A multicopy transposase-targeted qPCR assay for highly sensitive diagnosis of scrub typhus

Objectives: Scrub typhus, caused by the bacterium Orientia tsutsugamushi, is frequently underdiagnosed due to its non-specific clinical presentation and the frequent absence of eschar. Most molecular diagnostic assays target single-copy genes of O. tsutsugamushi, which can limit diagnostic sensitivity. We aimed to develop an ultra-sensitive quantitative PCR (qPCR) assay targeting a highly repetitive element in O. tsutsugamushi genome. Methodology: We developed a SYBR Green-based qPCR assay (TranScrub) targeting a multicopy transposase gene of O. tsutsugamushi and compared its performance with assays targeting the 56kDa (single-copy) and traD (multicopy) genes. Diagnostic performance was evaluated using clinical specimens and a panel of blood-borne pathogens. The limit of detection (LOD) was estimated using serial dilutions of quantified template. The assay was further applied to dried blood spot (DBS) samples from patients with acute febrile illness of unknown aetiology, with positives confirmed by Oxford Nanopore amplicon sequencing. Results: Targeting the multicopy transposase gene enabled highly sensitive detection of O. tsutsugamushi, outperforming the conventional 56-kDa assay and matching the traD assay. TranScrub achieved a 91% sensitivity (29/32) and 100% specificity (77/77) using blood-derived DNA, with no cross-reactivity. The LOD was 0.024 genome equivalents/μL. Among 81 DBS samples from acute febrile patients of unknown aetiology, 6 (7.5%) tested positive, all confirmed by sequencing. Conclusions: The transposase gene represents a novel target that improves molecular detection of scrub typhus. TranScrub enables sensitive and specific detection from both blood and DBS, supporting its use in clinical diagnosis and field surveillance.