Optimization of polymerase chain reaction for monitoring of Borrelia burgdorferi infection by ixodid ticks

Abstract

Determination of the infection rate of ixodid ticks with tick-borne borreliosis pathogens and determination of belonging to the pathogenic genotype by PCR is an important component for monitoring, risk assessment and control of the epizootic situation of Lyme borreliosis in different territories. The results of testing and optimization of the internal laboratory protocol of the classical polymerase chain reaction for the identification of Lyme disease pathogens are presented. Eight samples of extracted DNA from ixodid ticks collected from vegetation in the forest park tract "Golendernya", Bila Tserkva, Kyiv region, were examined by classical PCR. Samples were formed from pools of ten tick specimens: seven pools - ticks of the genus I. ricinus and one pool - ticks of the genus D. reticulatus. For detection of borrelia DNA, primer sets were used to detect DNA of Borrelia burgdorferi sensu lato complex; Borrelia burgdorferi and pathogenic borrelia: Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii. The protocol for nucleic acid extraction from ticks was modified using the commercial IndiSpin Pathogen Kit. Optimization of amplification temperature conditions was carried out by the annealing temperature gradient method for each primer pair. Based on the results of the study, internal laboratory protocols for classical PCR using specific oligonucleotide primers were developed. It was found that in each of the pools of I. ricinus and D. reticulatus there were infected tick specimens with the Borrelia burgdorferi sensu lato complex and Borrelia afzelii genus, and also identified the Borrelia burgdorferi sensu stricto genus in one of the pools of I. ricinus and D. reticulatus, DNA of the Borrelia garinii genus was not detected. The developed internal laboratory protocols of classical PCR will be further used to study the infection of ixodid ticks with tick-borne borreliosis pathogens: Borrelia burgdorferi sensu lato, Borrelia burgdorferi sensu stricto and Borrelia afzelii. Key words: Lyme borreliosis, Ixodes ticks, polymerase chain reaction, Borrelia burgdorferi sensu lato, Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii.