%0 Journal Article %A LI Dong-mei %A LI Qing-duo %A LI Shou-jiang %A LIU Qi-yong %A LU Liang %A RAO Hua-xiang %A SONG Xiu-ping %A ZHOU Ruo-bing %T Real-time detection of rodent-borne Bartonella by nanopore sequencing %D 2021 %R 10.11853/j.issn.1003.8280.2021.04.002 %J Chinese Journal of Vector Biology and Control %P 390-397 %V 32 %N 4 %X Objective To evaluate a real-time detection technology for rodent-borne pathogens based on the MinION high-throughput nanopore sequencing platform with Bartonella spp. as an indicator. Methods The whole-genome DNA was extracted from the spleen and lung tissues of wild rodents with positive Bartonella culture results. Sanger sequencing following conventional PCR amplification (using universal primers of 16S ribosomal RNA [16S rRNA] gene) and nanopore sequencing was performed and compared, and then evaluate the feasibility and accuracy of MinION nanopore sequencing technology in the detection of rodent-borne pathogens. Results By using the first-generation Sanger sequencing after the conventional 16S rRNA gene amplification, Bartonella was not identified in 12 samples. In contrast by using the third-generation nanopore sequencing, Bartonella was identified in all samples. The number of reads per sample used for taxonomic analysis ranged from 4 to 609 424, and the reads length was mainly 1 500 bp, with the mean accuracies ranging from 79.2% to 92.0%. Bartonella was the main pathogen detected, and its number of reads ranged from 1 to 77 833. By using cloud real-time analysis software EPI2ME, the identification results were generated within 30 minutes after the sequencing was started. In addition, Brucella spp. was also identified in some samples. The concentration of the original samples had an impact on the amount of sequencing data, with evidence showing that low-concentration samples had significantly less total data volume and number of reads. The cleaning procedure significantly reduced the residual nucleic acid fragments in the sequencing chip, but could not completely remove them, with a 1.40% residual ratio. Conclusion Nanopore sequencing of 16S rRNA gene amplicons can be used for direct and real-time detection and identification of pathogens in organs and tissues from rodents. Nanopore MinION sequencing provides convenience for on-site detection and pathogen monitoring with its portable and simple operation, and real-time data transmission and analysis. %U http://www.bmsw.net.cn/EN/10.11853/j.issn.1003.8280.2021.04.002