Enhanced 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 inhibits polymerase elongation. These were combined to selectively reduce the amplification of host's DNA from blood samples. The developed targeted NGS test successfully detected Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Babesia bovis in human blood samples spiked with as few as 1, 4, and 4 parasites per microliter, respectively. Validation study using field cattle blood samples revealed that this test could detect multiple Theileria species co-infections in the same cattle. The established parasite targeted NGS test using a portable nanopore platform enables comprehensive parasite detection with high sensitivity and accurate species identification.