RU2698662C1 - Test system for detecting rna of agent of arteritis virus in horses - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is a test system for detecting an RNA of an arteritis virus agent in horses, comprising a buffer for carrying out a polymerase chain reaction with fluorescent detection in real time, a mixture for carrying it out, consisting of deoxynucleoside triphosphates, primers and fluorescent probes specific for an infection virus agent and for an internal control sample; mixture of enzymes from DNA polymerase with antibodies inhibiting activity of TAQ POLYMERASE enzyme; RNA dilution buffer, internal control sample, negative control sample, positive control sample, according to the invention, the biological material extracted from the arteritis virus (Equine viral arteritis – EAV) infected horses is used for RNA isolation, wherein for internal control sample suspension of bacteriophage MS2 with concentration of 5×10³ copies of nucleotide sequences at 1 mcl, and for a positive control sample – a mixture of recombinant plasmid DNA containing a fragment of genome of arteritis virus (Equine viral arteritis – EAV) in horses and a fragment of genome of bacteriophage MS2, taken in ratio 1:1.

EFFECT: invention enables authentically diagnosing RNA of an arteritis virus agent in horses.

1 cl, 3 dwg, 4 tbl

Description

The invention relates to veterinary virology, and in particular to means for diagnosing viral and infectious diseases in animals, in particular to methods for detecting DNA of the causative agent of the arteritis virus in horses.

Horse viral arteritis (Arteriitis viralis equorum, Equine viral arteritis) is a contagious disease characterized by necrosis of the blood vessel wall, catarrh of the respiratory and digestive organs in foals, abortions in mares and impaired reproductive function in stallions.

It is known that the diagnosis of viral arteritis is established on the basis of epizootological, clinical data and, in the case of death of animals, pathomorphological studies. Laboratory diagnostic methods include the isolation of the virus in cell culture, retrospective serological studies. To isolate the virus, horse cell cultures are used, transplanted lines of BHK-21, Vero and others. The virus is identified in the neutralization reaction with a specific serum. Alternative (auxiliary) methods of laboratory research are RSK, ELISA. A polymerase chain reaction for the diagnosis of viral arteritis in horses is under study. http://www.liveanimal.ru/loshadi/veterinariya/zaraznye-zabolevaniya/infektsionnye-zabolevaniya/virusnyj-arteriit-loshadej

A known test system for detecting RNA of an infectious disease virus by conducting a real-time polymerase chain reaction using oligonucleotide primers specific for the genome of the pathogen infection, a fluorescently-labeled probe and control samples (RF patent No. 2515916, class C12N 15/11, 2014).

Also known is a test system for detecting the genome of an infectious agent using a multiplex polymerase chain reaction with real-time detection, including a buffer for polymerase chain reaction, a mixture for its conduct consisting of deoxynucleoside triphosphates, primers and fluorescent probes specific for coronavirus, a mixture of enzymes from DNA polymerase with antibodies inhibiting enzyme activity, TAQ POLYMERASE and reverse transcriptase MMLV REVERSE TRANSCRIPTASE; buffer for dilution of RNA in the form of deionized water, an internal control sample, a negative control sample, a positive control sample, (RF patent No. 2506317, C12Q 1/68, 2014 - prototype).

A common disadvantage of the known technical solutions is the inability to diagnose the DNA of the causative agent of the arteritis virus in horses.

The technical result is the expansion of functionality and obtaining reliable diagnostics using RT - PCR in real time.

The technical result is achieved by the fact that in the test system for detecting RNA of the causative agent of the arteritis virus in horses, which includes a buffer for polymerase chain reaction with fluorescence detection in real time, the mixture for its conduct consisting of deoxynucleoside triphosphates, primers and fluorescent probes specific for the virus pathogen infections and for the internal control sample; a mixture of enzymes from DNA polymerase with antibodies that inhibit the activity of the enzyme, TAQ POLYMERASE; RNA dilution buffer, internal control sample, negative control sample, positive control sample, according to the invention, biological material taken from horses infected with the pathogen of arteritis virus (Equine viral arteritis-EAV) is used to isolate RNA, while a bacteriophage suspension is used for the internal control MS2 with a concentration of 5 × 10 ³ copies of nucleotide sequences per 1 μl, and for a positive control sample, a mixture of recombinant plasmid DNA containing a fragment the genome of the arteritis virus (Equine viral arteritis-EAV) in horses and a fragment of the genome of the bacteriophage MS2, taken in the ratio 1: 1, with the following nucleotide sequences:

The novelty of the claimed technical solution lies in the fact that to obtain a reliable diagnosis of equine arteritis virus, a test system is used to conduct two sequential reactions: reverse transcription of viral RNA to obtain cDNA and polymerase chain reaction for amplification of a fragment of the obtained cDNA matrix. Both reactions are carried out sequentially in one PCR tube (one-tube) using oligonucleotide primers specific for the arteritis genome region, a fluorescently-labeled probe, and different types of control for which different forms of MS2 bacteriophage material are used: suspension and a fragment of the genome with primers specific to it and a probe. This real-time RT-PCR formulation shortens and simplifies the analysis procedure, reduces the risk of contamination and the possibility of errors when transferring cDNA to another tube for PNR. In addition, the detection of amplification products is carried out using the principle of cleavage of the fluorescent label at the 5 'end of the oligonucleotide probe.

The features that distinguish the claimed technical solution from the prototype are aimed at achieving a technical result and have not been identified in the study of this and related fields of science and technology and, therefore, meet the criterion of "inventive step".

The inventive test system is recommended for use in veterinary virology, and in particular to diagnostic tools for the pathogen of horse arteritis virus, which meets the criterion of "industrial applicability".

The invention is illustrated in the drawing, which shows screenshots of graphs and tables, in Fig. 1 - the FAM / Green channel is presented - for testing the presence of DNA of the causative agent of the arteritis virus virus in horses; in fig. Figure 2 shows the channel Cy5 / Red - for testing the signal of the internal control sample (CTP), in Fig. 3 is a table of quantitative data for Cycling A.Green (Equine arteritis virus) and A.Red (EKO).

An example of a specific use of a test system for the detection of horse arteritis virus pathogen RNA in horses

For research, RNA is isolated using the biological material of animals of their choice: in the form of secretions from the nose, eyes, feces, blood, semen and fragments of internal organs and tissues taken from horses infected with the causative agent of the arteritis virus (Equine arteritis virus). For the internal control sample, a suspension of bacteriophage MS2 with a concentration of 5 × 10 ³ copies of nucleotide sequences per 1 μl is used, and for a positive control sample, a mixture of recombinant plasmid DNA containing a fragment of the equine arteritis virus pathogen genome and a bacteriophage MS2 genome fragment taken in a ratio of 1: 1, with the following nucleotide sequences:

Then measure the accumulation of the fluorescent signal in the channels: FAM / Green for a specific signal; Cy5 / Red for the internal control signal and interpret the results based on the presence / absence of the intersection of the fluorescence curve with the threshold line (Threshold). If the fluorescence signal accumulation curves go out before the 35th cycle, then the reaction result is considered positive, and if the curves do not cross the threshold line or cross it after the 35th cycle, the reaction result is negative.

The use of different forms of the material of the bacteriophage MS2 for different types of control: suspension and a fragment of the genome with specific primers and a probe is due to the fact that this allows you to control the correct course of the reaction in each tube, and also controls the stage of RNA extraction from samples.

Sequence selection and calculation of the primary structure of oligonucleotide primers and probes.

Arteritis virus is a member of the family Arteriviridae, genus Arierivirus. Virions about 60 nm in size, spherical in shape. The type of symmetry is cubic; some of them have tail-like protrusions. They consist of a nucleocapsid and a lipid-containing supercapsid membrane of cell origin, on the surface of which there are protrusions 12-15 nm long. The genome is represented by 10 fragments of a 2-strand RNA. Its protein capsid consists of 7 structural proteins (36-120 kD).

Primers are highly specific for short conserved regions of the ORF5 gene (Equine arteritis virus strain SER-1 large envelope protein (ORF5) gene, partial cds, access code KX645659.1, region 70 and 390) and are not complementary to any regions of the genomes of other viruses. Primers were designed using Primer Express Software v3.0 (Applied Biosystems) and tested using BLAST to confirm their specificity. To detect amplification products, an EAVP oligonucleotide fluorescently-labeled probe (complementary to the nucleotide sequence region limited by the annealing positions of the EAVF and EAVR primers) was selected. The probe was labeled with a FAM dye. To quench spontaneous fluorescence at the 3'-end of the oligonucleotide probe, a BHQ-1 quencher is attached. Using the Oligo 6.0 program, the basic properties of the calculated oligonucleotides are described, which determined the possibility of their use in PCR.

Using the Oligo 6.0 program, the nucleotide sequence of the bacteriophage MS2 (Enterobacteria phage MS2 isolate ST4, complete genome. ACCESSION EF204940) was analyzed. The bacteriophage MS2 contains single-stranded positively oriented RNA of 3569 bases. As a result of the analysis within the maturation protein gene, a region between 200 and 350 nucleotides containing unique nucleotide sequences was selected; primary structures of oligonucleotide primers flanking the selected genome region were calculated. An MS2P oligonucleotide fluorescently labeled probe complementary to the nucleotide sequence region bounded by the annealing positions of the MS2F and MS2R primers was selected to detect amplification products. Using the program "Oligo 6.0" describes the basic properties of the calculated oligonucleotides, which determined the possibility of their use in PCR.

For research using the following material:

- Scrapes from the mucous conjunctiva of the eyes and oropharynx are taken with dry cotton probes;

- Feces weighing 5 g are collected in a sterile plastic container;

- Sperm, taken into a 1.5 ml plastic microtube

- To obtain serum, blood is taken into a test tube without an anticoagulant;

- Fragments of internal organs and tissue (trachea, lungs, spleen, brain, air sacs, intestines, lymph nodes) are placed in a sterile container.

Scrapes from the mucous membranes of the conjunctiva and oropharynx in physiological saline are examined without prior preparation.

Sperm is diluted with saline 1: 3, thoroughly mixed on a vortex. An aliquot of 0.1 ml of suspension is used for RNA extraction.

To obtain serum, blood tubes (without anticoagulant) are allowed to stand at room temperature for 30 minutes until a clot forms completely. It is then centrifuged at 600-1600 g (3000 rpm on a MiniSpin centrifuge, Eppendorf, Germany) for 10 minutes at room temperature. The serum is transferred by separate filter tips to sterile 1.5 ml tubes.

Small pieces of up to 1 g in weight are cut from tissues and organs. The samples are ground in separate porcelain mortars or homogenized on automatic homogenizers. Then prepare a 10% suspension in sterile saline or phosphate buffer. The suspension was transferred to a 1.5 ml tube and centrifuged at 9000 rpm on a MiniSpin centrifuge, Eppendorf, for 1 min. An aliquot of the supernatant (0.1 ml) is used for RNA extraction.

From feces (1-5 g) prepare a 10% suspension in sterile saline. A feces suspension is decanted for 5-10 minutes. 1 ml of supernatant was collected and transferred to a clean 1.5 ml tube, centrifuged at 5000 rpm on a MiniSpin centrifuge, Eppendorf, for 5 minutes. RNA extraction from clarified feces extract is carried out immediately, if possible. If necessary, the storage is frozen.

The analysis is carried out using a set of reagents "PCR-ARTERIIT-FACTOR" by RT-PCR RV:

The kit consists of a kit of reagents for conducting multiplex RT-PCR RV (Kit No. 1) and control samples (Kit No. 2).

The kit is available in two versions:

1) For analysis of 55 samples (including control samples)

2) For analysis of 110 samples (including control samples).

The composition of the kit is shown in Tables 1 and 2.

The kits are used in accordance with the instructions for the use of the PCR-ARTERIIT-FACTOR reagent kit to detect equine arteritis virus RNA in biological material from animals by the combined reverse transcription reaction and polymerase chain reaction with real-time fluorescence detection ( FROM RT PCR), TU 21.10.60-126-51062356-2017 (for in vitro diagnostics)

^* Light opalescence possible

http://www.vetfaktor.ru/.

The kit does not include reagents for the extraction of NK. RNA isolation can be carried out, for example, using sets based on the sorption method, which include silica or microcentrifuge columns, sets based on phenol-chloroform extraction, etc. It is recommended to use the DNA / RNA-S-FACTOR kit or similar.

The analysis consists of three stages:

- NK extraction (at this stage, reagents for extraction are additionally used, for example, the DNA / RNA-C-FACTOR kit);

- carrying out the RT-PCR reaction;

- accounting for the results of the analysis.

The extraction (isolation) of nucleic acids (NK) from the studied samples is carried out as follows.

The required number of 1.5 ml disposable tubes was selected, including a negative selection control. Pipette into all tubes, including a tube for negative control sample (OKO), 10 μl of internal control sample (EQA EQ), which is used as a suspension of bacteriophage MS2 with a concentration of 5 × 10 ³ copies of nucleotide sequences per 1 μl, if the concentration of copies of nucleotide sequences deviates up or down, then observed repetition of doubtful samples. For a positive control sample (EAV PEC), a mixture of recombinant plasmid DNAs containing a fragment of the equine arteritis virus pathogen genome and a bacteriophage MS2 genome fragment taken in a 1: 1 ratio with the following nucleotide sequences is used:

NK is isolated from the analyzed and control samples according to the manufacturer’s instructions for the NK extraction kit.

To prepare samples for PCR, the total volume of the reaction mixture was 25 μl, the volume of the RNA sample was 10 μl.

Successful completion of the reaction is monitored using components from sets 1 and 2 of the set of PAV EAV, VKO EAV and RNA Buffer.

In a separate tube, mix the kit components for each reaction: 10 μl PCR Buffer EAV 5 μl PCR MIX EAV 0.75 μl RT PCR ENZ

Vortex the mixture and mix the drops by short centrifugation. Select the required number of tubes for amplification of the NK of the studied and control samples and add 15 μl of the prepared reaction mixture to them.

Using tips with a filter in prepared tubes make:

a) in a test tube negative control PCR (K-) 10 μl of RNA buffer;

b) in a row of test tubes for the test samples - 10 μl of the corresponding sample of NK are introduced into each;

C) in a tube with a positive PCR control (K +) contribute 10 μl of PAV EAV.

Prepare the tubes prepared for PCR in the amplifier cells, program the device according to the manufacturer's instructions and interpret the analysis results

Next, carry out PCR RT with fluorescence detection using a device - amplifier type "Rotor-Gene Q" in the following modes shown in table 3.

The data obtained - the fluorescence signal accumulation curves are analyzed using the software of the device used for PCR in accordance with the manufacturer's instructions for the device. Analysis of the results of RT-PCR of the RT is carried out by the presence or absence of the intersection of the fluorescence curve with the threshold line set at the appropriate level (which corresponds to the presence or absence of the threshold cycle value “Ct” for the test sample).

The result is considered reliable if the positive and negative controls for amplification and extraction of NK are correctly passed in accordance with table 4.

The appearance of any Ct value in the results table for the negative control of the VK- extraction stage on the Fam / Green channel and for the negative control of the K- PCR stage on any of the channels indicates the presence of reagent or sample contamination. In this case, the results of the analysis for all samples are considered invalid. It is required to repeat the analysis of all samples, as well as take measures to identify and eliminate the source of contamination.

Samples for which the Ct value is absent or exceeds the 35th cycle on the Cy5 / Red channel (while the Ct value is absent on the Fam / Green channel), a repeated study is required (Fig. 2, 3). A delay in the threshold cycle values for the studied samples on the Cy5 / Red channel indicates the presence of inhibitors in the sample (s) or errors in the formulation of the RT-PCR reaction. A study is required starting from the stage of NK extraction.

The sample is considered positive, the horse arteritis virus RNA is present if an exponential signal growth is observed on the Fam / Green channel, while the Ct values of the control samples are within normal limits (see Table 3, Fig. 1, 3). If the Ct value for the test sample through the Fam / Green channel is determined after the 37th cycle with the correct passage of the positive and negative controls, it is considered controversial and re-examined from the stage of NC extraction. If during re-staging a similar result is observed (Ct on the Fam / Green channel is more than 37), re-sampling of the material from the same animal is required for PCR studies and / or the use of alternative diagnostic methods.

Claims (2)

A test system for detecting arteritis virus pathogen DNA in horses using real-time polymerase chain reaction with fluorescence detection, including a buffer for polymerase chain reaction, a mixture for its conduct, consisting of deoxynucleoside triphosphates, primers and fluorescent probes specific for the virus pathogen infections and for the internal control sample; a mixture of enzymes from DNA polymerase with antibodies that inhibit the activity of the enzyme, TAQ POLYMERASE; RNA dilution buffer, internal control sample, negative control sample, positive control sample, characterized in that for the internal control sample, a suspension of bacteriophage MS2 with a concentration of 5 × 10 ³ copies of nucleotide sequences per 1 μl is used, and for a positive control sample, a mixture of recombinant plasmid DNA containing a fragment of the genome of the arteritis virus (Equine viral arteritis - EAV) in horses and a fragment of the genome of the bacteriophage MS2, taken in a 1: 1 ratio, with the following nucleotide sequences vegetable features:

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