In silico evaluation of PCR - primers for detection of Lyme Borrelia
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Original versionSultan, N. In silico evaluation of PCR - primers for detection of Lyme Borrelia. Master thesis, University College of Southeast Norway, 2016
Lyme borreliosis (LB) or Lyme disease is the most prevalent vector-borne disease in US and Europe. The etiologic is some species of tick-borne spirochetes Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. The most common clinical symptoms of LB is the erythema migrans (EM). The pathogen is transmitted to humans through the tick bite of Ixodes species, and spread to cause more severe manifestations such as Acrodermatitis Chronica Atrophicans (ACA), Lyme arthritis, and neuroborreliosis. Although polymerase Chain reaction (PCR) assay is an important molecular analysis for detection of DNA-pathogen in infected organisms, an apparent discrepancy about its accuracy and reliability is still existing. In order to validate PCR assay for detection of LB, 77 PCRprimer pairs were assembled from previous publications, and investigated for specificity and sensitivity using bioinformatics applications. The primers targeted genes coding for outer surface proteins ospA (29), ospB (2), and ospC (11), flagellin flaB (25), outer membrane protein p66 (5), genetic recombination protein recA (2), and plasmid-specific sequence (3). Basic Local Alignment Search Tool (BLAST) was employed to search homology between sequence of primers and sequences of GenBank database to find out cross-reactivity with non-targeted taxa of Lyme Borrelia or relapsing fever Borrelia. The primer-set that showed similarity only for the targeted species was considered a specific primer-set, while the primerset that homologous with untargeted Borrelia species was in cross-reaction and was considered unspecific for the target species or PCR analysis. The sensitivity of primer-sets was rational designed and presented by proportion of specific hits for particular species to the total hits number for the same species, which is diagnostic sensitivity (coverage). The results showed 25 (32%) specific primer-sets, 40 (52%) unspecific primer-sets, and 12 (16%) specific primer-sets but showed limited cross-reaction with untargeted Borrelia species. High proportion of ospA- primers were specific, most of flaB-primers were unspecific, and half of ospC-primers was specific. Most primers for remaining genes showed specificity for the species of interest. The vast majority of primer-sets ranged from low to moderate sensitive for the species of interest. This study demonstrated most PCR-primers that have been used previously for detection of Lyme Borrelia were suboptimal to be specific for taxon of interest and unsuitable to be specifically used in PCR for detection B. burgdorferi sl complex.