Journal Article Serotyping dengue virus with isothermal amplification and a portable sequencer

Yamagishi, Junya  ,  Runtuwene, Lucky R.  ,  Hayashida, Kyoko  ,  Mongan, Arthur E.  ,  Lan Anh Nguyen Thi  ,  Linh Nguyen Thuy  ,  Cam Nguyen Nhat  ,  Limkittikul, Kriengsak  ,  Sirivichayakul, Chukiat  ,  Sathirapongsasuti, Nuankanya  ,  Frith, Martin  ,  Makalowski, Wojciech  ,  Eshita, Yuki  ,  Sugano, Sumio  ,  Suzuki, Yutaka

7p.3510 , 2017-06-14 , Nature Publishing Group
The recent development of a nanopore-type portable DNA sequencer has changed the way we think about DNA sequencing. We can perform sequencing directly in the field, where we collect the samples. Here, we report the development of a novel method to detect and genotype tropical disease pathogens, using dengue fever as a model. By combining the sequencer with isothermal amplification that only requires a water bath, we were able to amplify and sequence target viral genomes with ease. Starting from a serum sample, the entire procedure could be finished in a single day. The analysis of blood samples collected from 141 Indonesian patients demonstrated that this method enables the clinical identification and serotyping of the dengue virus with high sensitivity and specificity. The overall successful detection rate was 79%, and a total of 58 SNVs were detected. Similar analyses were conducted on 80 Vietnamese and 12 Thai samples with similar performance. Based on the obtained sequence information, we demonstrated that this approach is able to produce indispensable information for etiologically analyzing annual or regional diversifications of the pathogens.

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