RU2714045C1 - Oligonucleotide primers and a fluorescent-labeled probe, a method and a test system for detecting genome of virus of infective nodular dermatitis (nodular dermatitis) of cattle in real-time polymerase chain reaction - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is represented by oligonucleotide primers and a fluorescent-labeled probe for amplification and detection of a fragment LSDV044 of a genome of a virus of infectious nodular dermatitis of cattle, in which other representatives of the family Capripoxviridae have no deletion of 3 bp. in this locus, while it is absent in IND viruses. What is also presented is a method for detecting the virus genome of cattle IND using these primers and a probe and a PCR test system in real time for conducting it. PCR-RV is used to amplify fragment of LSDV044 gene of cattle IND virus. High-sensitivity kit consisting of a ready PCR mixture, Taq-DNA polymerase, positive control and negative control is developed, which enables to detect genome of the cattle IND virus genome in the shortest possible time. Invention can be used for detection of DNA of all genetic variants of viruses of CRM of cattle in samples of a wide spectrum of pathological and biomaterial.

EFFECT: disclosed are oligonucleotide primers and a fluorescent-labeled probe, a method and a test system for detecting the viral genome of infective nodular dermatitis (nodular dermatitis) of cattle in a real-time polymerase chain reaction.

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Description

The invention relates to veterinary virology, to molecular diagnostics, and in particular to the detection of the genome of the virus of infectious nodular dermatitis (nodular dermatitis) in cattle (cattle itching).

The itch virus of cattle belongs to the genus Capripoxvirus and is the causative agent of cross-border cattle disease, which poses a serious threat to cattle breeding not only in the Russian Federation, but also in other countries, causing significant economic losses to households and economies [1, 2].

Itching of cattle is accompanied by fever, the formation of skin lesions on the skin and mucosa [3]. Mostly, cattle pruritus was recorded in the countries of the African continent, showing a seasonal pattern with a maximum distribution with an abundance of bloodsucking insect populations [4]. For the first time on the territory of the Russian Federation, outbreaks of cattle itching were recorded in 2015 and were caused by a field isolate [5]. However, already in 2017, in a number of regions of the Volga Federal District, in the biomaterial from cattle outbreaks, laboratory studies revealed a vaccine-like isolate of the itch virus belonging to the Neethling genetic line, which differed in genetic properties from the field isolate [6]. Based on the foregoing, the use of such diagnostic methods is required that would allow screening studies for all possible genetic variants of the itching virus of cattle.

A known method for the diagnosis of cattle itching by the virological method of isolating the pathogen in sensitive cell cultures, based on the specific manifestation of the action of the virus in biological systems [7]. The disadvantages of this method are: the duration, the complexity and the need to use biological models.

There is a method for the diagnosis of capricoxvirus infections, which include cattle pruritus, through amplification of a fragment of the P32 gene of capricoxvirus genomes [8]. The disadvantage of this approach is that according to this conservative locus, it is impossible to differentiate between capricox viruses.

A known method, which is based on the detection of itch virus of cattle, smallpox virus of goats and smallpox virus of sheep by amplification of DNA fragments with subsequent analysis of their melting curves with high resolution [9]. The disadvantage of this method is the high dependence of the method on the quality and concentration of DNA in the test sample.

Closest to the claimed method is a test system for detecting the genome of field viruses of infectious nodular dermatitis of cattle using real-time PCR based on the EEV126 gene region [10]. Despite its high sensitivity and specificity, this method cannot detect vaccine-like isolates of the itch virus.

Thus, a technical problem is the development of a reliable method with the ability to study a wide range of biological material (internal organs, nodules, blood serum, whole blood, cell culture) that is sensitive and specific for the detection of genomes of all possible genetic variants of the cattle itch virus by polymerase chain reaction in real-time by constructing a kit containing specific oligonucleotide primers and a fluorescently-labeled probe, as well as selecting conditions for SIC PCR with them, ensuring minimal risk of contamination in the laboratory and eliminates subjectivity in assessing results.

The invention relates to a test system consisting of oligonucleotide primers and a fluorescently-labeled probe for specific express identification of the genome of all genetic variants of cattle pruritus by real-time polymerase chain reaction. The presented method includes sequences of oligonucleotides specific for the itch virus of cattle only and having the following nucleotide sequence:

zdf41n CAA + AAA + CAA + TCG + TAAC + TAATCCA

zdr41n TG + GAGTTTTTA + TG + TCATCGTC

zdpro41nl FAM TC + GT + CGT + CG + TT + TAA + AACTGA BHQ1

The “+” sign indicates modifications to the LNA. FAM dye is used as a fluorescence source at the 5 'end of the probe, and BHQ1 is used to quench fluorescence at the 3' end. Fluorescence is measured by the FAM channel. The intersection of the fluorescence curve of the threshold line indicates the presence of an itch virus in the test sample in the sample genome, the lower the "Ct" indicator, the higher the genome of the itch virus. The absence of an intersection of the fluorescence curve of the threshold line indicates the absence of the genome of the itchy cattle virus in the test material.

The invention can be used in veterinary medicine, clinical and laboratory diagnostics to detect DNA of the itch virus of cattle in samples of biological and cultural material.

The technical result of the invention is: high specificity with respect to all possible genetic variants of the itch virus of cattle; an expanded range of biological material suitable for research without the possibility of obtaining false positive results; the possibility of using a wide range of amplifiers that meet the minimum requirements for PCR; reduction of time for mass studies of samples for the presence of the genome of the itch of cattle virus, as well as the expansion of the arsenal of diagnostic tools for the itch of cattle.

The essence of the invention lies in the fact that the specific detection of DNA of the itch virus of cattle is carried out in the presence of related capricoxviruses (smallpox viruses of sheep and goats) without any influence of the latter. Using the indicated primers (zdf41n, zdr41n) and a probe (zdpro41nl), a real-time polymerase chain reaction (PCR - RV) is carried out, which allows the genome of the itchy cattle virus to be detected. As a target, a fragment of the LSDV044 gene encoding the transmembrane domain is present, which is present in all genetic variants of the itch Bovine virus. The high degree of specificity of the test system is confirmed by comparing the currently published sequences of capripoxviruses with similar nucleotide sequences of other capripoxviruses (GenBank database), as well as using laboratory tests.

The invention is illustrated in the graphic material:

FIG. 1 - the linearity of the PCR-RV results is presented when testing 10-fold dilutions of the isolated DNA of the RED cattle virus (the strain of the nodular dermatitis virus “VND Bovine / Dagestan / 2015”);

FIG. 2 - amplification curves are presented when testing five consecutive 10-fold dilutions of the isolated DNA of the cattle itch virus (nodular dermatitis virus strain "GNI cattle / Dagestan / 2015")

The amplification products are detected by recording the fluorescence generated as a result of the destruction of the hybridization probe located at the 5'-end of the FAM fluorophore, and at the 3'-end of the BHQ1 quencher. In the absence of a target, the fluorophore and the quencher are close, and only slight fluorescence is observed, since the quencher absorbs the radiation emitted by the fluorophore. When specific products are accumulated during PCR, the probe hybridizes to an amplicon, which leads to its destruction due to the 5'-exonuclease activity of Taq polymerase. As a result, the fluorophore is separated from the quencher and its radiation can be detected. Thus, the increase in fluorescence is directly proportional to the amount of synthesized PCR product.

Primers and probe are designed for amplification and detection of the LSDV044 fragment, in which other representatives of this family. Capripoxviridae missing 3 bp deletion at this locus, and it is present in isolates of the itch of cattle viruses.

For the development of primers, genome sequences of capripox viruses were obtained from the GenBank database. The sequences were aligned using the Bioedit program, then conservative sites were visually evaluated, based on which a number of primers were obtained. As a result of testing for various parameters, an optimal pair of primers and probe was obtained, which is used in the test system.

The kit for detecting the genome of the itch virus of cattle in PCR-RV consists of the following components:

No. 1 finished PCR mixture, volume 550 μl - 2 tubes;

No. 2 enzyme Taq-DNA polymerase, volume 12.5 ml - 1 tube;

No. 3 positive control sample, volume 100 μl - 1 tube;

No. 4 negative control sample, volume 100 μl - 1 tube.

PCR-RV is carried out in one step using the finished PCR mixture (No. 1), which includes 5x PCR-RV buffer (5 μl), 25 mM magnesium chloride (3.5 μl), triphosphate deoxyribonucleotide (0.5 μl) for one reaction 10 pmol), oligonucleotide primers (1 μl of each primer 15 pmol), probe (1 μl 2.5 pmol) and Taq-DNA polymerase enzyme in a programmable amplifier. Before starting the reaction, it is necessary to defrost all the necessary components of the reaction, shake on a shaker, then centrifuge for several seconds on a low-speed centrifuge.

The reaction mixture for PCR-RV is prepared in vitro per reaction (V = 25 μl) as follows:

- finished PCR mixture No. 1 20 μl - enzyme Taq-DNA polymerase No. 2 0.25 μl - isolated DNA (negative control No. 3 or positive control No. 4) 5 μl

The PCR reaction mixture prepared in separate 1.5 ml tubes was transferred to 0.2 ml tubes of 20 μl and 5 μl of total DNA was added. Isolated DNA samples, negative and positive controls are also added to the appropriate samples. The total volume of the mixture is 25 μl.

Set the tubes in the amplifier for the PCR-RV formulation, note the location and characteristics of the samples in the program, select a working dye (FAM), set the temperature-time profile of the reaction in the program.

After initial denaturation at 95 ° C for 10 min, real-time PCR is carried out under the following conditions: 95 ° C - 15 s, 60 ° C - 60 s - 45 cycles.

The results are interpreted on the basis of the presence or absence of the intersection of the fluorescence curve with the threshold line, which corresponds to the presence or absence of the threshold cycle value "Ct" in the corresponding column in the table of reaction results derived from machine analysis.

The result is considered reliable only in the case of passing the positive (Ct≤35) and negative (Ct not defined) amplification controls.

The sample is considered positive for the presence of DNA of the RED herd virus, if the Ct value is not more than 35. However, if the Ct value for the samples is in the range from 36 to 40, it is necessary to repeat the reaction in order to confirm or deny the presence of virus DNA in the test sample.

A sample is considered negative for the presence of virus DNA if there is no Ct value for it.

The results are not subject to accounting (considered invalid):

- in the absence of registration of fluorescence growth in the sample with positive control, which may indicate errors made at the stage of PCR-RV;

- in the case of registration of the Ct value in the table of results on the computer screen for a negative control sample, which indicates the presence of contamination.

In any of these cases, it is necessary to repeat the analysis of all samples, and when re-identifying the contamination of the negative control, take measures to identify and eliminate the source of contamination.

The essence of the invention is illustrated by examples of its use, which do not limit the scope of the invention.

Example 1. Assessment of the specificity of the test system.

To assess the specificity of the test system, we used the following samples of homologous and heterologous viruses deposited in the collection of microorganism strains of FSBI “ARRIAH”: DNA of the itch virus of the strain “GNI KRS / Dagestan / 2015” (diagnostic), DNA of the virus of nodular dermatitis strain “E-95” , DNA of vaccine strain Neethling, DNA of smallpox virus of sheep vaccine strain "Afghan", DNA of smallpox virus of sheep vaccine strain "ARRIAH", DNA of virus of smallpox goat virus of strain "Primorsky 2003", DNA of smallpox virus of goats strain "ARRIAH 2003", DNA of small ruminant plague virus strain "ARRIAH" DNA vi baleen strain of vesicular stomatitis "ARRIAH" cowpox virus DNA strain "ARRIAH".

Work with DNA was carried out under the conditions regulated by the Methodological Instructions of MU 1.3. 2569-09 “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV” [11].

The procedure for DNA extraction from the test material was carried out using a set of reagents based on membrane columns.

Real-time PCR was carried out in the reaction mixture of the following composition (for 1 study):

- finished PCR mixture No. 1 20 μl - enzyme Taq-DNA polymerase No. 2 0.25 μl - isolated DNA (negative control No. 3 or positive control No. 4) 5 μl

Real-time PCR and recording of the results was carried out in a PCR-RV device according to the program described above.

The results were interpreted based on the presence (or absence) of the intersection of the fluorescence curve with the threshold line set at the corresponding level, which corresponds to the presence (or absence) of the threshold cycle Ct in the corresponding column in the results table. The result was considered positive if the fluorescence accumulation curve for the corresponding sample had a characteristic "sigmoid" shape and crossed the threshold line.

When testing the specificity of the test system using DNA of homologous and heterologous viruses, false-positive and false-negative results were not found.

Example 2. Assessment of the sensitivity of the test system.

To assess the sensitivity of the test system, the DNA of the itch virus of cattle of the GNI / Dagestan / 2015 strain with a titer of 5.23 lg TCD ₅₀ / cm ^{3 was used} [12]. The test system detected viral DNA with a sensitivity of 0.23 lg TCD ₅₀ / cm ³ . To evaluate the amplification efficiency, 3 repeated experiments were carried out and Ct values were obtained, which were used to calculate the efficiency. A linear regression was obtained with an amplification efficiency (E) value of 93%. The data obtained are shown in a graphic image - FIG. 1.

Example 3. Evaluation of the reproducibility of the test system.

The reproducibility of the test system was determined using the standard deviation (SD) for each series of dilutions using the obtained Ct values. SD ranged from 0.02 to 0.16 over five 10-fold dilutions (Fig. 2). The coefficient of determination g ₂ amounted to 0.999.

Thus, the invention can be used in veterinary practice to identify the genetic material of the itchy cattle virus in biological and cultural samples for diagnosis and to clarify the diagnosis, to solve research problems in monitoring the spread of the itchy virus of cattle among susceptible animals. The use of specific primers and a probe makes it possible to identify the genetic material of all genetic variants of cattle itching in the studied samples by the method of polymerase chain reaction (PCR) with hybridization-fluorescence detection in real time (RV).

Sources of information taken into account when drawing up the description of the invention to the application for the grant of a patent of the Russian Federation for the invention “Oligonucleotide primers and fluorescently-labeled probe, method and test system for detecting the genome of the virus of infectious nodular dermatitis (nodular dermatitis) cattle in the polymerase chain reaction in real time. "

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Claims (6)

1. A kit comprising oligonucleotide primers and a fluorescently-labeled probe for amplification and detection of a fragment of the gene for the outer capsid protein EEV of the virus of infectious nodular dermatitis in cattle zdf4ln CAA + AAA + CAA + TCG + TAAC + TAATCCA zdr4ln TG + GAGTTTTTA + TG + TCATCGTC zdpro4lnl FAM TC + GT + CGT + CG + TT + TAA + AACTGA BHQ1. 2. A method for detecting the genome of the field virus of RED cattle using oligonucleotide primers and a probe according to claim 1 in the PCR-PB reaction, consisting of the following steps: initial denaturation at 95 ° C for 10 min and thermal cycling at 95 ° C for 15 s , 60 ° С - 60 s - 45 cycles; at the same time, the sample is considered positive for the presence of DNA of the itch virus of cattle, if the Ct value is not more than 35, the sample is considered negative for the presence of virus DNA, if the Ct value is absent or more than 37 and if the Ct value for the samples is in the range from 36 to 40, it is necessary to repeat a reaction to confirm or deny the presence of virus DNA in the test sample. 3. A test system for detecting the genome of the itchy cattle virus in the PCR-RV reaction, including the finished PCR mixture, Taq-DNA polymerase enzyme, positive control sample, negative control sample, kit according to claim 1.

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