biostudies-literatureSugi TJapan Agency for Medical Research and DevelopmentJapan Society for the Promotion of Science Core-to-Core program41249https://www.ebi.ac.uk/biostudies/studies/S-EPMC12639079biostudies-literatureUnknown15(1)Microscopic examination is commonly used for blood parasite detection in resource-limited settings due to its low cost and simplicity. However, it requires microscopy experts and has poor species-level identification. This study proposes a targeted next-generation sequencing (NGS) approach using a portable nanopore platform to enable accurate and sensitive parasite detection in such settings. To improve species identification on the error-prone nanopore sequencer, we designed a DNA barcoding strategy targeting the 18S rDNA V4-V9 region, which outperformed the commonly used V9 region. To enrich parasite DNA and reduce host contamination, two blocking primers were developed: a C3 spacer-modified oligo competing with the universal reverse primer and a peptide nucleic acid (PNA) oligo that inhScientific reportsEnhanced blood parasite species identification using V4-V9 18S rDNA barcoding by universal primers on a nanopore platform.PMC12639079JPJSCCB20230007JP24ym0126801JP20wm0125008Hayashida KGaithuma AKKawai NIshiguro NReteng PNamangala BSugi TMutengo MMYamagishi JfalseEnhanced blood parasite species identification using V4-V9 18S rDNA barcoding by universal primers on a nanopore platform.Microscopic examination is commonly used for blood parasite detection in resource-limited settings due to its low cost and simplicity. However, it requires microscopy experts and has poor species-level identification. This study proposes a targeted next-generation sequencing (NGS) approach using a portable nanopore platform to enable accurate and sensitive parasite detection in such settings. To improve species identification on the error-prone nanopore sequencer, we designed a DNA barcoding strategy targeting the 18S rDNA V4-V9 region, which outperformed the commonly used V9 region. To enrich parasite DNA and reduce host contamination, two blocking primers were developed: a C3 spacer-modified oligo competing with the universal reverse primer and a peptide nucleic acid (PNA) oligo that inh2025-01-01T00:00:00Z2025 Nov2026-06-05T17:19:45.74Z2026-05-19T03:11:49.434ZS-EPMC126390794127182610.1038/s41598-025-25108-y