Development of gold nanoparticle based probes for the detection of pathogenic leptospires in canine

Abstract

Leptospirosis is a globally important zoonosis caused by the pathogenic Spirochaetes of the genus Leptospira which belong to the family Leptospiraceae. The clinical manifestations of leptospirosis are neither specific nor consistent, hence misdiagnosis is common. Different antigen detection tests have been developed but none of them are sensitive enough to be routinely used. Whereas antibody detection methods possess low sensitivity at the acute stage of the disease. Molecular detection methods involve expensive enzymes, harsh conditions and sophisticated instruments. Development of DNA detection methods with high sensitivity and specificity are extremely important in clinical diagnosis. Currently, no diagnostic technique is completely satisfactory. newlineA pair of DNA hairpins were designed by generating a script specific to Leptospira target using NUPACK, an online web tool for hybridization chain reaction. The products of HCR were visualized by colorimetric detection and agarose gel electrophoresis. For colorimetric detection, gold nanoparticles were synthesized and characterized. For efficient HCR, parameters such as hybridization buffer, hybridization temperature and volume of sodium citrate for AuNP synthesis were optimized. Among the buffers, 1 M Nacl, 50 mM Na2HPO4 propagated the chain newline newlinereaction at 25°C temperature. Hybridization chain reaction was carried out using the clinical samples and reference leptospiral strains to analyze HCR s performance in the environmental samples. Their products were visualized by agarose gel electrophoresis in 6% agarose gel and colorimetric detection using 40 nM gold nanoparticles. HCR- AuNP detection and HCR-Agarose gel electrophoresis detection were subjected for sensitivity evaluation and compared those with the sensitivity of conventional PCR. newline newline newline