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首页> 外文期刊>Forensic science international >Detection of diverse aquatic microbes in blood and organs of drowning victims: First metagenomic approach using high-throughput 454-pyrosequencing
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Detection of diverse aquatic microbes in blood and organs of drowning victims: First metagenomic approach using high-throughput 454-pyrosequencing

机译：溺水受害者血液和器官中各种水生微生物的检测：采用高通量454焦磷酸测序的第一种宏基因组学方法

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Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims.
机译：当前的454焦磷酸测序技术可实现大规模并行测序。我们使用这项技术调查了两个溺水受害者的14个标本（血液和器官）以及从发现地点采集的两个水样中水生微生物的多样性。微生物的16S核糖体RNA（rRNA）基因通常用于鉴定物种（或属），具有9个高度可变的区域（V1-V9），每个区域均被保守区域包围。保守区域内的某些部分在微生物域上很常见，例如在细菌和藻类之间（藻类叶绿体基因组上的16S rRNA基因）。因此，我们使用基于保守区的定制设计引物，通过PCR同时扩增了溺水受害者血液和器官中各种微生物的目标区域（V7和V8）。然后，我们使用基因组测序仪FLX Titanium系统（Roche-454 Life Sciences）通过焦磷酸测序详尽地分析了PCR产物。该方法在发现地点的受害人血液和器官以及水中发现了包括蓝藻和藻类在内的多种细菌，包括杆菌科（硅藻），隐藻科，Dictyochophyceae，Chrysophyceae和Trebouxiophyceae。我们的数据进一步表明，当常规的硅藻肺检法不能提供足够的水吸入证据时，通过454焦磷酸测序法检测各种外源微生物对于支持溺水死亡的结论非常有用。据我们所知，这是首次尝试使用新一代测序仪来调查溺水受害者的血液和封闭器官中的各种水生微生物。

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