Design, optimization and evaluation of a polymerase chain reaction for detection of borrelia spp.

BACKGROUND

Lyme borreliosis and relapsing fever are important zoonotic diseases worldwide and the improvement of diagnostic strategies is a prioritized task considering the morbidity of these diseases in some areas. PCR based methods appear to be of utmost importance because of the high sensitivity and specificity of these assays.

OBJECTIVES

To obtain a molecular method based on PCR for the detection of the genus Borrelia infection in different specimens.

RESULTS

Sets of reported primers were evaluated "in silico" and they did not fulfill the proposal parameters. On the other hand, the two new, designed sets of primers were theoretically efficient for Borrelia DNA amplification. PCR procedures with these primers were standardized with borrelial DNA and optimum annealing temperatures, primer concentrations and reaction cycle numbers were determined. The PCR analytical sensitivity was 10 genomes per reaction for each technique. Both PCR were highly specific to different Borrelia species DNA and to samples (sera, cerebrospinal liquids and hard ticks) infected artificially with a Borrelia strain, visualizing the amplification of the expected DNA fragment. No amplification was obtained when other microorganisms were used. 36 human clinical samples were negatives in a preliminary study.

CONCLUSIONS

Both sets of primers with their respective PCR protocols showed similar results, which suggest that each one can be used indistinctly in detecting Borrelia spp., mainly in countries where the situation of these diseases are unknown.