RU2700449C1 - Method for detecting dna of rhinotracheitis virus (bovine herpes virus 1, bohv-1) in cattle - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is a method for detecting DNA of rhinotracheitis virus (bovine herpes virus 1, BoHV-1) in cattle, involving DNA recovery by sorption method, staged one-stage polymerase chain reaction with simultaneous amplification cycles with real-time detection using oligonucleotide primers, probes, dyes and control samples specific for genome segment of rhinotracheitis virus (bovine herpes virus I, BoHV-1) in the form of internal and positive, measurement of specific signal and control signal via channels of corresponding dyes and interpretation of results, according to the invention, fluorescent detection is carried out, measuring the JOE(HEX)/Yellow accumulation of the fluorescent signal for a specific DNA signal of rhinotracheitis virus (bovine herpes virus 1, BoHV-1), and FAM/Green channel signal internal control sample and interpretation of results is carried out based on presence/absence of intersection of fluorescence curve with threshold line, if the accumulation curves of the fluorescent signal reach 35th cycle, the result of the reaction is considered to be positive, and if the curves do not cross the threshold line or cross it after 35 cycles, the reaction result is negative, wherein for internal control sample suspension of bacteriophage T4 with concentration of 5×10³ copies of nucleotide sequences at 1 mcl, and for a positive control sample there used is a mixture of recombinant plasmid DNA containing a fragment of genome of DNA of rhinotracheitis virus (bovine herpes virus 1, BoHV-1) and a fragment of genome of bacteriophage T4, taken in ratio 1:1.

EFFECT: invention enables authentically diagnosing a causative agent of cattle rhinotracheitis virus DNA.

1 cl, 3 dwg, 4 tbl

Description

The invention relates to veterinary microbiology, in particular for laboratory diagnosis of infectious pathogens, namely, means for diagnosing infection in animals.

, комплиментарных высоко консервативной области генома вируса ринотрахеита у крупного рогатого скота гена ти-мидинкиназы (tk) (Kibenge F.S. et al, "Amplification of strains of bovine herpesvirus 1 by use polymerase chain reaction with primers in the thymidine kinase region", American Journal of Veterinary Research, 1994, Sep; 55(9):1206-1212).A known method for detecting rhinotracheitis virus DNA in cattle using a single-stage PCR using a pair of primers

complementary to the highly conserved region of the rhinotracheitis virus genome in cattle of the thymidine kinase (tk) gene (Kibenge FS et al, "Amplification of strains of bovine herpesvirus 1 by use polymerase chain reaction with primers in the thymidine kinase region", American Journal of Veterinary Research, 1994, Sep; 55 (9): 1206-1212).

There is also a method for detecting cattle rhinotracheitis virus DNA using specific oligonucleotide primers in the polymerase chain reaction (PCR), which involves the extraction of DNA from biological material from infected animals by the sorption method, the formulation of a one-stage polymerase chain reaction with simultaneous carrying out no more than 40 amplification cycles with detection in real time using oligonucleotide primers, probes, cr carriers and control samples in the form of internal and positive, measurement of a specific signal and a control signal through the channels of the corresponding dyes and interpretation of the results (patent RU No. 2259398, class C12N 15/38, C12Q 1/68, 2005, prototype).

However, in the known method, the sequence is directly read by electrophoregram. The length of the fragment that can be decoded by this method is limited by the resolution of the gel electrophoresis method, in addition - limited functionality.

A common disadvantage of the known technical solutions is the lack of the possibility of obtaining reliable diagnostics for identifying the genome of the rhinotracheitis virus in cattle.

The technical result is to obtain a reliable diagnosis of the pathogen DNA virus of rhinotracheitis in cattle. The technical result is achieved by the fact that in the method for detecting DNA of rhinotracheitis virus (bovine herpes virus 1, BoHV-1) in cattle, including DNA extraction by the sorption method, setting up a one-stage polymerase chain reaction - with simultaneous amplification cycles with real-time detection with the use of oligonucleotide primers, probes, dyes, and control samples in the form of internal and positive oligonucleotide primers specific for the genome of the rhinotracheitis virus (bovine herpes virus 1, BoHV-1) measurement of a specific signal and the control signal through the channels of the corresponding dyes and the interpretation of the results according to the invention carry out fluorescence detection, measure the accumulation of the fluorescent signal for the specific DNA signal of the rhinotracheitis virus (bovine herpes virus 1, BoHV-1) on the JOE (HEX) / Yellow channel, and on the FAM / channel Green signal of the internal control sample, and the results are interpreted based on the presence / absence of intersection of the fluorescence curve with the threshold line, if the fluorescence signal accumulation curves go out before the 35th cycle, then the result is the action 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, while for the internal control sample, a suspension of bacteriophage T4 with a concentration of 5 × 10 ³ copies of nucleotide sequences per 1 μl is used, and for positive the control sample uses a mixture of recombinant plasmid DNA containing a fragment of the DNA genome of the rhinotracheitis virus (bovine herpes virus 1, BoHV-1) and a fragment of the bacteriophage T4 genome taken in a 1: 1 ratio, with the following nucleotide E sequences:

The novelty of the claimed technical solution lies in the fact that to obtain a reliable diagnosis of the causative agent of the DNA pathogen of rhinotracheitis virus (bovine herpes virus 1, BoHV-1), a reaction is carried out in one PCR tube (one-tube) using site-specific rhinotracheitis virus (bovine herpes virus 1, BoHV-1) of oligonucleotide primers of a fluorescently labeled probe and different types of control for which various forms of T4 bacteriophage material are used: a suspension and a fragment of the genome with specific primers and a probe. Such a real-time PCR setup reduces and simplifies the analysis procedure, reduces the risk of contamination. In addition, fluorescence 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, as it relates to diagnostic tools for the rhinotracheitis virus (bovine herpes virus 1, BoHV-1), which meets the criterion of "industrial applicability".

The invention is illustrated by drawings, which show screenshots of graphs, in FIG. 1 shows the FAM / Green channel of the internal control signal, FIG. 2 - channel JOE (HEX) / Yellow accumulation of a fluorescent signal for a specific DNA signal of rhinotracheitis virus (bovine herpes virus 1, BoHV-1), FIG. 3 is a quantitative data table of quantitative data for Cycling A.Yellow (bovine herpes virus 1, BoHV-1) and A. Green (BKO).

Method for detecting rhinotracheitis virus DNA (bovine herpes virus 1, BoHV-1)

in cattle, it is carried out as follows: Preliminarily, from the biological material that is taken: sperm, swabs from the vagina or from the nasal mucosa, tissues and organs (spleen, lungs) and lymph nodes secrete DNA by the sorption method. A one-stage polymerase chain reaction is carried out with simultaneous carrying out no more than 40 amplification cycles with real-time fluorescence detection using oligonucleotide primers, probes, fluorophore dye-specific oligonucleotide samples, specific for the rhinotracheitis virus genome site (BoHV-1) as internal and positive, the accumulation of a fluorescent signal for a specific DNA signal of rhinotracheitis virus (bovine herpes virus 1, BoHV-1) is measured by the JOE (HEX) / Yellow channel, and the signal is measured by the FAM / Green channel Cored oil control. Then, the results are interpreted based on the presence / absence of intersection of the fluorescence curve with the threshold line, if the fluorescence signal accumulation curves go out before the 35th cycle, then the reaction result is considered positive, i.e. DNA of rhinotracheitis virus (bovine herpes virus 1, BoHV-1) is present, and if the curves do not cross the threshold line or cross it after the 35th cycle, then the DNA of rhinotracheitis virus is absent i.e. the result of the reaction is negative. For the internal control sample, a suspension of T4 bacteriophage with a concentration of 5 × 10 ³ copies of nucleotide sequences per 1 μl is used, if the concentration of copies of the nucleotide sequences deviates up or down, then replicates of doubtful samples are observed, and for a positive control sample, a mixture of recombinant plasmid DNAs is used. containing a DNA genome fragment of the rhinotracheitis virus (bovine herpes virus 1, BoHV-1) and a fragment of the bacteriophage T4 genome taken in a 1: 1 ratio, if the ratio is from lonyatsya up or down, then there is a second suspicious samples.

A fragment of the rhinotracheitis virus DNA genome (bovine herpes virus 1, BoHV-1) and a fragment of the bacteriophage T4 genome are represented by the following nucleotide sequences:

The use of different forms of T4 bacteriophage material for different types of control: a 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 DNA extraction from samples.

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

DNA-specific primers of Infectious bovine rhinotracheites virus were selected based on the conserved region of the glycoprotein gene (Herpesvirus type 1 glycoprotein gene, complete cds. M23257) and were designed using Primer Express Software v3.0 (Applied Biosystems). Using BLAST, primers were tested for lack of homology with sequences of other viruses and the human genome. Thermodynamic analysis of the selected primers was performed using Vector NTI. The calculated length of the specific fragment was 120 pairs of nucleotides.

To detect amplification products, an oligonucleotide fluorescently labeled probe IRT-P was selected (complementary to the portion of the nucleotide sequence limited by the annealing positions of the IRT-F and IRT-R primers). The probe was labeled with dye R6G. To quench spontaneous fluorescence, a BHQ-1 quencher is attached at the 3'-end of the oligonucleotide probe. The main properties of the calculated oligonucleotides, which determined the possibility of their use in PCR, were described using the Oligo 6.0 program.

Bacteriophage T4 having genomic DNA of the order of 169-170 thousand nucleotide pairs (Enterobacteria phage T4T, complete genome GenBank: HM137666.1) was used as an internal control. As a result of the analysis, a region between 400 and 500 nucleotides containing unique nucleotide sequences was selected; primary structures of oligonucleotide primers flanking the selected genome region were calculated. Primers were designed using Primer Express Software v3.0 (Applied Biosystems) and tested using BLAST to confirm their specificity.

To detect amplification products, an oligonucleotide fluorescently labeled T4P probe was selected that is complementary to the portion of the nucleotide sequence limited to the annealing positions of the T4F and T4R primers. The probe was labeled with Fam. 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 DNA of rhinotracheitis virus (bovine herpes virus 1, BoHV-1) in cattle.

For research using the following biological material of choice:

- semen is taken in a volume of at least 2 ml in sterile tubes.

- swabs from the vagina are taken with a sterile probe using sterile gynecological instruments. The collected material is placed in a test tube containing 500 μl of sterile saline.

- smears from the nasal mucosa are taken with a sterile probe, placing the collected material in a test tube containing 500 μl of sterile saline.

- 1 × 1 × 1 cm pieces are cut from tissues and organs (spleen, lungs) and placed in a sterile container. The lymph nodes are taken as a whole. The preparation of the test material is carried out as follows:

Smears from the vagina and from the nasal mucosa are used without prior preparation.

Sperm samples are mixed in a ratio of 1: 4 with saline. For the extraction of DNA using 100 μl of the resulting mixture.

Samples of tissues and organs are homogenized using sterile porcelain mortars and pestles, then a 10% suspension is prepared in sterile saline or phosphate buffer. The suspension is transferred into a 1.5 ml tube and centrifuged at 12 thousand rpm for 2 minutes. The supernatant is used for DNA extraction. Next, an analysis consisting of three stages is carried out:

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

- real-time PCR with fluorescence detection;

- accounting for the results of the analysis.

For analysis, a kit is used in accordance with the instructions for use of the PCR-RINOTRAKHEIT-KRS-FACTOR reagent kit for the detection of rhinotracheitis virus DNA (bovine herpes virus 1, BoHV-1) in biological material by polymerase chain reaction with real-time fluorescence detection time (RT PCR), TU 21.10.60-125-51062356-2016, for in vitro diagnostics, (http://www.vetfaktor.ru/.).

The kit consists of a kit of reagents for conducting multiplex RT-PCR (kit No. 1) and a set of 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 kit does not include reagents for extraction of nucleic acids. DNA can be isolated, for example, using kits based on the sorption method, which include silica or microcentrifuge columns, as well as kits based on phenol-chloroform extraction, etc. It is recommended to use the DNA / RNA-S-FACTOR kit or similar. The composition of the kit is shown in tables 1 and 2.

* Light opalescence possible

The 1st stage of the analysis is the extraction (isolation) of nucleic acids (NK) from the studied samples.

The required number of 1.5 ml disposable tubes was selected, including a negative selection control. Pipette 10 μl of the BoHV-1 internal control sample (EQA) into all tubes with test samples, including a test tube for negative control (OKO). As an internal control sample (CTP) BoHV-1, a suspension of bacteriophage T4 with a concentration of 5 × 10 ³ copies of nucleotide sequences per 1 μl is used.

Enter the test samples in volume according to the instructions, to the kit for the selection of NK, in the test tube of the negative control of isolation, instead of the test sample, add the OKO (designate the tube as VK-).

DNA is isolated from the analyzed and control samples according to the protocol of the manufacturer's instructions for the kit for the selection of NK.

Isolated DNA can be stored for one week at a temperature of 2 ° C to 8 ° C or for a year at a temperature not exceeding minus 16 ° C.

Next, prepare samples for PCR. The total volume of the reaction mixture is 25 μl, the volume of the DNA sample is 10 μl.

Successful completion of the reaction is monitored using a positive control sample (PKO) BOHV-1, VKO BOHV-1 and DNA buffer. As a positive control sample (FFP), a mixture of recombinant plasmid DNAs containing a fragment of the rhinotracheitis virus DNA genome (bovine herpes virus 1, BoHV-1) and a bacteriophage T4 genome fragment taken in a 1: 1 ratio is used; if this ratio is not observed, duplicates of doubtful samples.

The components of the kit are mixed in a separate tube based on each PCR reaction:

5 μl PCR MIXTURE BOHV-1

10 μl PCR buffer BUFER BOHV-1

0.5 μl TAQ POLYMERASE

Vortex the mixture and mix the drops by short centrifugation.

Select the required number of tubes for amplification of the DNA of the investigated and control samples. Add 15 μl of the prepared reaction mixture.

Using tips with a filter in prepared tubes make:

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

b) in a series of test tubes for the studied samples - add 10 μl of the DNA of the corresponding sample obtained in accordance with clause 7.1 (including the VK- sample) to each;

c) in vitro positive PCR control (K +) 10 μl PKO BOHV-1

The 2nd stage of the analysis is the PCR reaction of RS with fluorescence detection using the Rotor-Gene Q device.

The parameters of the temperature-time mode of amplification on the device "Rotor-Gene Q" are presented in table 3.

Place prepared tubes for PCR in amplicator cells. Program the device according to the manufacturer's instructions.

After completion of the amplification program, the waste tubes are disposed of in accordance with MU 1.3. 2569 -09.

3rd stage - accounting for analysis results, interpretation of analysis results. The data obtained - the fluorescence signal accumulation curves are analyzed using the software of the device used for real-time PCR in accordance with the manufacturer's instructions for the device.

The results of PCR analysis are taken into account 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 DNA amplification and extraction controls are correctly passed in accordance with Table 4, FIG. 1, 2, 3

The appearance of any Ct value in the results table for negative control of the VK- extraction stage on the JOE (HEX) / Yellow channel and for 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 FAM / Green channel has no Ct value or exceeds the 35th cycle (and the Ct value is absent for the JOE (HEX) / Yellow channel) require repeated testing. The delay in the threshold cycle values for the studied samples on the FAM / Green channel (Figs. 1, 3) indicates the presence of inhibitors in the sample (s) or errors in DNA extraction or in the formulation of the reaction. Research is required starting from the DNA extraction step.

The sample is considered positive (cattle rhinotracheitis virus DNA is present) if an exponential signal growth is observed on the JOE (HEX) / Yellow channel, while the Ct values are within normal limits (Table 4, Figs. 2, 3). If the Ct value for the test sample through the JOE (HEX) / Yellow 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 JOE (HEX) / Yellow channel 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.

To prove the effectiveness of using PCR with fluorescence detection in real time, a comparative analysis of the sensitivity of the claimed technical solution with a prototype was carried out, in which the method of PNR with electrophoretic detection was used. The sensitivity of PCR with fluorescence detection when detecting the genome of the cattle rhinotracheitis virus DNA was found to be 3.0-3.3% higher than PCR with electrophoretic detection.

Claims (7)

A method for detecting rhinotracheitis virus DNA (bovine herpes virus 1, BoHV-1) in cattle, including DNA extraction by the sorption method, setting up a one-stage polymerase chain reaction with simultaneous real-time amplification cycles using specific rhinotracheitis virus genome ( bovine herpes virus 1, BoHV-1) oligonucleotide primers, probes, dyes and control samples in the form of internal and positive, measurement of a specific signal and a control signal through the channels corresponding dividing lines and interpreting the results, characterized in that they carry out fluorescence detection, measure the accumulation of the fluorescent signal for the specific DNA signal of the rhinotracheitis virus (bovine herpes virus 1, BoHV-1) using the JOE (HEX) / Yellow channel, and the internal signal through the FAM / Green channel the control sample and the interpretation of the results is carried out on the basis of the presence / absence of intersection of the fluorescence curve with the threshold line, 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, then the reaction result is negative, in this case, a suspension of bacteriophage T4 with a concentration of 5 × 10 ³ copies of nucleotide sequences per 1 μl is used for the internal control sample, and a mixture of recombinant plasmid DNA is used for the positive control sample, containing a DNA genome fragment of the rhinotracheitis virus (bovine herpes virus 1, BoHV-1) and a fragment of the bacteriophage T4 genome, taken in a 1: 1 ratio, with the following nucleotide sequences: IRT-F: TCGCCGACACGGGTCTGCTC direct primer IRT-R: CATATTGCCGTCCGTCTTGACC reverse primer IRT-P: R6G CATTGACCGCGTGGTCAAGACGG BHQ1 probe T4F TACATATAAATCACGCAAAGC T4R TAGTATGGCTAATCTTATTGG T4P FAM ACATTGGCACTGACCGAGTTC BHQ1 probe.

Images