Design and application of a loop-mediated isothermal amplification (lamp) assay for detection of tick borne relapsing fever

Faezeh Houman sadr,1,* Mohammad soleimani,2 Gholamreza javadi,3 Saied reza naddaf,4

1. Department of Biology Cellular and Molecular, Science and Research branch, Islamic Azad University, Tehran, Iran.
2. Department of microbiology , Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran.
3. Department of Biology Cellular and Molecular, Science and Research branch, Islamic Azad University, Tehran, Iran.
4. Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran

Abstract

Introduction

Tick borne relapsing fever (tbrf) caused by borrelia microti is transmitted via bites of the soft tick ornithodoros erraticus that primarily inhabits caves and small niches. the disease is reported in north and south america, africa, asia, and europe. there is a real need for a rapid, inexpensive, and easy to operate tool to facilitate accurate disease diagnosis and surveillance for better management of tbrf.

Methods

We successfully established a loop-mediated isothermal amplification (lamp) assay which exhibited a markedly higher sensitivity than previously developed detection methods for tbrf. the lamp was performed using specific primers designed based on the sequence of the glycerophosphodiester phosphodiesterase (glpq) of tbrf borreliae. analytical specificity and sensitivity (limit of detection, lod) of the lamp were evaluated.

Results

In this study, we developed a lamp assay targeting the elongation glpq gene sequence for visual detection of tbrf borreliae. the lamp reaction was performed at 60-65°c for 45 min with glpq gene of borrelia microtti. the amplification obtained with the lamp was detected by visual inspection of turbidity and confirmed by gel electrophoresis. the lod of the assay for visual detection of turbidity and agarose gel electrophoresis were 32 fg and 0.32 fg respectively. the lamp assay was highly specific and no amplification products were observed from the non-borrelia organisms.

Conclusion

The results of this study indicated that the glpq-lamp is a simple, rapid, sensitive and specific technique for detection of borrelia spp. that may improve diagnostic potential in clinical laboratories.

Keywords

Tbrf, glpq, lamp, borrelia microtti