High-throughput and Efficient Assay for Central Nervous System Infection with Targeted Nanopore Sequencing Technology

Ye Shi,1,2 Zhongdong Lin,3 Zhanguo Chen,4 Chuyuan Ye,3 Jian Yu,4 Jianan Xi,3 Yaya Geng,1,2 Man Zou,1 Haitao Ren,1,2 Lanni Wang,1,2 Bing Wang,1,2 Feng Xu,1,2 Xiaoqun Zheng,1,2 Guangxin Xiang1,2 1School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 2Key Laboratory of Laboratory Medicine, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 3Department of Neurology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 4Department of Clinical Laboratory, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of ChinaCorrespondence: Guangxin Xiang, Email [email protected] Xiaoqun Zheng, Email [email protected]: Central nervous system (CNS) infections represent a significant global public health concern and are characterized by high morbidity and mortality rates. In this study, we developed an integrated diagnostic approach for CNS infections by combining high-throughput nanopore sequencing with multiplex PCR amplification, designated targeted nanopore sequencing (tNPS).Methods: The tNPS assay employed a dual detection strategy incorporating pathogen-specific primers targeting 17 prevalent CNS pathogens (seven bacteria, one fungus and nine DNA viruses), with universal primers for the comprehensive amplification of full-length 16S ribosomal RNA (16S rRNA) and internal transcribed spacer (ITS) regions.Results: Analytical validation of tNPS was successfully carried out using the 12 positive reference strains (seven bacteria, one fungus, and four DNA viruses) individually, the ZymoBIOMICS microbial community (eight bacteria and two fungi), the laboratory synthetic community of bacteria and fungi (seven bacteria and one fungus), and the laboratory synthetic community of viruses (five DNA viruses). With accelerated turnaround time within 8 h, the tNPS also assayed 11 clinical cerebrospinal fluid (CSF) samples, which further confirmed the feasibility of precise identification of CNS pathogens compared to CSF culture and metagenomic next-generation sequencing.Discussion: Our tNPS as a culture-independent diagnostic assay offered enhanced efficiency, high-throughput capability, and an expanded pathogen detection spectrum, facilitating potential implementation in molecular diagnosis of CNS infection.Keywords: central nervous system infection, cerebrospinal fluid, assay development, nanopore sequencing, targeted sequencing, multiplex PCR