify. MtDNA was extracted and the PCR amplification reaction wasoptimized by two pairs of primers in COI gene. The PCR products wereobtained and purified through agar gel electrophoresis and sequenced onABI Model377DNA sequencers. Sequences of498bp and841bp in COIgene were acquired by multiple-alignment DNAMAN (version6.0) andthe sequence alignments were disposed. BLAST search and homologyalignment was carried out in the GenBank database. Phylogenetic treeswere constructed by the MEGA5.1package.
Results28of30sarcosaphagous fly samples collected from5different regions were identified in correct alignment rate of93.33%by
sequence I (489bp) of COI gene. The divergence of sequence I within andbetween species was0.1%～1.6%and2.2%～11.2%respectively.Different species can be identified successfully except2samples of luciliacuprina (Wiedemann) and lucilia sericata (Meigen).35of36sarcosaphagous fly samples collected from3diffrent regions wereidentified in correct alignment rate of97.22%in sequence II (841bp) ofCOI gene. The divergence of sequence II within and between species was0.2%～1.5%and2.7%～10.6%respectively. All the different species canbe identified successfully by the phylogenetic trees.
Conclusion Methods of sequence homology alignment andphylogenetic tree of COI gene was an important supplementary means inthe species identification of sarcosaphagous flies, which realized the rapid,accurate and easy identification in forensic science application.
PART II: Effect of feeding on different tissues on larva developmentof Chrysomya megacephala (Diptera: Calliphoridae)
Objective To observe the effect of dietary factors on the developmentof Chrysomya megacephala larvae and provide assistance for the entomology research and estimating of post mortem interval (PMI) inforensic science application.
Methods The pig’s brain, heart, liver and kidney were collected tomake different substrates for the subsequent experiment. Fresh pure leanpork and subcutaneous fat was minced and mixed for5groups substratesand the proportion of fat in the mixture was0%,10%,30%,50%,80%byweight. The Chrysomya megacephala larvae were reared on the differentsubstrates at a constant temperature of25℃. Length and weight of larvaeand pupae were measured at12h interval16h after eclosion.10larvae orpupae were collected each time. The time of development, mortality, andsex ratio of adults were recorded for statistical analysis of the5replicatedexperiments.
Results Variation in the type of tissue that larvae feed on canproduce marked differences in developmental rate (P<0.01). The larvaefed on liver grew slowly and the time of reaching maximum length andweight was delayed for about36hours compared with other groups(P<0.01). Duration of second instar, third instar and total larvadevelopment was longer than that of other groups (P<0.01). More dietaryfat content was beneficial for development of larvae in the early stage butit was adverse in the later larval stages (P<0.01). Larvae fed on high-fatdiet reached the wandering stage earlier and the mortality of larvae andpupae was also hiher (P<0.01). There was no significant difference in the sex ratio between the four groups (P﹥0.05).
Conclusion The body size, development duration and mortality of
Chrysomya megacephala were different due to feed on the different tissuesor diets. This results should be considered in the entomology experimentsand forensic science application.
PART III: Determination of clozapine in samples of Chrysomyamegacephala by SPME-GC-MS method
Objective To explore and establish effective and reliably quantitativedetection methods of clozapine in samples of necrophagous flies so as tooffer a new detection methods and forensic medical analysis strategies forthis kind of poisoning cases.
Methods Method including standard curve of Solid-phasemicroextraction (SPME)-Gas Chromatography-Mass Spectrometry (GC-MS) was established to determine clozapine concentration in samples.Clozapine was extracted by100m polydimethylsiloxane (PDMS) fiber.Loxapine was used as internal standard and detected by GC-MS inselected ion monitoring (SIM). Samples of Chrysomya megacephala fedon different diet substrate which added a certain amount of clozapine wasdetected and statistical analyzed at different developmental stages including larvae and pupae.
Results The analytical results showed that SPME-GC-MS can beused in the samples detection and the detection limit of clozapine was0.1ng/mL. The standard curve was linear in the range of5～5000ng/mL. Theaverage recovery of clozapine in the range of93%～104%in differentconcentration. The precisions (RSD) of within-day and between-day wasless than8%. The concentration of CLP in samples was related with theconcentration of diet which Chrysomya megacephala fed on.
Conclusion This method was validated with respect to simplicity,accuracy and precision and provided a new potential effective detectionstrategies. It can be used for in necrophagous flies samples in this kind oftoxic poisoning cases.
引文
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