Abstract：
Leptospirosis is a potentially life-threatening zoonosis caused by pathogenic Leptospira and for rapid diagnostics, direct detection is desirable. LipL32 protein is the most suitable biomarker for direct detection. DNA aptamers are sought to be generated against LipL32 by Systemic Evolution of Ligands via Exponential Enrichment (SELEX). LepDapt-5a is the most potent aptamer candidate among all the candidates, as determined by direct Enzyme-linked Aptasorbent Assay (ELASA). LepDapt-5a was predicted to form a G-quadruplex structure as predicted by QGRS Mapper and validated experimentally by direct ELASA. The diagnostic potential of the aptamer was further tested on a direct and sandwich ELASA platform. A LOD of 106 mL-1 and 105 mL-1 were estimated by direct and sandwich ELASA platforms, respectively, which are within the range associated with leptospiremia levels. The dot blot assay developed was able to attain a LOD of 104 CFU mL-1 against pathogenic Leptospira, which is also within the leptospiremia level. This is the first-ever DNA aptamer and hybrid-heterodimeric aptamer constructed against LipL32. The diagnostic potentiality of the LepDapt-5a DNA aptamer was proven on three major diagnostic platforms, which are direct ELASA, sandwich ELASA, and aptamer-based dot assay.
摘要：
钩端螺旋体病是由致病性钩端螺旋体引起的潜在威胁生命的人畜共患病，用于快速诊断，直接检测是可取的。LipL32蛋白是最适合直接检测的生物标志物。试图通过经由指数富集(SELEX)的配体的系统进化来产生针对LipL32的DNA适体。LepDapt-5a是所有候选者中最有效的适体候选者，如通过直接酶联Aptasorbent测定(ELASA)所测定。预测LepDapt-5a形成G-四链体结构，如QGRSMapper所预测的，并通过直接ELASA进行实验验证。在直接和夹心ELASA平台上进一步测试适体的诊断潜力。通过直接和夹心ELASA平台估计的LOD为106mL-1和105mL-1，分别,在与钩端螺旋体血症水平相关的范围内。所开发的斑点印迹测定法能够针对致病性钩端螺旋体获得104CFUmL-1的LOD，这也在钩端螺旋体血症的水平内。这是第一个针对LipL32构建的DNA适体和杂合异二聚适体。LepDapt-5aDNA适体的诊断潜力已在三个主要诊断平台上得到证实，这是直接的ELASA，三明治ELASA,和基于适体的点测定。
