A Preliminary Study Of Single-cell Sequencing-based Technology In The Pathogen Detection

In recent years, new infectious diseases outbreak in succession, such as severe acute respiratory syndrome (SARS), the epidemic of H7N9avian influenza and so on. These diseases seriously threaten the people’s health, sustainable development of social economic and national security, and bring severe challenges to the control of infectious diseases. The pathogen detection is a important part of infectious disease control, and more and more new technologies have applied in it. For example, the next-generation of high-throughput sequencing technology has made remarkable achievements in the actual work. But there are still a few of shortcomings and active demands in the detection of a single infection cell and a single pathogen. At present, the booming development of single-cell sequencing-based technology brings the dawn to these problems. Single-cell sequencing-based technology was evaluated as one of the six notable realms of science by Science in2013. Now this technology is concentrated mainly on comparative genomics between single tumor cells or haploid cells, but there are few research reports in the area of pathogen detection.Based on the pathogen detection requirements of infectious diseases, this research aims at preliminary exploring the application of single-cell sequencing-based technology in the area of pathogen detection by using the single-cell sequencing-based technology and the next-generation of high-throughput sequencing technology, and the starting point is the high-throughput detection of viruses in a single cell that infected by the virus.The original materials of the research are rhabdomyosarcoma cells infected by coxsackievirus A16which isolated by our own lab. We got the single cells by using laser capture microdissection. After that we used random reverse transcription PCR to amplify the viral genome. Finally, we carried on high-throughput sequencing analysis with the help of Ion Torrent ion semiconductor sequencing technology. As a result, we received42.25%total genomic coverage. The genomic coverages of sequences we got in the region of5’UTR, VP4, VP1,2A,2C and3C were all more than60%, and we also discovered eight nucleotide mutations. We could identified the infected pathogen and its type with these results. Thereby, we preliminary explored the application of single-cell sequencing-based technology in the area of pathogen detection. Furthermore, this research also studied the inhibition of enterovirus71replication by7-hydroxyflavone (HF) and diisopropyl-flavon7-yl phosphate (FIP). We found that both HF and FIP had strong effects of inhibition to the replication of enterovirus71by molecular simulation, the inhibition assay of protease activity in vitro, antiviral assay and so on. These discoveries would contribute to the research of anti-enterovirus71drugs.