Analysis of the polymorphism of the LIPL32 gene of pathogenic leptospira for the development of a real-time polymerase chain reaction test system

The purpose of this work was to study the polymorphism of the LipL32 gene of various pathogenic leptospira serovars, as well as to develop a prototype test system for the indication of this gene using the real-time polymerase chain reaction method with an assessment of its stability. The objects of the study were 245 nucleotide sequences of the LipL32 gene of Leptospira interrogans, DNA samples of leptospira serovars Pomona, Grippotyphosa, Canicola, Bataviae and Tarassovi. Based on the results of bioinformatic analysis of 245 nucleotide sequences of the LipL32 gene, it was established that this gene is a highly conserved region of the genome of the causative agent of leptospirosis. To indicate the pathogen under study for this gene by RT-PCR, a marker locus (lipoprotein L32 region) with a size of 173 nucleotide pairs was determined. Within this site, oligonucleotide primers and a probe were developed, which, according to multiple alignment of the amplified locus, turned out to be identical for most of the analyzed nucleotide sequences of the LipL32 L. interrogans gene. BLAST analysis of the sequenced nucleotide sequences of the pathogenic leptospira serovars Pomona, Grippotyphosa, Canicola, Bataviae and Tarassovi showed that the identity of the lipoprotein L32 gene region amplified by engineered oligonucleotides of the above isolates with DNA sequences of the causative agent of leptospirosis stored in the NCBI database ranged from 95.74 to 98.95%. Using the developed oligonucleotides and a fluorescent probe, a prototype test system for the detection of pathogenic leptospires by PCR-RV was constructed. It was found that the specificity of the test kit presented in this work was 100%, and the sensitivity could reach 5·102 copies of DNA per ml of the test material. The stability assessment of the test system showed that the variation coefficients of the minimum threshold values did not exceed 10%, indicating that the kit components were stable during long-term storage.

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