A field-deployable high-resolution SNP genotyping platform for rapid surveillance of Yersinia pestis

Yersinia pestis, the causative agent of plague, poses a persistent global health threat due to its rapid transmission and high mortality. To enable rapid, field-deployable surveillance, we developed a high-resolution, culture-independent genotyping assay for precise identification of Y. pestis lineages and sublineages. We curated and validated 25 canonical single nucleotide polymorphisms (canSNPs) to resolve 24 global lineages, along with 12 region-specific canSNPs distinguishing 9 predominant sublineages circulating in Inner Mongolia, China. By integrating ARMS-HANDS PCR with multicolor melting curve analysis, we established a hierarchical multiplex assay capable of simultaneously genotyping 37 canSNPs in three real-time PCR reactions. The system exhibited high sensitivity (detection limit: 50 genome copies per reaction) and achieved 100% accuracy in a double-blind evaluation of 166 Y. pestis strains. Incorporated into a portable "sample-in, result-out" device, the assay enabled direct lineage identification from infected gerbil liver samples within 120 min, accurately detecting the epidemiologically critical sublineage 2.MED3.1.4 in field-collected specimens. This robust and deployable genotyping platform addresses key limitations in current plague surveillance efforts, offering a transformative solution for real-time outbreak investigation and epidemiological tracking in resource-constrained settings.