Abstract:
Objective To develop and evaluate a rapid detection assay for Leptospira interrogans serogroup icterohaemorrhagiae based on loop-mediated isothermal amplification (LAMP) combined with nanoparticle-based lateral flow biosensor (LFB).
Methods The LAMP primers targeting the glycotransferase gene (gtf) in the O-antigen gene cluster of L.serogoup icterohaemorrhagiae were designed. After specific label of primers (FIP-FAM and LF-Biotin) and condition optimization, we evaluated the sensitivity, specificity and feasibility of gtf-LAMP-LFB assay. A total of 53 strains of Leptospira were identified by using LAMP-LFB assay, PCR and microscopic agglutination test (MAT), and the results were analyzed to evaluate the practicality of LAMP-LFB assay.
Results Our data showed that the detection limit of the assay was 100 fg/μL for genomic DNA of reference strain of L. icterohaemorrhagiae (56601), and the specificity was 100% because there were no cross reactions with nucleic acids of other Leptospira serogroups and non-Leptospira strains. For the application examination, LAMP-LFB and MAT identification results were completely consistent, while the sensitivity of LAMP-LFB assay was higher than PCR. In addition, the color of CL and TL bands could be observed directly to determine the results by using LFB to detect LAMP amplicons.
Conclusion The LAMP-LFB assay developed based on LAMP technique has high repeatability, sensitivity and specificity, which can be used for the rapid and accurate identification of the strains of L.interrogans serogroup icterohaemorrhagiae, and can be used as a potential screening and diagnosis tool for L.interrogans serogroup icterohaemorrhagiae.
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