RU2732626C1 - System of oligonucleotide primers and probe for detecting dna mycoplasma bovigenitalium - Google Patents

Abstract

FIELD: biotechnology; veterinary science.

SUBSTANCE: invention relates to microbiology, biotechnology and veterinary science. Disclosed is a system of oligonucleotide primers and a probe for identifying Mycoplasma bovigenitalium DNA in biological material from cattle, raw material for biological industry and nutrient mediums for operation with cell cultures by polymerase chain reaction in real time. Pair of oligonucleotide primers has structure: M.bgen2 F 5'-TTGTTGCGCACCTTGGATCT-3' M.bgen2 R 5'-GGTCGATTCCACCAGCTCTA-3'. Fluorescent labeled probe for identification of amplicons synthesized on the Mycoplasma bovigenitalium DNA matrix during the PCR reaction in real time has the following structure: M.bgen2 Pr 5'-FAM CCATCATATGAGCTGAATCCTACTGT BHQ1-3'.

EFFECT: invention enables to differentiate Mycoplasma bovigenitalium from closely related Mycoplasma bovis mycoplasmas and can be used to detect carrier Mycoplasma bovigenitalium in cattle, quality control of raw material for biotechnological industry and culture nutrient media for research work.

1 cl, 3 ex, 2 dwg

Description

The invention relates to the field of microbiology, biotechnology and veterinary medicine, and can be used for the diagnosis of animal diseases of viral, bacterial and other etiology, as well as for the detection of the carriage of Mycoplasma bovigenitalium in cattle, quality control of raw materials for the biotechnological industry and culture media for research work ...

The proposed system for detecting Mycoplasma bovigenitalium DNA includes a pair of oligonucleotide primers complementary to the species-specific region in the ackA acetate kinase gene of Mycoplasma bovigenitalium, and possessing the activity of forward and reverse primers and having the structure

and a fluorescently labeled hydrolysis probe for assessing the accumulation of amplicons on the Mycoplasma bovigenitalium DNA template during the real-time PCR reaction, complementary to the species-specific region in the ackA gene of Mycoplasma bovigenitalium, located in the sequence flanked by the above-mentioned primers

The use of primers makes it possible to detect M. bovigenitalium DNA in biological material from cattle and to differentiate mycoplasmas of this species from phylogenetically closely related M. bovis mycoplasmas.

The invention can be used in veterinary medicine to identify the genetic material of one of the causative agents of mycoplasmosis in cattle - M. bovigenitalium, in order to diagnose associated infectious diseases and identify cases of asymptomatic carriage, and the developed test system can be used for scientific research.

The invention can be used in biotechnology to control the contamination of raw materials of animal origin and its derivatives for contamination with mycoplasmas of the species M. bovigenitalium.

M. bovigenitalium is an etiological agent for mastitis, pneumonia, arthritis and keratoconjunctivitis in cattle. Also, mycoplasmas of this species often colonize the reproductive tract of animals, and can be associated with various diseases of the reproductive organs, abortion and barrenness. Infections of the reproductive system of cows are often asymptomatic or accompanied by the development of vulvovaginitis. The pathogen is also excreted in cows from the vagina and cervical canal during ineffective insemination (Hillman R., 2008).

With the colonization of M. bovigenitalium of the reproductive system of cows, further spread of the pathogen with discharge from the animal's vagina is possible, while mycoplasmas often fall on the surface of the udder, which leads to the development of mastitis.

M. bovigenitalium is more often than other mycoplasma species detected in infectious diseases of the reproductive system of bulls. The distal reproductive tract is usually affected. At the same time, the persistence of M. bovigenitalium in the reproductive system of bulls can be asymptomatic, while carriage is detected in 9 -63% of bulls in different countries. With the spread of infection, the development of epididymitis and testiculitis is possible, which affects the quality of sperm, expressed, first of all, in a change in the morphology of spermatozoa and a decrease in their mobility (Kirkbride SA, 1987).

Infection with M. bovigenitalium can occur through sexual contact, contact and airborne droplets. Specific measures for the prevention of diseases associated with this pathogen have not yet been developed, which determines the importance of timely detection of cases of infection and carriage among the cattle population.

Also M. bovigenitalium can be present in the serum of clinically healthy cattle and act as a contaminant of biological raw materials for the biological industry.

M. bovigenitalium is an auxotrophic microorganism; therefore, its cultivation requires nutrient media enriched with various substances. For the energy metabolism of these mycoplasmas, alcohols and fatty acids must be present in the medium. The need for fatty acids or alcohols is also characteristic of M. bovis mycoplasmas. These bacteria, like M. bovigenitalium, are the causative agents of diseases in cattle. M. bovis and M. bovigenitalium have a high level of similarity, while diseases caused by M. bovis are often characterized by a more severe course. Also, the severe course of the disease can be associated with mixed infections caused by the two indicated types of mycoplasma. Thus, it is advisable to carry out differential diagnosis of infections caused by mycoplasmas of these species, as well as cases of mixed infection.

The polymerase chain reaction (PCR) method provides the detection of M. bovigenitalium DNA with greater accuracy than cultural methods for the detection of this pathogen (Justice-Allen A., 2011). This method is based on the process of specific matrix synthesis of DNA strands, which leads to multiple copying of fragments of the target DNA, if present in the material. The process of matrix synthesis is carried out by the enzyme DNA polymerase in the reaction mixture.

A distinctive feature of the modification of PCR, known as real-time PCR, is the ability to assess the course of the reaction during its course. In this case, the assessment is carried out by measuring the accumulation of fluorescence emitted by the fluorescein (FAM) associated with the probe in the system. A prerequisite for the emission of fluorescence by fluorescein is the destruction of the probe, which occurs when it binds to the complementary sequence in the presence of the DNA polymerase enzyme, leading to spatial separation of fluorescein from BHQ-1 molecules bound to the same probes. In this case, the activity of the cleavage of probes and the release of fluorochromes is in direct proportion to the content of the target amplicons in the reaction mixture.

Ensuring the specificity of the DNA amplification reaction is determined by the selection of primers for the target DNA sequences that recognize unique nucleotide structures inherent only to them. DNA copying is possible only when the primer binds to single-stranded DNA, and the process begins exclusively at the 5 'end of the primer. Primers are essential components of the reaction mixture. By their chemical structure, they are oligonucleotides complementary to highly conserved and highly specific sequences in the target DNA. In test systems for the diagnosis of infectious diseases, the target DNA is the DNA of the corresponding pathogen.

Oligonucleotide probes in the reaction mixture provide the ability to detect accumulation of PCR product in real time. The possibility of detecting the PCR product is achieved due to the release of fluorochromes when the probes are destroyed in the presence of specific amplicons. The increase in specificity is determined by the fact that the probes do not interact with other DNA sequences in the test material.

Thus, the choice of a fragment for the selection of the primer system and probe is decisive for the specificity of the PCR test system for the diagnosis of infectious diseases. This fragment should be a sequence in the genome of the pathogen, the detection of which is aimed at the test system, which has a conservative structure highly specific for this particular pathogen and not detected in the genome of other organisms.

The closest analog of the presented system of primers and probe for detecting M. bovigenitalium DNA are specific systems of oligonucleotide primers and probes aimed at detecting the sequences of the 16S-23S rRNA intergenic spacer in the test material (Boonyayatra S., 2012; Parker A.M., 2017). We used the designed primer and probe systems in real-time PCR to identify M. bovigenitalium. Their use made it possible to identify M. bovigenitalium in clinical material from cattle, as well as to differentiate M. bovigenitalium from other mycoplasmas that cause diseases in cattle. The difference between the proposed system of primers and probes from those described is the choice of a species-specific target, namely, a region within the ackA acetate kinase gene.

An object of the present invention is to provide a system comprising oligonucleotide primers and a probe for identifying M. bovigenitalium by real-time polymerase chain reaction.

The goal is achieved by constructing a system of specific primers and a probe for identifying DNA of one of the pathogens of mycoplasmosis in cattle M. bovigenitalium, having the following structure:

Characterization of oligonucleotide primers and a region of amplified DNA.

Based on the analysis of the genomic sequence of M. bovigenitalium, presented in the international database GenBank NCBI (National Center for Biotechnology Information), primers were selected, designated M.bgen2 F and M.bgen2 R, complementary to the gene fragment, the product of which is acetate kinase, ackA M. bovigenitalium and having forward and reverse primer activity. An oligonucleotide probe M.bgen2 P was also selected, complementary to the region of the M. bovigenitalium genome sequence located between the regions recognized by the primers M.bgen2 F and M.bgen2 R.

In the course of experiments on the development and testing of the test system, samples of clinical material from cattle were used as a positive control. The material was selected during the examination of animals at livestock enterprises, where cases of bronchopneumonia of calves and mastitis with symptoms characteristic of infections associated with M. bovis, M. bovigenitalium and other mycoplasmas were detected. DNA was isolated from the material under study, and the obtained preparations of purified DNA were used in the experiments.

Examples of specific implementation.

Example 1. Procedure for constructing a system of oligonucleotide primers and a probe for identifying DNA of the causative agent of mycoplasmosis in cattle M. bovigenitalium using real-time PCR

Based on the study of the genome-wide sequence of the causative agent of mycoplasmosis in cattle M. bovigenitalium, presented in the international database Genbank, for the design of primers, a sequence of the ackA acetate kinase gene was selected, suitable for identification of M. bovigenitalium. The size of this gene is 1359 bp. (GenBank NCBI AP017902.1). As part of the selected genetic structure, a DNA segment was identified that had nucleotide differences from the genomic sequences of other mycoplasmas that cause diseases in cattle and other microorganisms. The calculated length of the DNA fragment, which can be amplified using the selected primers, was 126 bp. As part of this fragment, a sequence specific for M. bovigenitalium was selected to create an oligonucleotide probe. Fluorescein (FAM) and BHQ-1 molecules were chosen as the components of the oligonucleotide probe providing detection of DNA amplification. In this case, the photons emitted by fluorescein upon excitation, and having a wavelength of about 518 nm, are completely absorbed by the BHQ-1 quencher.

Evaluation of the homology of the region recognized using the proposed primers with similar sequences in the genomes of other organisms was carried out in silico by comparing this region with genetic structures deposited in the international GenBank database. The comparison was carried out using the BLASTN algorithm on the web-server of the National Center for Biotechnological Information (NCBI) (bttp: //www.ncbi.nlm.mh.gov/BLASTA. At the time of in silico analysis, no homology of the selected sequence with DNA sequences of other organisms was revealed It was.

Example 2. Amplification of specific DNA fragments of M. bovigenitalium with detection of the accumulation of a specific DNA fragment by real-time PCR.

To carry out real-time PCR using the developed system of primers and probes, reaction mixtures were prepared, which included 10 pmol of each of the components of the system for identification of the pathogen of mycoplasmosis in cattle M. bovigenitalium, as well as other components necessary for carrying out this type of reaction.

The total volume of the reaction mixture is 25 μl per sample. To prepare it, the following components were mixed in a test tube:

Ready mixture for PCR brand qPCRmix-HS (OOO "Evrogen", Moscow) - 5 μl

Primer M.bgen2 F (10 pM / μl) - 1 μl

Primer M.bgen2 R (10 pM / μl) - 1 μl

M.bgen2 Pr probe (10 pM / μl) - 1 μl

deionized water - 15 μl

investigated DNA sample - 2 μl.

To carry out real-time PCR using the software of the CFX96 thermocycler (Bio-Rad, USA), a temperature regime was set, which included the stage of preliminary DNA denaturation at 94 ° C for 5 min, then DNA denaturation at 94 ° C for 50 cycles C - 15 sec; annealing of primers at 60 ° С - 30 sec; chain elongation at 70 ° C - 30 sec. The assessment of the level of fluorescence in the sample was carried out at the end of each cycle. In the presence of M. bovigenitalium DNA in the sample, an increase in fluorescence was observed during the course of the reaction, which is reflected in the graph based on the data on the accumulation of fluorescence collected and processed by the CFX-Manager program (Bio-Rad, USA) (Fig. 1). 15 curves on the graph, reaching a plateau at RFU values (relative fluorescence units) along the y-axis of 350-770 reflect the process of accumulation of the fluorescent signal when performing PCR with samples containing DNA from Mycoplasma bovigenitalium. The lines passing in the area not higher than 40 on the y scale reflect the absence of accumulation of the fluorescent signal when performing PCR with samples that do not contain Mycoplasma bovigenitalium DNA.

Example 3. Evaluation of the analytical specificity of the amplification reaction using the developed oligonucleotide primers to detect the DNA of the causative agent of mycoplasmosis in cattle M. bovigenitalium.

To determine the analytical specificity, real-time PCR was performed with DNA isolated from clinical samples obtained from cattle, in which the presence of closely related pathogens of cattle M. bovis and Ureaplasma diversum, as well as with DNA samples isolated from cultures was established. M. hominis, U. parvum and U. urealyticum, pathogenic for humans.

The PCR reaction was carried out in accordance with the procedure described in example 2. When PCR was performed with the material containing the DNA of the above pathogenic mycoplasmas, no accumulation of the product was observed (Fig. 2). The curve reaching a plateau at RFU values (relative fluorescence units) along the y-axis of> 250 reflects the process of accumulation of the fluorescent signal when performing PCR with a sample containing Mycoplasma bovigenitalium DNA. Five curves reflecting the absence of fluorescent signal accumulation when performing PCR with samples that do not contain Mycoplasma bovis DNA, containing DNA of Mycoplasma hominis, Mycoplasma bovis, Ureaplasma diversum, Ureaplasma urealyticum and Ureaplasma parvum do not cross the threshold line of the 30 RFU line.

The proposed system of primers and probe is suitable for use for identification of M. bovigenitalium, they allow to differentiate it from a phylogenetically close species M. bovis and determine the presence of M. bovigenitalium DNA within 3-4 hours in biological material from animals, and differs from existing analogues by using as a genetic target of a site within the ackA acetate kinase gene.

Claims (2)

The system of oligonucleotide primers and probe for the identification of Mycoplasma bovigenitalium DNA in biological material from cattle, raw materials for the biological industry and nutrient media for working with cell cultures using the real-time polymerase chain reaction, characterized in that it includes primers and a probe, complementary to a fragment of the ackA acetate kinase gene from Mycoplasma bovigenitalium, having the following structure:

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