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二代测序技术检测宏基因组18s rDNA V4用于溺死检验的研究

发表时间:2018-03-15  浏览量:1208  下载量:256
全部作者: 陈晓刚,张霁,杜吉佩,赵苑村,赵小红,侯一平
作者单位: 四川大学华西基础医学与法医学院
摘 要: 目的:探索通过使用二代测序结合DNA条形码技术检测溺死实验家兔样品及水样中浮游生物的种属信息进行溺水诊断的新方法。方法:在不同河流、湖泊取样,percoll分离水样及溺死实验家兔肺脏和心血样品中的浮游生物。抽提DNA后分别用D534f/D729r引物对PCR扩增以及D29f/D1198r联合D534f/D729r引物对巢氏PCR扩增;扩增产物二代测序(Illumina MiSeq平台)。测序数据用MOTHUR软件包进行生物信息学分析后,进行相似性分析(analysis of similarity,ANOSIM)、主成分分析(principal component analysis,PCA)和现代相似性技术(modern analog technique,MAT)分析。结果:溺死家兔肺脏内浮游生物DNA用D534f/D729r引物对PCR扩增,二代测序检出的优势真核浮游生物在取样点水样中检出。肺脏样品中检出的真核浮游生物信息能反映淹溺水样中真核浮游生物构成的差异。溺死家兔心血中收集的浮游生物量少,DNA经巢氏PCR扩增,二代测序检出的优势原生生物、植物界物种均为淹溺水样中的物种。因检出物种少,无法进行心血样品与水样一致性的分析。结论:本研究建立的方法能成功检测出溺死过程中进入实验动物体内的真核浮游生物的种属信息,并据此对溺死动物样品与淹溺水样之间的关系进行分析判定。
关 键 词: 法医学;真核浮游生物;二代测序;18s核糖体DNA第4高变区(18s rDNA V4);溺死
Title: Study on the application of 18s rDNA V4 metagenomic next-generation sequencing in the diagnosis of drowning
Author: CHEN Xiaogang, ZHANG Ji, DU Jipei, ZHAO Yuancun, ZHAO Xiaohong, HOU Yiping
Organization: West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University
Abstract: Objective: The species information on assemblage of a variety of planktons mixed in specimens taken from drowned body will be valuable to the diagnosis of drowning and inference of drowning place. This study aimed to investigate whether next-generation sequencing (NGS) can support and enhance the rapid identification of eukaryotic planktons that was aspired during drowning. Methods: The specimens were taken from two rivers and a fluvial lake. The lungs and blood specimens were collected from the drowned experimental rabbits or rabbits immersed after death. Planktons in lungs and blood specimens were isolated with percoll. Genomic DNA from small amount of planktons was subjected to either one round of PCR amplification with primer pair D534f/D729r or two rounds of PCR amplification with primer pairs D29f/D1198r and D534f/D729r. NGS was proceeded on the Illumina MiSeq platform. The FASTQ files were then processed using MOTHUR software package. The analysis of similarity (ANOSIM), principal component analysis (PCA) and modern analog technique (MAT) analysis were performed. Results: The genera of dominant eukaryotic planktons detected by NGS in the lung specimens from the drowned rabbits after one round of PCR amplification with primer pairs D534f/D729r were found in the water specimen from the same site. The assemblages of plankton collecting from lungs of the drowned rabbits were correlated with that of corresponding water specimens. The genera of planktons detected in the blood specimens from the drowned rabbits after two rounds of PCR amplification with primer pairs D29f/D1198r and D534f/D729r were quite a few and insufficient to conduct statistical analysis. Within the genera detected in the blood specimens, the dominant protists and plantae species detected in NGS were found in the water specimen form the same site. Conclusion: Using the method in our study, the assemblages of planktons collected from the drowned rabbits in our animal test could be detected. Based on the analysis of the results, the relationship of the specimens from the drowned animal and the water could be decided.
Key words: forensic science; eukaryotic plankton; next-generation sequencing; 18s rDNA V4; drowning
发表期数: 2018年3月第5期
引用格式: 陈晓刚,张霁,杜吉佩,等. 二代测序技术检测宏基因组18s rDNA V4用于溺死检验的研究[J]. 中国科技论文在线精品论文,2018,11(5):457-466.
 
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