RU2802065C1 - Set of specific oligodeoxyribonucleotide primers and a fluorescently labeled probe for the orf1 (non-structural protein 1) gene fragment of feline calicivirus (fcv) rna (cdna) for the detection of feline calicivirus (fcv). - Google Patents

Abstract

FIELD: biotechnology; veterinary medicine.

SUBSTANCE: set of highly specific primers and a fluorescently labeled probe for the detection of genetic material (RNA, cDNA) of Feline calicivirus (FCV) for the detection of Feline calicivirus (FCV) is proposed. The presented set of oligodeoxyribonucleotide primers and a fluorescently labeled DNA probe contains a pair of oligonucleotides with the activity of forward and reverse primers in the polymerase chain reaction, as well as one fluorescently labeled DNA probe.

EFFECT: invention allows more reliable detection of the genetic material (RNA, cDNA) of Feline calicivirus (FCV).

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Description

Field of technology to which the invention relates:

The invention relates to the field of biotechnology and concerns a set of highly specific primers and a fluorescently labeled probe for detecting the genetic material (RNA, cDNA) of Feline calicivirus (FCV) for detecting Feline calicivirus (FCV). The presented set of oligodeoxyribonucleotide primers and a fluorescently labeled DNA probe contains a pair of oligonucleotides with the activity of forward and reverse primers in the polymerase chain reaction, as well as one fluorescently labeled DNA probe. The present invention allows more reliable and reliable detection of the genetic material (RNA, cDNA) of Feline calicivirus (FCV) for the detection of Feline calicivirus (FCV) and can be used in veterinary medicine. EFFECT: method allows detecting a fragment of the conservative region of the ORF1 (Non structural Protein 1) gene of RNA (cDNA) of Feline calicivirus (FCV), which makes it possible to identify animals infected with Feline calicivirus (FCV) at any stages of the disease.

The invention relates to primer sets for detecting the genetic material (RNA, cDNA) of the ORF1 gene fragment (Non structural Protein 1) RNA (cDNA) of Feline calicivirus (FCV) in clinical samples, sectional samples, culture fluids and other biological products for the purpose of setting diagnosis, correction of treatment, epidemiological investigation, as well as for solving research problems of the properties of Feline calicivirus (FCV), and can be used in veterinary medicine.

Feline calicivirus belongs to the genus Vesivirus of the Caliciviridae family, it is a small non-enveloped virus with a spherical capsid and a bowl-shaped recess in it (calici - cup (lat.)). The capsid makes the virus sufficiently stable in the environment and allows easy spread through fomites, including pet owners and veterinary clinic staff. The virus genome is a single-stranded RNA molecule. Feline calicivirus is widespread throughout the world; infection is characterized by long-term carriage and excretion in apparently healthy cats (up to 25% of healthy livestock). Isolation of feline calicivirus occurs with the secretion of the oropharynx, conjunctiva and nose. Virus transmission is most effective through direct contact between a calicivirus carrier and a susceptible animal; aerosol transmission plays a minor role as droplets of secretion rarely spread more than 1 meter during sneezing. The main source of feline calicivirus is asymptomatic recovered cats, which can continuously shed calicivirus into the environment for many years and even for life.

Feline calicivirus is quite stable in the external environment, its survival under favorable conditions reaches 28 days, in the dried state it can remain active for several months. As a result of the increased resistance of the calicivirus, fomites play a significant role in infecting susceptible cats. Fleas are also capable of spreading calicivirus, presumably through their faeces, when cats are infected when they are groomed.

Features of the development of calicivirus depend both on the strain of the virus and on the immune status of the animal. Infection with calicivirus leads to the development of a transient viremia leading to widespread dissemination of the virus, dissemination of the virus, in most cases, is not accompanied by clinical signs. Feline calicivirus primarily affects the epithelial cells of the upper respiratory tract, oral cavity, and conjunctiva, unlike feline herpesvirus, calicivirus is not associated with corneal involvement. Exposure to calicivirus leads to necrosis of the epithelium, the most characteristic lesions are found in the oral cavity of cats, they are characterized by the formation of vesicles that develop into ulcers (the marginal areas of the tongue are more often affected). In addition to the development of calicivirus on the mucous membranes, it sometimes affects the skin adjacent to the oral cavity (lips, nose). Feline calicivirus can also infect the lower respiratory tract (alveolar epithelium), and some strains of the virus are prone to severe interstitial pneumonia. Rarely, the virus can spread outside the respiratory tract and infect the joints of cats, with synovitis and lameness occurring. Separate strains of the virus are capable of causing a virulent systemic manifestation of feline calicivirus, while animals get sick regardless of vaccination status, this syndrome is characterized by generalized vasculitis, multiorgan failure and high mortality. Also, in this course of feline calicivirosis, damage to the skin and mucocutaneous junctions is often noted, accompanied by the formation of ulcers on the ears, muzzle and paws. The underlying pathogenesis of this virulent clinical manifestation of calicivirus is presumably based on viral mutation leading to hypervirulence, but the exact mechanism of calicivirus mutation remains unclear.

The clinical manifestations of feline calicivirus vary greatly, from a mild upper respiratory tract infection to severe viral pneumonia with a fatal outcome, this largely depends on the strain of the virus and the immune status of the animal. The incubation period for calicivirus infection is 2-6 days, at a veterinary appointment, typical manifestations are outflow from the eyes and nose (usually serous), the formation of erosions of ulcers in the oral cavity with a possible spread to the lips, nasal mirror and periungual space (viral paronychia). The main reason for the contact of the owner to the veterinary clinic is the sneezing of the animal, lacrimation, increased salivation, general depression and fever. A characteristic lesion for feline calicivirus is the formation of erosions and ulcers in the oral cavity, especially at the tip and along the edge of the tongue. Eye lesions may consist of chemosis, blepharospasm, and serous effusions; signs of conjunctival involvement in calicivirus infection are usually mild and self-limiting. Lesions of the nasal mucosa in this disease are usually mild, may consist of sneezing and serous expiration.

In kittens after vaccination or during the period of illness with calicivirus, lameness may develop due to inflammation of the joints (synovitis). A number of authors find a relationship between infection with calicivirus and the development of chronic lymphoplasmacytic gingivostomatitis, however, in this disease, in addition to calicivirus, other pathogens are also isolated.

With the development of a systemic infection, adult cats are more likely to be affected, regardless of vaccination status, a single virulent strain of feline calicivirus is responsible for the development of systemic and severe manifestations. In addition to the characteristic signs of calicivirus (mouth ulcers, lacrimation, salivation), with a systemic lesion, there is high fever, swelling of the muzzle and paws, the formation of ulcers on the skin (muzzle, ears, skin of the fingers), shortness of breath due to the development of pneumonia, jaundice due to the lesion liver and bleeding from the mucous membrane of the large intestine due to disseminated intravascular coagulation (DIC). The course of calicivirosis in cats with systemic lesions is characterized by lightning speed, the death of the animal can be observed within the first day after the development of clinical signs, mortality reaches 60%. Cats with a systemic virulent disease that have died from calicivirus disease often have liver cell necrosis, pancreatic involvement, interstitial pneumonia, and dual cavity effusions (thoracic and abdominal) on necropsy.

With a favorable course of the disease, the period of clinical recovery from calicivirus is 2-3 weeks. In some cats, after recovering from calicivirus, a persistent infection develops with a very long shedding of the virus, sometimes for life. These animals do not have any clinical manifestations of calicivirus, but they continue to be latent carriers of the infection, and are an important source of the virus in the population, and can also serve as a source of a new, mutated virus.

The presence of erosions and ulcers, together with characteristic signs (fever, oppression, hypersalivation, etc.), is an important indicator of feline calicivirus infection, but these signs can be observed both in the normal course of calicivirus infection and in systemic virulent infection. The presence of IgG antibodies to the virus in the blood of sick animals is usually of little diagnostic value, since most cats are seropositive. The gold standard for diagnosis is the identification and isolation of the virus, but this type of study is not available in all veterinary clinics. Recently, the identification of the nucleic acid of the virus by means of the polymerase chain reaction (PCR) has been increasingly used; this research method has high sensitivity and specificity. None of the diagnostic methods is able to distinguish between a common virus and a systemic virulent disease virus, the distinction between these forms is based on the characteristics of the clinical course of feline calicivirus.

Background of the invention: When developing a set of oligodeoxyribonucleotide primers and a fluorescently labeled probe for diagnosing RNA (cDNA) of the ORF1 (Non structural Protein 1) gene fragment of Feline calicivirus (FCV) RNA (cDNA), a comparative analysis of the structure of the nucleotide sequences of the complete calicivirus genomes was carried out. Feline calicivirus (FCV) cats, hosted on the NCBI web resource https://www.ncbi.nlm.nih.gov/nucleotide/, and then the primers and fluorescently labeled probe were designed by computer simulation using computer programs Beacon Designer v . 8.14 PREMIER Biosoft International (San Francisco, CA 94131-2175, USA) and Vector NTI 11 (Invitrogen, USA). Alignment of genomic sequences was carried out by the Clustal W method, phylogenetic analysis was performed by the method of unweighted pairwise average - UPGMA. Quality control and thermodynamic analysis of the selected primers and fluorescently labeled probe were performed using the OLIGO DNA/RNA primer analysis software, v.5.0 and BLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi (USA).

The amplifiable cDNA region, being a marker, makes it possible to identify the viral agent in the test sample. Effective real-time PCR requires a fluorescently labeled DNA probe and DNA primers - primers (synthetic oligonucleotides) - strictly specific to the cDNA of the viral genome. The complexity of the choice of primers and probe is due to the requirement of their strict species specificity. The primers must be complementary to the DNA nucleotide sequences, limiting the amplifiable region on the right and left in such a way that DNA synthesis by DNA polymerase takes place strictly in the selected region. The fluorescently labeled DNA probe, in turn, must lie within the DNA region delimited by the primers. The correct choice of primers allows an exponential increase in the number of copies of the target DNA region. The correct choice of a combination of a pair of primers and a DNA probe allows real-time detection of the accumulation of amplification products. In general, the specificity of the performed RT-PCR, and hence the reliability of the study, depends on the correct choice of oligodeoxyribonucleotide primers and probes.

In the computer design of primers and a fluorescently labeled DNA probe, the main criteria were: the absolute degree of homology (complementarity) with the selected gene region; the absence of self-complementary regions in the structure of primers and their complementarity to each other in order to prevent the emergence of stable secondary structures (dimers); maximum closeness of primer annealing temperatures.

Disclosure of the invention: The technical result of the claimed invention is the creation of a set of oligodeoxyribonucleotide primers and a fluorescently labeled probe that allows real-time identification of a fragment of the ORF1 gene (Non structural Protein 1) of RNA (cDNA) of Feline calicivirus (FCV) and has a higher homology to currently circulating strains of Feline calicivirus (FCV), which increases the validity and reliability of the assays.

This technical result is achieved by the development of a set of highly specific oligodeoxyribonucleotide primers for detecting a fragment of the ORF1 gene (Non structural Protein 1) RNA (cDNA) of Feline calicivirus (FCV) containing a pair of oligonucleotides that have the activity of forward and reverse primers in a polymerase chain reaction, as well as fluorescent -labeled DNA probe. These oligonucleotides have the following structure:

Sequences of oligodeoxyribonucleotide primers and a fluorescently labeled probe to the ORF1 gene fragment (Non structural Protein 1) RNA (cDNA) of Feline calicivirus (FCV).

Forward primer 5`→3`

VT-Z-Ca-F2 5'- gAACTACCCgCCAATCAACATgTg-3'

Reverse primer 5`→3`

VT-Z-Ca-R2 5'- AgCACATCATATgCggCTCTgAT -3'

Fluorescently labeled DNA probe (Probe) 5`→3`

VT-Z-Ca-P2 5'- (R6G)- gTgTTTgATTTggCCTgggCTCTTCg -(BHQ2) -3'

Table 1. Characterization of the developed primers and fluorescently labeled DNA probe Index VT-Z-Ca-F2 VT-Z-Ca-R2 Fluorescently labeled DNA probe VT-Z-Ca-P2 Structure from 5' to 3' gAACTACCCgCCAATCAACATgTg AgCACATCATATgCggCTCTgAT (R6G)- gTgTTTgATTTggCCTgggCTCTTCg -(BHQ2) Length, nucleotides 24 23 26 T of annealing taking into account T of melting, °C 60 60 70 GC composition, % 50 48 54 T of enzyme work, °C 68.7 68.8 77.6

The design of the primers and probe proposed for patenting includes all data from the international GenBank database on the nucleotide sequences of the complete RNA genomes of Feline calicivirus (FCV) as of May 2022. The primers and probe used in the work have greater homology to currently circulating Feline calicivirus (FCV) strains of feline calicivirus, which in turn increases the sensitivity of the proposed set of diagnostic primers and probe. The target for the primers and probe used in this work is the highly conserved region of the ORF1 (Non structural Protein 1) gene of genomic DNA.

Oligodeoxyribonucleotide primers and a fluorescently labeled DNA probe submitted for patenting make it possible to detect in an RNA (cDNA) sample a fragment of the ORF1 (Non structural Protein 1) gene of genomic RNA (cDNA) of Feline calicivirus (FCV) in real time, as well as to amplify the fragment DNA, which makes it possible to sequence the resulting amplicon, with which molecular biological work can be carried out, and, consequently, a deeper study of the properties of Feline calicivirus (FCV) in cats. In addition, the use of a plasmid construct carrying a specific insert (as described below) as a positive control allows the development of a quantitative PCR, which in turn makes it possible to assess the viral load in the test sample.

The primers were tested using a biotechnological construct based on a plasmid with the insertion of a specific DNA fragment. It was experimentally shown that the selected primers and DNA probe provide reliable synthesis of target DNA fragments. The specificity of the amplification was further confirmed by sequencing.

Positive control samples were obtained by TOPO-T/A cloning of virus-specific DNA duplexes into the PC DNA 3.1 plasmid (Invitrogen, USA). After that, competent E. coli cells of the TOP 10 line (Invitrogen, USA) were transformed with the obtained plasmid carrying the type-specific fragment of the ORF1 gene (Non structural Protein 1) of the genomic RNA (cDNA) of Feline calicivirus (FCV).

It is important to note that the optimization of PCR conditions was carried out using sets of commercially available reagents, instruments and enzymes intended for mass use in laboratory practice, which allows the rapid and reliable application of this invention in medical and research laboratories.

Analysis of the transformation efficiency was carried out by real-time PCR in accordance with the protocol described below, where 1 × TE buffer containing recombinant plasmids with the insertion of a virus-specific synthesized DNA duplex was added to the reaction mixture as a positive sample. 1×TE buffer was added to the reaction mixture as a negative control.

Amplification conditions were optimized for the concentration of magnesium ions, the concentration of primers and probes in the reaction mixture, and the primer annealing temperature.

The composition of the reaction mixture was modeled in such a way that the concentration of MgCl ₂ ions ensured the optimal speed and accuracy of the operation of the Mut-3 Taq polymerase enzyme, the dNTP concentration did not exceed 0.4 mM, the primer concentration was 10 pmol/μl, the fluorescently labeled DNA probe 5 pmol/µl, and the sample volume was 5 µl (to increase the specificity of the reaction).

Table 2. PCR mixture for the reaction (based on one tube): Component Volume for 1 reaction (20 µl), µl PCR buffer ×10 2.5 dNTP (25 mM) 0.2 MgCl ₂ (50 mM) 1 Taq-pol Mut-3 polymerase (5 U/µl) 0.5 Forward primer - VT-Z-Ca-F2 (10 pmol/µl) 1 Reverse primer - VT-Z-Ca-R2 (10 pmol/µl) 1 Fluorescently labeled DNA probe VT-Z-Ca-P2 (5 pmol/µl) 1 Sample (DNA sample) 5 diH ₂ O (bidistilled) 6

Table 3 Temperature-time regime of PCR Stage T, °С Time, sec. Number of repetitions Plate Type Amplifier Rotary cycler Initial denaturation 95 300 300 1 Denaturation 95 10 10 45 cycles with fluorescence detection Annealing 58 25 dimension 25 dimension Elongation 72 25 40 Storage 4 ∝ ∝

PCR conditions were tested using the developed primers and a fluorescently labeled DNA probe using plate type amplifiers "DT-96", "DT-Prime", "DT-Light" ("DNA-Technology", Russia) and rotary type "Rotor Gene Q" (QIAGEN, Germany).

To determine the sensitivity of the method, isolated DNA samples (smears and swabs) were subjected to tenfold dilutions from 10 ^-1 to 10 ^-5 . The resulting dilutions were analyzed by PCR using the developed specific oligonucleotide primers and a fluorescently labeled DNA probe.

The specificity of the developed method was tested on samples of feline calicivirus Feline calicivirus (FCV), as well as on samples of biological material obtained from intact and seronegative animals against feline calicivirus Feline calicivirus (FCV). and also using the Sanger sequencing method on the automatic sequencer ABI PRISM 3500 (USA) figure 1, figure 2, figure 3 in the application "Scheme".

Claims (1)

Set of specific oligodeoxyribonucleotide primers VT-Ca-F2 5'-gAACTACCCgCCAATCAACATgTg-3', VT-Ca-R2 5'-AgCACATCATATgCggCTCTgAT-3' and fluorescently labeled probe VT-Ca-P2 5'-(R6G)-gTgTTTgATTTggCCTgggCTCTTCg-( BHQ2 )-3' to the ORF1 RNA (cDNA) gene fragment of Feline calicivirus (FCV) for detection of Feline calicivirus (FCV).