Development of A Loop-Mediated Isothermal Amplification (LAMP) Assay for Detection of Relapsing Fever Borreliae.

BACKGROUND

This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of tick-borne relapsing fever in resource-limited areas.

METHODS

A set of six primers were designed based on the conserved regions of the Glycerophosphodiester phosphodiesterase () gene of species. For sensitivity assay, serial dilutions of a recombinant plasmid containing a 219bp sequence of the glpQ were prepared and used as the template DNA. The LAMP reactions containing the six primers and the reagents required for amplification were incubated at 60-65 °C for 60min in a Loopamp real-time turbidimeter. For the specificity test, DNA from 14 other bacteria were included in the assays, and double-distilled water was used as the negative control. Also, DNA from dried blood spots (DBSs) of spirochetemic mice, and blood samples from relapsing fever-suspected patients were examined by the LAMP along a -specific nested PCR that targets the IGS region.

RESULTS

The LAMP detected as low as 90 copies in reactions. The primers reacted with DNA from DBS of spirochetemic mice showing spirochete concentrations of ≤ one per a 1000X microscopic field. In clinical samples, the LAMP assay showed a higher sensitivity compared to nested-PCR. The LAMP specificity was 100%, as the primers did not react with other bacteria DNA.

CONCLUSION

The high sensitivity and specificity of the test, along with the simplicity of the DNA extraction procedure, make the LAMP a reliable and adaptable tool for the diagnosis of tick-borne relapsing fever in rural endemic areas.