Gong Liang, Huang Yao-Ting, Wong Chee-Hong, Chao Wen-Cheng, Wu Zong-Yen, Wei Chia-Lin, Liu Po-Yu
Genome Technologies, The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, United States of America.
National Chung Cheng University, Department of Computer Science and Information Engineering, Chia-Yi, Taiwan.
PLoS One. 2018 Jan 9;13(1):e0190853. doi: 10.1371/journal.pone.0190853. eCollection 2018.
The identification of microbial species has depended predominantly upon culture-based techniques. However, the difficulty with which types of organisms are cultured implies that the grown species may be overrepresented by both cultivation and plate counts. In recent years, culture-independent analysis using high-throughput sequencing has been advocated for use as a point-of-care diagnostic tool. Although it offers a rapid and unbiased survey to characterize the pathogens in clinical specimens, its accuracy is reduced by the high level of contamination of human DNA. In this paper, we propose using a culture-independent analysis for a Klebsiella pneumoniae clinical strain within a liver abscess using nanopore sequencing. Owing to the highly-contaminated cell population within a liver abscess, we managed to reduce the confounding effects of human DNA through the use of DNase and differential centrifugation. Genomic DNA was sequenced through the use of Nanopore MinION sequencer and analyzed using a suite of bioinformatics approaches. K. pneumoniae was successfully identified along with antibiotic-resistant genes. Our results indicate that, by integrating real-time nanopore sequencing and bioinformatics software, real-time pathogen identification in a liver abscess can be achieved.
微生物物种的鉴定主要依赖于基于培养的技术。然而，培养生物体类型的困难意味着培养出的物种在培养和菌落计数中可能被过度代表。近年来，有人主张使用基于高通量测序的非培养分析作为即时诊断工具。尽管它能提供快速且无偏差的检测来鉴定临床标本中的病原体，但其准确性因人类DNA的高度污染而降低。在本文中，我们提议使用纳米孔测序对肝脓肿内的肺炎克雷伯菌临床菌株进行非培养分析。由于肝脓肿内细胞群体的高度污染，我们通过使用脱氧核糖核酸酶和差速离心法成功降低了人类DNA的干扰影响。通过使用纳米孔MinION测序仪对基因组DNA进行测序，并使用一套生物信息学方法进行分析。成功鉴定出肺炎克雷伯菌及其耐药基因。我们的结果表明，通过整合实时纳米孔测序和生物信息学软件，可以实现肝脓肿中病原体的实时鉴定。
