Design of primers for indication of pathogenic Leptospira by nested polymerase chain reaction in real time

Leptospirosis is a widespread natural focal zoonotic disease that threatens epidemic and epizootic well-being. Bacteriological, serological and molecular genetic methods exist for laboratory diagnostics of leptospirosis. In samples of the studied material obtained from animals and from the environment, pathogenic leptospires are in most cases present in small quantities, based on this, the nested polymerase chain reaction (PCR) method is one of the optimal methods for detecting these pathogens due to its higher sensitivity and specificity. In this study, a bioinformatics analysis of the nucleotide sequences of the lipL32 gene of pathogenic leptospires of the Canicola, Grippotyphosa, Hebdomadis, Icterohaemorragiae, Pomona, Tarassovi and Sejroe serogroups was carried out. Based on the results of bioinformatics analysis, the most specific regions were selected and a combination of external and internal primer pairs, as well as a fluorescently labeled oligonucleotide probe, were developed. with the following characteristics: GC composition (%) and melting temperature (Tm) of the primers varied from 44,0 to 59,1% and from 55,1 to 61,6 ºС, respectively. Using the multiple alignment method, it was found that the primer annealing zones for most sequences, except for AY609330, AY461909, AY609332 and JN886738, do not have nucleotide substitutions. However, these substitutions are single in these sequences and should not reduce the amplification efficiency of the desired lipL32 gene locus.