RU2700481C1 - Method for detecting rna of an arteritis virus agent in horses - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is a method for detecting RNA of an agent of arteritis virus in horses, involving RNA extraction from biological material of infected individuals by a sorption method, synthesis of cDNA on an RNA matrix by single-step staging with addition of internal and positive controls of samples of a multiplex reverse transcription reaction and polymerase chain reaction with 45 cycles of amplification with real-time detection using oligonucleotide primer-specific oligonucleotide primers, a fluorescent-labeled probe and control samples, measuring accumulation of a fluorescent signal through channels of corresponding fluorescent dyes and interpreting results based on presence or absence of intersection of the fluorescence curve with a threshold line – Threshold, according to the invention, the biological material of animals is used for RNA extraction by choice: in the form of discharge from nose, eyes, faeces, blood, sperm and fragments of internal organs and tissues taken from horses infected with the arteritis virus (Equine arteritis virus) agent, wherein for internal control sample suspension of bacteriophage MS2 with concentration of 5×10³ copies of nucleotide sequences per 1 mcl, and for a positive control sample, a mixture of recombinant plasmid DNA containing a fragment of the genome of the horse arteritis virus (Equine arteritis virus) and a fragment of the bacteriophage MS2 genome, taken in ratio 1:1, FAM/Green for a specific signal; Cy5/Red for internal control signal, if the accumulation curves of the fluorescent signal reach 35th cycle, the result of the reaction is considered positive, and if the curves do not cross the threshold line or cross it after 35 cycles, the reaction result is negative.

EFFECT: invention allows to expand functional capabilities and to conduct reliable diagnostics of the disease.

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/virusnyi-arteriit-loshadej

There is also a known method for detecting infections by multiplex PCR with real-time detection (RF patent No. 2460803, C12Q 1/68, 2014 - prototype), including the isolation of RNA from biological material of infected individuals by the sorption method, cDNA synthesis on the RNA matrix by staging one-stage with the addition of internal and positive controls of samples of the multiplex reverse transcription reaction and polymerase chain reaction with 45 amplification cycles with real-time detection using specific for portion of the genome of the viral pathogen oligonucleotide primers fluorescently-labeled probe and reference samples, measuring the accumulation of the fluorescent signal on channels corresponding fluorescent dyes and interpretation of the results based on the presence or absence of the intersection of the curve with the fluorescence threshold line -Threshold.

The disadvantage of the prototype is the lack of the possibility of obtaining reliable diagnosis of the arteritis virus in horses.

The technical result is to obtain reliable diagnosis of the disease and the expansion of functionality.

The technical result is achieved by the fact that in the method for detecting RNA of the causative agent of the arteritis virus in horses, including the extraction of RNA from biological material of infected individuals by the sorption method, cDNA synthesis on the RNA matrix by staging one-step with the addition of internal and positive control samples of the multiplex reverse transcription reaction and polymerase chain reaction with 45 cycles of amplification with real-time detection using site-specific viral pathogen genome ligonucleotide primers, fluorescently-labeled probe and control samples, measuring the accumulation of the fluorescent signal through the channels of the corresponding fluorescent dyes and interpreting the results based on the presence or absence of the intersection of the fluorescence curve with the threshold line - Threshold, according to the invention, horses use the biological material of their choice: discharge from the nose, eyes, feces, blood, semen and fragments of internal organs and tissues taken from horses of infected pathogens m virus arteritis (Equine arteritis virus), wherein for the internal control sample is a suspension of bacteriophage MS2 at a concentration of 5 × ¹⁰ March copies of nucleotide sequences to 1 l, and for the positive control sample is a mixture of recombinant plasmid DNA containing a fragment of the genome of the pathogen viral arteritis horses (Equine arteritis virus) and a fragment of the genome of the bacteriophage MS2 taken in a 1: 1 ratio, with the following nucleotide sequences:

wherein the accumulation of the fluorescent signal is measured by the channels: FAM / Green for a specific signal; Cy5 / Red for the internal control signal, 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 novelty of the claimed technical solution lies in the fact that to obtain a reliable diagnosis of equine arteritis virus, two sequential reactions are used: 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 PCR tube. 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 method is recommended for use in veterinary virology, and in particular to diagnostic tools for the causative agent of the horse arteritis virus, which meets the criterion of "industrial applicability".

The invention is illustrated by 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).

A method for detecting RNA of the causative agent of the arteritis virus in horses is as follows

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 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 1: 1 ratio, 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, a BHQ-1 quencher is attached at the 3'-end of the oligonucleotide probe.

Using the program "Oligo 6.0" describes the basic properties of the calculated oligonucleotides, 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, the 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.

An example of a specific implementation of the method for detecting RNA of an arteritis virus pathogen in horses.

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 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.

* Light opalescence possible

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) 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 a negative control sample (OKO), 10 μl of an 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, and for a positive control sample (PAV EAV) use a mixture of recombinant plasmid DNA containing a fragment of the genome of the causative agent of the horse arteritis virus (Equine arteritis virus) and a fragment of the genome of the bacteriophage MS2 taken in a 1: 1 ratio, with the following nucleotide sequence awns:

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

To prepare samples for the NDP, the total volume of the reaction mixture is 25 μl, the volume of the RNA sample is 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 test tube with a positive PCR control (K +) contribute 10 μl of PCO EAV.

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

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

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 RNA 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 (8)

A method for detecting DNA of the causative agent of arteritis virus in horses, including the extraction of RNA from biological material of infected individuals by the sorption method, the synthesis of cDNA on the RNA matrix by staging one-step with the addition of internal and positive control samples of a multiplex reverse transcription reaction and polymerase chain reaction with 45 amplification cycles with detection in real time using oligonucleotide primers specific for the viral pathogen genome region, fluorescence ennogo probe and reference samples, measuring the accumulation of the fluorescent signal on channels corresponding fluorescent dyes and interpretation of the results based on the presence or absence of the intersection of the fluorescence curve with the threshold line - Threshold, characterized in that for the internal control sample is a suspension of bacteriophage MS2 at a concentration of 5 × ¹⁰ March copies of nucleotide sequences per 1 μl, and for a positive control sample using a mixture of recombinant plasmid DNA containing a fragment the genome of the causative agent of equine arteritis virus (Equine arteritis virus) and a fragment of the genome of the bacteriophage MS2, taken in the ratio 1: 1, with the following nucleotide sequences:

- direct primer

- reverse primer

- probe wherein the accumulation of the fluorescent signal is measured by the channels: FAM / Green for a specific signal; Cy5 / Red for the internal control signal, 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.

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