Isolation of DNA aptamers by Tripartite-hybrid-high throughput SELEX (Tripartite-hybrid-HT SELEX) for the detection of pathogenic Leptospira

Leptospirosis is a zoonosis caused by the pathogenic Leptospira, having an estimated global epidemiology of 1.03 million cases. Early diagnosis of leptospirosis is crucial to prevent its progression into more severe form known as Weil’s syndrome. A pragmatic approach for early diagnosis of leptospirosis necessitates the direct detection pathogenic Leptospira itself by a specific molecular recognition element. To achieve this, we have isolated two aptamers against the target LipL32 protein of the pathogenic Leptospira, LepDapt-9149 and LepDapt-5561 by a modified SELEX strategy known as the Tripartite-hybrid-HT SELEX. These two aptamers have K_d values of 64.59 ± 11.25 nM and 48.36 ± 8.03 nM, respectively, estimated by direct ELASA. On the other hand, the K_d values obtained using filter binding assay-qPCR analysis were 39.35 ± 0.12 and 18.03 ± 0.23 nM for LepDapt-9149 and LepDapt-5561, respectively. Integrating these aptamer pair into sandwich ELASA, the developed assay is highly specific against the pathogenic Leptospira without displaying any cross-reactivity against the non-pathogenic Leptospira and NS1 protein of dengue virus. The assay also reached a LOD of 55 CFU/ mL in 1% serum, which is sensitive enough to reach the clinical range of leptospirosis, which is between 10⁴ to 10⁶ CFU/ mL. The sandwich ELASA assay integrated with the isolated aptamer pair has an enormous potential for the diagnosis of leptospirosis.