RU-2861443-C1 - SET OF OLIGONUCLEOTIDE PRIMERS FOR SNP TYPING OF BRUCELLA ABORTUS STRAINS

RU2861443C1RU 2861443 C1RU2861443 C1RU 2861443C1RU-2861443-C1

Abstract

FIELD: biotechnology; molecular biology. SUBSTANCE: set of oligonucleotide primers for SNP typing of Brucella abortus strains by capillary DNA sequencing is proposed. Primers having the structure shown in SEQ ID NO: 1-6 are used for amplification and subsequent detection in specific regions of brucella genes of specific single nucleotide polymorphism (SNP) determining the belonging of the strain to subgenotype C1, C2 or C4 of genetic line C in the structure of the global B. abortus population. EFFECT: possibility of using an additional genotyping method for solving epidemiological surveillance tasks, establishing the probable geographical origin of the strain, possible routes of brucellosis introduction, and accumulating knowledge about the phylogenetic structure of the B. abortus population. 1 cl, 2 dwg, 3 tbl, 2 ex

Inventors

Kovalev Dmitrij Anatolevich
KUZNETSOVA IRINA VLADIMIROVNA
PISARENKO SERGEJ VLADIMIROVICH
Shapakov Nikolaj Andreevich
Krasovskaya Tatyana Leonidovna
Zhirov Andrej Mikhajlovich
Safonova Nadezhda Sergeevna
Dementeva Elena Nikolaevna
Ponomarenko Dmitrij Grigorevich

Dates

Publication Date: 20260505
Application Date: 20250912

Claims (1)

A set of oligonucleotide primers for SNP typing of Brucella abortus strains using capillary DNA sequencing, characterized in that primers having the structure presented in SEQ ID NO: 1-6 are used for amplification and subsequent detection in certain regions of Brucella genes of a specific single polymorphism (SNP) that determines the strain's belonging to the subgenotype C1, C2 or C4 of the genetic line C in the structure of the global B. Abortus population.

Description

Field of technology The invention relates to molecular biology, in particular to laboratory diagnostics of infectious diseases, and can be used for molecular genetic typing of B. abortus strains in solving epidemiological surveillance problems, as well as for scientific research purposes. State of the art More than 2 million new cases of brucellosis infection are registered annually in humans worldwide. Up to 70-80% of brucellosis cases occur in regions with developed livestock farming in the Middle East, Mediterranean, Africa, Southeast Asia, and South and Central America. The brucellosis situation is particularly acute in the Eastern Mediterranean, North Africa, and Central and East Asia [1, 2]. The Russian Federation has experienced an unstable epidemiological situation over the past 10 years, with 3,537 cases of brucellosis identified. In 2022-2023, a trend toward an increase in human brucellosis incidence of 30-50% relative to long-term averages was noted, associated with the emergence of cattle epizootics [3]. In this regard, differentiation of endemic Brucella isolates and identification of imported cases of brucellosis are of paramount importance, and genetic profiling of territories serves as an effective tool for epizootological and epidemiological surveillance of the infection. Recently, whole-genome analysis of the distribution of single nucleotide polymorphisms (wgSNP) has been widely used as a tool for phylogenetic analysis, which is one of the most effective and accurate among modern methods for studying the evolutionary history and diversity of pathogens [4]. Based on wgSNP typing data, 4 main genetic lines, designated A, B, C and D, were identified in the structure of the global B. abortus population, and it was determined that the strains isolated in the Russian Federation belong to two genetic lines: C, widespread in the countries of Europe and East Asia (subgenotypes C1, C2 and C4), and, in isolated cases, D, to which most of the known vaccine strains of B. abortus belong (subgenotype D4) [5, 6]. A search of patent databases and scientific and technical sources of information revealed a lack of information on oligonucleotide primers designed to differentiate subgenotypes of B. abortus genetic lines using capillary electrophoresis. The scheme of multilocus analysis of the variable number of tandem repeats (VNTR) copies (MLVA) was chosen as a prototype of the invention, which consists of setting up PCR with specific primers to 16 VNTR loci, and taking into account the obtained results by electrophoresis in agarose gel [7]. The MLVA method is less effective for studying evolutionary relationships and accurately determining the origin of strains, since the corresponding loci are characterized by a relatively high frequency of mutations and manifestations of homoplasy. Another disadvantage of this scheme is that when conducting gel electrophoresis, difficulties may arise in the subjectivity of the interpretation of the results; there is a high risk of contamination, which requires large expenses for its elimination. Disclosure of invention The technical objective of the invention is to develop a set of oligonucleotide primers for a rapid, efficient and economically accessible method for genetic typing of B. abortus strains, based on the identification of clearly discriminable single polymorphisms in the Brucella genome in order to determine the probable geographic and phylogenetic origin of the strains. The technical result is achieved by isolating DNA from a pure culture of the strain under study, performing a polymerase chain reaction (PCR) with specific primers, and determining the nucleotide sequence of the amplification products to detect the presence/absence of SNPs using Sanger sequencing technology (capillary sequencing). This method is based on identifying specific single polymorphisms (SNPs) in specific regions of Brucella genes, which determine the strain's affiliation with subgenotype C1, C2, or C4 of the C genetic lineage within the global B. abortus population. Implementation of the invention Example 1. Design and testing of a set of oligonucleotide primers. The selection of primers, optimal annealing temperature and the probability of non-specific interactions between oligonucleotides were carried out using the Primer-BLAST program (version 2.5) [8]. The developed set of oligonucleotide primers was tested on a sample of collection strains of B. abortus of the Stavropol Anti-Plague Institute of Rospotrebnadzor. The study material consisted of DNA isolated from a pure two-day culture of B. abortus strains. Disinfection and sample preparation were carried out in accordance with SanPiN 3.3686-21 "Sanitary and Epidemiological Requirements for the Prevention of Infectious Diseases" and MU 1.3.2569-09 "Organization of Laboratories Using Nucleic Acid Amplification Methods When Working with Material Containing Microorganisms of Pathogenicity Groups I-IV." The isolated DNA was amplified