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Reduction of Human DNA Contamination in Clinical Cerebrospinal Fluid Specimens Improves the Sensitivity of Metagenomic Next-Generation Sequencing.
Journal of Molecular Neuroscience ( IF 2.8 ) Pub Date : 2020-01-31 , DOI: 10.1007/s12031-019-01472-z
Xin-Chao Ji 1 , Lin-Fu Zhou 2 , Chao-Yang Li 1 , Ya-Jun Shi 1 , Meng-Li Wu 1 , Yun Zhang 1 , Xiao-Fei Fei 1 , Gang Zhao 1
Affiliation  

Metagenomics next-generation sequencing (mNGS) is increasingly available for the detection of obscure infectious diseases of the central nervous system. However, human DNA contamination from elevated white cells, one of the characteristic cerebrospinal fluid (CSF) features in meningitis patients, greatly reduces the sensitivity of mNGS in the pathogen detection. Currently, effective approaches to selectively reduce host DNA contamination from clinical CSF samples are still lacking. In this study, a total of 20 meningitis patients were enrolled, including 10 definitively diagnosed tuberculous meningitis (TBM) and 10 definite cryptococcal meningitis (CM) cases. To evaluate the effect of reduced human DNA in the sensitivity of mNGS detection, three specimen-processing protocols were performed: (i) To remove human DNA, saponin, a nonionic surfactant, was used to selectively lyse white cells in CSF followed by DNase treatment prior to the extraction of DNA; (ii) to reduce host DNA, CSF was centrifuged to remove human cells, and the supernatant was collected for DNA extraction; and (iii) DNA extraction from the unprocessed specimens was set as the control. We found that saponin processing significantly elevated the NGS unique reads for Cryptococcus (P < 0.01) compared with the control but had no effects for Mycobacterium tuberculosis (P > 0.05). However, detection of centrifuged supernatants improved the NGS unique reads for both TBM and CM compared with controls (P < 0.01). Our results demonstrate that the use of mNGS of centrifuged supernatants from clinical CSF samples in patients with TBM and CM is a simple and effective method to improve the sensitivity of pathogen detection.

中文翻译:

减少临床脑脊液标本中的人类DNA污染可提高下一代基因组测序的敏感性。

元基因组学下一代测序(mNGS)越来越多地用于检测中枢神经系统的隐匿性传染病。但是,脑膜炎患者脑脊液(CSF)的特征之一,即白细胞升高引起的人类DNA污染大大降低了mNGS在病原体检测中的敏感性。目前,仍然缺乏有效的方法来选择性减少临床CSF样品中的宿主DNA污染。在这项研究中,总共招募了20名脑膜炎患者,其中包括10例确诊的结核性脑膜炎(TBM)和10例明确的隐球菌性脑膜炎(CM)病例。为了评估减少的人类DNA对mNGS检测灵敏度的影响,进行了三种标本处理方案:(i)为了除去人类DNA,皂苷,一种非离子表面活性剂,在提取DNA之前,先用DNA裂解CSF中的白细胞,再用DNase处理。(ii)为了减少宿主DNA,将CSF离心以除去人细胞,并收集上清液用于DNA提取;(iii)从未处理的样品中提取DNA作为对照。我们发现皂苷加工显着提高了NGS的独特阅读隐球菌P  <0.01)与对照组相比,但对结核分枝杆菌没有影响(P  > 0.05)。然而,与对照相比,离心上清液的检测提高了TBM和CM的NGS独特读数(P  <0.01)。我们的结果表明,在TBM和CM患者中使用临床CSF样品的离心上清液的mNGS是提高病原体检测灵敏度的简单有效的方法。
更新日期:2020-01-31
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