RU2586504C1 - Synthetic oligonucleotide primers and method for thereof for indicating immunodeficiency viruses and leukaemia of cats in clinical material by multiplex polymerase chain reaction - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and a set of synthetic oligonucleotide primers and method for use thereof. Proposed set comprises two pairs of primers having following structure - FIV F: 5′-AAGAGTCCCAAATATGCCATAGG-3′ and FIV R: 5′-TCCATCCAAATTGCTACTGTTC-3′; FeLV F: 5′-GAATAAACCTCTTGCTGTTTGC-3′ and FeLV R: 5′-AATCAGATCGAATGACAGAGACAC-3′. Presented primers do not contain palindromic repetitions of nucleotides, do not form expressed secondary structure and do not have extended G-C portions, and temperature of annealing is 55 °C for both pairs of oligonucleotides. Described method involves preparation of reaction mixture by mixing buffer of 10-fold for PCR - 2.5 mcl, a dNTP (25 mM) - 0.4 mcl, primers FIV F, FIV R, FeLV F and FeLV R (15 pmol/mcl) in 1 mcl, Taq polymerase (5 units/mcl) - 0.2 mcl MgCl₂ (50 mM) - 1.5 mcl bidistilled water - 11.4 mcl and test DNA 5 mcl, amplification is carried out in following mode: total denaturation 95 °C - 3 minutes, cycle denaturation (95°C - 20 or 60 s) - annealing (55 °C - 20 or 60 s) - elongation (72 °C - 40 or 60 s), cycle of denaturation-annealing-elongation is repeated 35 times, final elongation 72 °C - 5 minutes, storing 4 °C - 10 minutes, and detection of obtained results is carried out by 1.5 % agarose gel. Presence or absence of amplified fragments of nucleotide sequences length 397 base pairs for FIV (feline immunodeficiency virus) and 221 base pairs for FeLV (feline leukemia virus) indicates presence or absence of viruses in organism cat.

EFFECT: invention can be used in scientific research for detecting a genetic material of immunodeficiency viruses - FIV and leukemia FeLV cats by multiplex polymerase chain reaction (PCR) in animal blood.

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Description

The invention relates to the field of biotechnology, molecular biology, molecular genetic diagnosis of animal viral diseases, and can be used in scientific research to detect the genetic material of FIV immunodeficiency viruses and feline leukemia virus leukemia feline leukemia multiplex polymerase chain reaction (PCR) in the blood of animals.

The causative agents of diseases - FIV and FeLV - belong to the family Retroviridae. These viruses are characterized by a high degree of homology and a similar genome structure: they have three main structural genes encoding viral proteins in the following order: 5′-gag-pol-env-3 ′: gag (group specificanti gen - encode core proteins), pol (encode reverse transcriptase - polymerase and integrase) and env (encode envelope proteins - envelope).

Within the group of FIV strains isolated from domestic cats, 5 genetic variants with a certain variability of genomes can be distinguished. Separation into types and subtypes is carried out on the basis of the analysis of modifications of the structure of the env gene (Martins A.N. et all, 2008). FeLV is also characterized by significant variability in the structure of genes. According to the results of the analysis of viral genomes, the least variable were: the gag FIV gene and the region located immediately in front of the gag-pro-pol FeLV gene.

The life cycle scheme of retroviruses includes: binding of the virion to the receptor of the host cell, entry into the cell, reverse transcription with the formation of cDNA integration into the host genome (formation of the provirus) (Supotnitsky MV Evolutionary pathology. On the issue of HIV infection and HIV / AIDS pandemics among other infectious, epidemic and pandemic processes. - M.: University Book, 2009. - 400 p.). In accordance with the biological characteristics of viruses, methods aimed at detecting antibodies are less informative than those that can detect the virus itself.

Viruses of leukemia and immunodeficiency in cats may not manifest themselves in the infected body for many years, making it a source of infection. They are transmitted horizontally and vertically, from mother to fetus. Often these diseases occur in combination, that is, one animal is a carrier of both viruses. These infections are widespread among both domestic and stray cats (Krasnikov A.V., Larionova O.S., Marusheva Yu.A. Analysis of infection of cats with retroviral infections in the Saratov region // Agrarian scientific journal No. 2, 2015. S . 14-16).

Viruses are lymphoneurotropic, infection leads to deep immunosuppression, manifested by the development of secondary infections and tumor processes. The animal has been on maintenance therapy all his life. At the same time, treatment regimens for viral leukemia and immunodeficiency in cats are different, because despite the fact that viruses primarily affect lymphocytes, FIV causes an immune disease - a decrease in the population of CD4-T lymphocytes, and FeLV provokes an oncological disease - cloning of virus-modified B-lymphocytes (Sulimov A. A. Viral diseases of cats. - M .: KolosS, 2004. - S. 40-48, 66-70).

In Russia, there are two PCR test systems for diagnosing these infections, one for detecting leukemia and the other for cat immunodeficiency (InterLabService LLC, Russia). Both are designed for real-time PCR. A device on which analysis can be carried out costs from 2 million rubles and more. This makes the diagnosis of these diseases unavailable for ordinary veterinary hospitals and laboratories. Where there is such a device, one analysis costs from 600 rubles. Given the fact that there are two infectious agents and in some cases the study needs to be repeated, the owners are not able to pay for a high-quality diagnosis. Therefore, there is a need to develop and put into practice more accessible and economical methods.

The PCR method is a direct highly sensitive and highly specific method by which it is possible to identify fragments of proviral DNA FIV and FeLV in lymphocytes of peripheral blood of cats. The specificity of the method is determined by primers - oligonucleotides that flank the desired site, joining on the principle of complementarity on denatured DNA (annealing), and, being a seed for polymerase, initiate the elongation of a new chain on the desired DNA fragment. The main parameters for the efficient passage of the amplification reaction, which provide specificity and sensitivity, are the correct choice of the genome region of the identified agent, the structure and temperature regime of primer annealing.

A known method for the determination of antibodies to FIV and FeLV in animal blood by enzyme-linked immunosorbent assay (ELISA), based on the detection of a specific antigen-antibody complex using a color reaction due to the fermentation of the substrate with an enzyme associated with the conjugate (BMC Vet Res. 2013; 9: 2. Comparison of the geographical distribution of feline immunodeficiency virus and feline leukemia virus infections in the United States of America (2000-2011). Bimal K Chhetri, Olaf Berke, David L Pearl, and Dorothee Bienzle). The disadvantage of this method is the low information content in the case of examination of a vaccinated animal (false positive results) and in the early stages of infection, when the blood level of antibodies is not high enough (false negative results).

A known method for detecting FIV in the blood of cats by one-step PCR based on the detection of feline immunodeficiency virus DNA of cats, as well as a method for detecting FeLV in the blood of cats by one-step RT-PCR based on the detection of feline leukemia virus RNA (Vet Res Forum. 2014 Fall; 5 (4): 255-61. Molecular and clinical study on prevalence of feline herpesvirus type 1 and calicivirus in correlation with feline leukemia and immunodeficiency viruses. Hamideh Najafi, Omid Madadgar, Shahram Jamshidi, Arash Ghalyanchi Langeroudi, and Mahdieh Darz ) The disadvantages of the method are multioperational analysis, that is, the need for at least three sequential reactions on one biomaterial to identify the above pathogens, each separately.

A known method for detecting FIV and FeLV in cats blood using the Real-time PCR method, based on the detection of each pathogen individually using double-labeled fluorescent probes (Krasnikova E.S., Krasnikov A.V., Agoltsov V.A. Diagnostic value assessment polymerase chain reaction and immunochromatographic analysis in some prevailing retroviral infections of cats // Agrarian scientific journal. 2013. No. 2. P. 23-25). The disadvantages of this method is the inability to detect FIV and FeLV at the same time, which leads to an increase in research time and increased consumption of materials, in addition, the need to use fluorescent probes to account for the results contributes to a rise in price.

Closest to the claimed invention is the method described in patent RU No. 2340673 dated 04/10/2007, published on 12/10/2008: “A set of oligonucleotide primers for identification of rabies virus RNA in samples”. In which the design of the primers was carried out by comparing the nucleotide sequences of various strains of lissaviruses deposited in the international GeneBank database (http://www.ncbi.nlm.nih.gov/genbank). In this case, two pairs of oligonucleotide primers for the conserved region of the nucleoprotein gene were calculated and melting point 52 ° C. For testing the primers, the following PCR parameters were used: 94 ° C for 10 sec, 56 ° C for 30 sec with a temperature decrease of 1 ° C per cycle, 72 ° C for 1 min - 6 cycles, then 94 ° C for 10 sec., 50 ° C 15 sec. 72 ° C 30 sec. 35 cycles, the final synthesis - 7 min 72 ° C, provides the synthesis of DNA fragments of the calculated size (558 bp) under the conditions of RT-PCR. The specificity of amplification products was confirmed by direct sequencing.

But this method is not designed to detect rabies viruses in animals by multiplex PCR and cannot be used to detect immunodeficiency viruses and leukemia due to the unspecificity of primers for FIV and FeLV, and it is also necessary to carry out a reverse transcription reaction before PCR and sequencing to take into account the results , which is quite laborious and costly.

The technical task is to develop an effective, fast and inexpensive method for the detection of DNA of immunodeficiency virus and leukemia of cats by multiplex PCR by constructing two specific pairs of oligonucleotide primers and selecting the conditions for conducting multiplex PCR with them.

The technical result of the invention is achieved by constructing a diagnostic set of primers for the conserved region of the FIV and FeLV genomes and developing a method for their use in multiplex PCR taking into account the result by agarose gel electrophoresis, which ensures simplicity, informativeness and accessibility of studies for ordinary laboratories.

The specified technical result is achieved by creating a set of oligonucleotide primers for the identification of immunodeficiency viruses and leukemia of cats by comparing the nucleotide sequences of different strains of FIV and FeLV, deposited in the international GeneBank database. Moreover, in conservative regions of the FIV and FeLV genomes that do not have polindromic nucleotide repeats, two pairs of oligonucleotide primers were calculated and synthesized that do not form pronounced secondary structures, do not have extended GC sites, and differ in that they have an annealing temperature of 55 ° C for both pairs of oligonucleotides, the following structure is FIV F: 5′-AAGAGTCCCAAATATGCCATAGG-3 ′ and FIV R: 5′-TCCATCCAAATTGCTACTGTTC-3 ′; FeLV F: 5′-GAATAAACCTCTTGCTGTTTGC-3 ′ and FeLV R: 5′-AATCAGATCGAATGACAGAGACAC-3 ′, with the formation of one pair (FIV) of a fragment of 397 bp in length, as a result of the work of another pair (FeLV) - of length 221 bp For the use of primers, multiplex PCR is used, which differs from the prototype in that there is no need to carry out RT, as well as the composition of the reaction mixture, which is prepared by mixing a 10-fold buffer for PCR - 2.5 μl, dNTP (25 mm) - 0.4 μl primers FIV F, FIV R FeLV F and FeLV R (15 pmol / μl) 1 μl, Taq polymerase (5 units / μl) 0.2 μl, MgCl ₂ (50 mm) 1.5 μl bidistilled water - 11.4 μl and DNA samples - 5 μl. In this case, amplification is carried out in the following mode: total denaturation 95 ° C - 3 min, denaturation cycle (95 ° C - 20 or 60 sec) - annealing (55 ° C - 20 or 60 sec) - elongation (72 ° C - 40 or 60 sec), and the denaturation-annealing-elongation cycle is repeated 35 times, the final elongation is 72 ° C for 5 minutes, storage 4 ° C for 10 minutes. In this case, the results obtained are detected by electrophoresis in a 1.5% agarose gel, where, by the presence or absence of amplified fragments of nucleotide sequences 397 bp in length. for FIV and 221 bp for FeLV, the presence or absence of viruses in cats is judged.

The figure 1 presents the structure of the genomes of the viruses of immunodeficiency and leukemia of cats, where it is shown that the viruses contain similar genes and have approximately the same genome size. In figure 1, the oval marks the areas on which the design of the primers was carried out.

The figure 2 presents the electrophoregram of the results of a blood test of FIV and FeLV positive cats by multiplex PCR using developed primers to determine the size of amplified fragments. Samples 1, 4, 5, 7, and 8 are FIV and FeLV positive, and samples 2, 3, and 6 contain only FeLV DNA.

The figure 3 presents an electrophoregram of the results of the study of clinical material from cats in a developed way. Samples 1, 2, 3, 4, 5, 9 - FIV and FeLV - positive, samples 6, 7, 10 contain only FeLV DNA, sample 8 - negative.

The construction of two pairs of synthetic oligonucleotide primers used to isolate DNA from immunodeficiency viruses and feline leukemia, and the development of a method for the diagnosis of viral immunodeficiency and feline leukemia using them was carried out in five stages:

- analysis of the structure of the FIV and FeLV genomes;

- design of primers;

- selection of two pairs of primers that are optimally combined with each other;

- modeling the composition of the reaction mixture of the method for the diagnosis of viral immunodeficiency and leukemia in cats using selected pairs of primers;

- verification of the effectiveness of selected primers with clinical material.

Analysis of the structure of the FIV and FeLV genomes. Analysis of the FIV and FeLV genomic sequences hosted on the NCBI resource was carried out by computer simulation using proprietary computer programs: “Nucleotide sequences and characteristics of genomes, variable tandem repeats, oligonucleotide primers and probes of pathogens of bacterial and viral infections of pathogenicity groups I-II” ( Certificate of state registration of the database №2011620585 (authors - Yashechkin Yu.I., Naydenova E.V., Scherbakova S.A.)) and "Program for the calculation of oligonucleotide primers on th the homologous genomes of RNA-containing viruses for their detection in PCR and database formation ”(Certificate on state registration of computer programs No. 20133613699 (authors - Yashechkin Yu.I., Naydenova EV, Scherbakova SA)). Alignment of genomic sequences was carried out using the ClustaW method, the cluster tree was modeled using the UPGMA method.

Design primers. Using the GeneRunner computer program, the FASTA virus genome was used to select primers in the gIV FIV gene region and in the region immediately in front of the gag-pro-pol FeLV gene, as the most conserved genomes of these viruses.

Selection of two pairs of primers that are optimally combined with each other. Quality control and thermodynamic analysis of the selected primers was performed using OLIGO DNA / RNA primer analysis software, v. 5.0. (http://molbiol-tools.ca/molecular_biology_freeware.htm#Primer%20design) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). When designing primers, the main requirements were: the degree of homology (complementarity) with the selected gene region; the lack of self-complementary sites within the primers and complementarity to each other in order to prevent the emergence of stable secondary structures (dimers); the proximity of the annealing and melting temperatures of the primers.

Based on the computer analysis, two pairs of primers were selected, the characteristics of which are shown in table 1.

The selected two pairs of oligonucleotide primers (FIV F and FIV R; FeLV F and FeLV R) have the optimal size (22-24 N.) and structure (lack of self- and mutual complementarity), as evidenced by the Gibbs energy indicators at the 3 ′ end (0 cal / mol) and Gibbs energy of dimeric structures (-1 cal / mol), optimal GC composition (42%) and annealing temperature for both primer pairs 55 ° C. As a result of the work of one pair (FIV F and FIV R), a fragment with a length of 397 bp is formed, as a result of the work of another pair (FeLV F and FeLV R) of a length of 221 bp, GC the composition of the products is 40% and 56%, respectively the melting point of the product is 74 ° C and 79 ° C, respectively.

Modeling the composition of the reaction mixture of the method for the diagnosis of viral immunodeficiency and leukemia in cats using selected pairs of primers. Isolation and purification of nucleic acids from the blood of cats was carried out by the method of nucleosorption on silica gel using the DNA sorb-B kit (LLC InterLabService, Russia). PCR was performed in the volume of the reaction mixture - 25 μl per 1 sample. The composition of the reaction mixture are presented in table 2.

The composition of the reaction mixture was selected so that the concentration of MgCl ₂ ions (1.5 mM) ensured the optimal speed and accuracy of the Taq polymerase enzyme, the concentration of dNTP - 0.4 mm, the concentration of primers - 15 pmol / μl and the sample volume - 5 μl , which helps to increase the specificity of the reaction. When using an amplifier with a lid without heating, 20 μl of PCR mineral oil is layered onto the mixture. When using an amplifier with a heated lid, mineral oil is not used.

PCR using the developed primers was performed on amplifiers “AMPLI 4” (“Biocom”, Russia) and “T100” (“BioRad”, USA). The temperature-time regime of the reaction for amplifiers is presented in table 3.

For amplifiers with active temperature control in a solution in a test tube, for example, T100, the passage time of all stages of amplification (denaturation-annealing-elongation) is shorter than for amplifiers with matrix temperature control, for example, AMPLI 4.

Accounting was carried out by gel electrophoresis in a 1.5% agarose gel with the addition of ethidium bromide as an intercolating dye for DNA. The result was taken into account on the equipment of the BioRad company (USA). To determine the size of amplicons after PCR with the developed primers, the molecular weight marker “1 kb DNA ladder” (“Stratagene”, USA) was used (Fig. 2). In electrophoretic paths of samples 1, 4, 5, 7, and 8, there are luminous bands at levels of 397 bp and 221 bp, which indicates the presence of FIV and FeLV proviruses in DNA samples. In the electrophoretic paths of samples 2, 3, and 6, luminous bands are present only at the level of 221 bp, which indicates the presence of only FeLV provirus DNA in the samples.

Verification of the effectiveness of selected primers with clinical material. As a positive control, we used DNA isolated from the blood of cats in which the presence of FIV and FeLV were confirmed by immune chromatography (Animal Genetics, inc., Korea) and Real-time PCR (LLC InterLabService, Russia). For research, blood samples were taken from cats suspected of being infected with viral immunodeficiency and leukemia (Fig. 3). Samples 1, 2, 3, 4, 5, 9 - contain DNA of proviruses FIV and FeLV, the electrophoretic mobility of amplicons of which is 397 bp and 221 bp accordingly, it coincides with the sizes of amplicons of the positive control (K +). Samples 6, 7, 10 contain only FeLV provirus DNA, the amplicon electrophoretic mobility of which is 221 bp, sample 8 is negative. In the electrophoretic track corresponding to the negative control (K-), there are no bands.

The claimed invention is an affordable, reliable, highly sensitive and highly specific method for detecting fragments of proviral DNA of immunodeficiency virus and leukemia of cats in animal blood lymphocytes in a short time. Moreover, computer analysis showed a lack of response to homologous and heterologous organisms.

The proposed method was tested with positive results and regular reproducibility of these results in 2015 on 18 DNA samples obtained from the blood of cats belonging to private owners and contacting the UNIVERSITY Veterinary Hospital FSBEI HPE Saratov State Autonomous Administration. The work was carried out on the basis of the interdepartmental educational and research laboratory "Genome" of the Federal State Budget Educational Establishment of Higher Professional Education "Saratov State Agrarian University" and the Federal Public Health Institution of RosNIPCHI "Microbe" (Saratov).

Claims (2)

1. A set of synthetic oligonucleotide primers used to isolate DNA from immunodeficiency proviruses and feline leukemia obtained by computer programs by analyzing the structure of viruses, selecting conserved regions of the viral genomes, selecting two optimal pairs of primers that lack polindromic nucleotide repeats that do not form expressed secondary structures, and do not have extended GC sites and differ in that they have an annealing temperature of 55 ° C for both pairs of oligonucleotides and the following structure - FIV F: 5′-AAGAGTCCCAAATATGCCATAGG-3 ′ and FIV R: 5′-TCCATCCAAATTGCTACTGTTC-3 ′; FeLV F: 5′-GAATAAACCTCTTGCTGTTTGC-3 ′ and FeLV R: 5′-AATCAGATCGAATGACAGAGACAC-3 ′, resulting in the use of a fragment of 397 bp for FIV and 221 bp for FeLV. 2. A method for the diagnosis of viral immunodeficiency and leukemia in cats using two pairs of synthetic oligonucleotide primers, which consists in conducting multiplex PCR, characterized in that there is no need for RT, the reaction mixture is prepared by mixing a 10-fold buffer for PCR - 2.5 μl, dNTP (25 mM) - 0.4 μl, a set of primers according to claim 1 (15 pmol / μl) 1 μl, Taq polymerase (5 units / μl) - 0.2 μl, MgCl ₂ (50 mm) - 1 5 μl, double-distilled water - 11.4 μl and DNA samples - 5 μl, while amplification is carried out in the following mode: total denaturation 95 ° С - 3 min, denaturation cycle (95 ° С - 20 or 60 sec) - annealing (55 ° С - 20 or 60 sec) - elongation (72 ° С - 40 or 60 sec), and denaturation-annealing cycle elongation is repeated 35 times, final elongation is 72 ° C for 5 minutes, storage is 4 ° C for 10 minutes, and the results are detected by electrophoresis in a 1.5% agarose gel, where, by the presence or absence of amplified fragments of nucleotide sequences of length 397 bp for FIV and 221 bp for FeLV, the presence or absence of viruses in a cat is judged.

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