Isolation of a novel DNA aptamer against LipL32 as a potential diagnostic agent for the detection of pathogenic Leptospira

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 10 6 mL -1 and 10 5 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 10 4 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.

Establishment and application of quantitative detection of Bacillus velezensis HMB26553, a biocontrol agent against cotton damping‐off caused by Rhizoctonia

“…Several powerful detection methods for identifying target microorganisms had been developed and were widely used, including culture‐dependent techniques with an antibiotic‐resistant mutant, enzyme‐linked immunosorbent assay (ELISA), loop‐mediated isothermal amplification (LAMP), and nucleic acid amplification‐based methods like polymerase chain reaction (PCR) and quantitative PCR (qPCR) 4–9 . Among them, qPCR has reliably detected and quantified species such as Plectosphaerella cucumerina , Metarhizium clade 1, Clonostachys rosea , 10–12 and specific strains such as Pantoea agglomerans strain CPA‐2, Trichoderma asperellum strain icc012, Trichoderma gamsii strain icc080, Trichoderma atroviride strain SC1, B acillus subtilis strain QST713, B acillus amyloliquefaciens strain D747, Chaetomium globosum , Fusarium oxysporum strain Fo47, and Pseudomonas fluorescens strain EPS62e 13–20 .…”

“…Several powerful detection methods for identifying target microorganisms had been developed and were widely used, including culturedependent techniques with an antibiotic-resistant mutant, enzymelinked immunosorbent assay (ELISA), loop-mediated isothermal amplification (LAMP), and nucleic acid amplification-based methods like polymerase chain reaction (PCR) and quantitative PCR (qPCR). [4][5][6][7][8][9] Among them, qPCR has reliably detected and quantified species such as Plectosphaerella cucumerina, Metarhizium clade 1, Clonostachys rosea, [10][11][12] and specific strains such as Pantoea agglomerans strain CPA-2, Trichoderma asperellum strain icc012, Trichoderma gamsii strain icc080, Trichoderma atroviride strain SC1, Bacillus subtilis strain QST713, Bacillus amyloliquefaciens strain D747, Chaetomium globosum, Fusarium oxysporum strain Fo47, and Pseudomonas fluorescens strain EPS62e. [13][14][15][16][17][18][19][20] In genus Bacillus, many studies have focused on the specific detection and quantification of B. cereus group due to its human pathogenicity by qPCR, [21][22][23][24][25] but few have reported critical markers for the specific detection and quantification of other Bacillus species, [26][27][28] specifically for strain-specific detection in Bacillus velezensis.…”