使用纳米孔测序的基于散弹枪宏基因组学的血流感染诊断的关键步骤。

IF 2.2 4区 医学 Q4 IMMUNOLOGY
Apmis Pub Date : 2025-01-14 DOI:10.1111/apm.13511
Amelia Björnberg, David Nestor, Nilay Peker, Bhanu Sinha, Natacha Couto, John Rossen, Martin Sundqvist, Paula Mölling
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引用次数: 0

摘要

霰弹枪宏基因组学为病原体的快速血流感染提供了广泛的病原体检测,但在临床样本中,病原体数量往往很低,而人类背景DNA含量却很高,这让人很头疼。本研究旨在开发一种散弹枪宏基因组学方案,使用加有各种细菌的血液,并评估细菌DNA提取效率与人类DNA消耗。血液病原体试剂盒(Molzym)用于从edta全血(WB)和血浆样本中提取DNA,通过牛津纳米孔测序和基于pcr的文库制备人工血液样本进行散弹枪宏基因组学诊断。结果显示,WB中细菌读数高于血浆。与大肠杆菌相比,金黄色葡萄球菌和肺炎链球菌的差异更为明显。血浆样品具有更好的方法重现性,对人类DNA的微滴数字PCR结果更加一致。研究发现,提取革兰氏阳性菌的效率高于革兰氏阴性菌,这表明人类DNA的消耗对革兰氏阴性菌产生了负面影响。总之,霰弹枪宏基因组学需要进一步优化,以提高细菌DNA的恢复和提高病原体检测的灵敏度。本研究强调了使用纳米孔测序的基于散弹枪宏基因组学的血流感染诊断方法中的一些关键步骤。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Critical Steps in Shotgun Metagenomics-Based Diagnosis of Bloodstream Infections Using Nanopore Sequencing

Critical Steps in Shotgun Metagenomics-Based Diagnosis of Bloodstream Infections Using Nanopore Sequencing

Shotgun metagenomics offers a broad detection of pathogens for rapid blood stream infection of pathogens but struggles with often low numbers of pathogens combined with high levels of human background DNA in clinical samples. This study aimed to develop a shotgun metagenomics protocol using blood spiked with various bacteria and to assess bacterial DNA extraction efficiency with human DNA depletion. The Blood Pathogen Kit (Molzym) was used to extract DNA from EDTA-whole blood (WB) and plasma samples, using contrived blood specimens spiked with bacteria for shotgun metagenomics diagnostics via Oxford Nanopore sequencing and PCR-based library preparation. Results showed that bacterial reads were higher in WB than plasma. Differences for Staphylococcus aureus and Streptococcus pneumoniae were more pronounced compared to Escherichia coli. Plasma samples exhibited better method reproducibility, with more consistent droplet digital PCR results for human DNA. The study found that extraction was more efficient for Gram-positive bacteria than Gram-negative, suggesting that the human DNA depletion exerts a negative effect on Gram-negative bacteria. Overall, shotgun metagenomics needs further optimisation to improve bacterial DNA recovery and enhance pathogen detection sensitivity. This study highlights some critical steps in the methodology of shotgun metagenomic-based diagnosis of blood stream infections using Nanopore sequencing.

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来源期刊
Apmis
Apmis 医学-病理学
CiteScore
5.20
自引率
0.00%
发文量
91
审稿时长
2 months
期刊介绍: APMIS, formerly Acta Pathologica, Microbiologica et Immunologica Scandinavica, has been published since 1924 by the Scandinavian Societies for Medical Microbiology and Pathology as a non-profit-making scientific journal.
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