Integrative assessment of species-level genetic markers for the diagnosis of pathogenic Leptospira.

BACKGROUND

Leptospiral isolation and whole-genome sequencing are often unfeasible for genotyping; therefore, single-locus culture-independent sequencing is widely used. However, primer sets remain broadly applied without comprehensive quantitative or qualitative performance evaluation.

OBJECTIVE

This study integrated in silico primer performance, phylogenetic benchmarking, and barcoding gap analysis to identify Leptospira markers that best delimit species, support diagnostics, and enable standardized BLAST identity cut-off thresholds.

METHODS

An integrative analysis evaluated 10 primer pairs targeting seven loci: secY inner, secY outer, lfb1, flaB, rpoB, gyrB, and lipL32. New primers for 16S rRNA and flaB were designed and included in the framework. Primer hybridization and theoretical amplification were assessed in silico in genomes of five pathogenic Leptospira species. The best-performing markers were then tested for congruence with core-genome and full-ppk phylogenies. Global and species-specific barcoding gap analyses assessed genetic distances, and BLAST identity cut-offs were proposed for markers performing consistently across all steps.

RESULTS

Only five markers showed good theoretical amplification performance: 16S rRNA, secY outer, flaB, rpoB, and lipL32. However, only the phylogenies generated from secY outer, redesigned flaB, and lipL32 were similar with core-genome and ppk trees. Barcoding analyses further showed that only secY outer and redesigned flaB could discriminate both species and intraspecific strains, with proposed BLAST identity cut-offs of 96 and 95%, respectively.

DISCUSSION: The present study extends barcoding gap analysis to bacterial marker evaluation within an integrative framework, an approach previously applied mainly to Metazoa. It also highlights an important limitation in the widespread use of genotyping markers that lack reliable in silico amplification performance and sufficient intra- and interspecific discriminatory power. Together, these findings support the combined use of secY outer and redesigned flaB for molecular surveillance of the main pathogenic leptospires, although further in vitro validation remains needed.