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

  • Faezeh Houmansadr Department of Cellular and Molecular Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
  • Mohammad Soleimani Department of Microbiology, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran, Tasnim Biotechnology Research Center, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran
  • Saied Reza Naddaf Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran
Keywords: Relapsing fever; LAMP; glpQ; Iran

Abstract

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 (glpQ) gene of Borrelia 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 Borrelia-specific nested PCR that targets the rrs-rrl-IGS region.Results: The LAMP detected as low as 90glpQ 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.

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Published
2019-08-03
How to Cite
1.
Houmansadr F, Soleimani M, Naddaf SR. Development of A Loop-Mediated Isothermal Amplification (LAMP) Assay for Detection of Relapsing Fever Borreliae. J Arthropod Borne Dis. 14(1):47–55.
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Original Article