RU2416648C1 - Oligonucleotide primers, method and test system for identifying genome of rabbit viral hemorrhagic disease by reverse transcription - polymerase chain reaction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to synthetic oligonucleotide primers, complementary to high conservative VP60 gene region of a genome of rabbit viral hemorrhagic disease virus, to a method for identifying rabbit viral hemorrhagic disease virus and to a test system for identifying RNA of rabbit viral hemorrhagic disease virus. The offered invention can be used in veterinary virology. The method for identifying rabbit viral hemorrhagic disease virus involves sample preparation, RNA recovery from the biological material. It is followed with conducting a polymerase chain reaction with using primers 5'-caa cgt get cca gtt ttg gta cg-3', 5'-att ctg tct ggt tgg ggc gtg t-3'. Further, viral RNA is amplified. Then, the reaction is assessed by agarose gel electrophoresis, with a reaction result considered as positive if the PCR product corresponds to the size of 398 base pairs.

EFFECT: invention allows higher sensitivity of the method, as well as reduced time of diagnostic manipulations with organ and blood samples of the infected animals.

3 cl, 4 tbl, 4 ex

Description

The invention relates to veterinary virology, and in particular to diagnostic tools.

Viral hemorrhagic rabbit disease (HBV) is an acute, highly contagious disease characterized by hemorrhagic diathesis in all organs, especially in the lungs and liver [1].

High mortality of rabbits with viral hemorrhagic rabbit disease necessitates the development of rapid diagnostic methods for HCVF in order to quickly and timely take quarantine measures and preventive measures. One of these methods is polymerase chain reaction (PCR), which has already found wide application in diagnostic studies in veterinary medicine.

Many methods of laboratory diagnosis of HBVC are known, such as the hemagglutination reaction (RGA), the long-term complement fixation reaction (RDSK), the hemagglutination inhibition reaction (RTGA), and the enzyme-linked immunosorbent assay (ELISA) [2]. However, in some cases, the application of these methods does not give clear results, for example, when studying decomposed material or objects of veterinary surveillance. In addition, before setting up the reaction, preliminary preparation of an organ suspension is required, which is rather laborious.

A known method for detecting HBV virus by the method of PNR, in which for the isolation of RNA using the method of phenol-chloroform extraction using proteinase K [3, 4]. The need to work with toxic reagents and the duration of the procedure limits its widespread use. In addition, this method includes the step of preparing a suspension of organs, which increases the reaction time.

The aim of the invention is to develop a more specific, sensitive express method and the corresponding test system for the detection of HBVV virus using the nested RT-PCR variant using a pair of synthesized oligonucleotide primers complementary to the conserved region of the VP60 gene of the virus genome, which allows amplification of a 398 complementary DNA fragment by.

To achieve this goal, specific primers were selected and synthesized to identify the causative agent of HBVC, which are used for the synthesis of cDNA and PCR:

5 '- saa cgt get cca gtt ttg gta cg-3'

5 '- att ctg tct ggt tgg ggc gtg t-3'

A method for detecting HBV virus RNA in organ samples, blood samples of infected rabbits includes a preliminary stage of RNA denaturation and annealing of primers at 75 ° C for 5 min followed by cDNA synthesis at 37 ° C for 30 min. The reaction of the NDP includes 42 amplification cycles: 94 ° C - 30 sec, 60 ° C - 30 sec, 72 ° C - 30 sec. Then, the results of the reaction are evaluated and taken into account without preliminary restriction of the obtained product using the electrophoresis kit.

The test system consists of three separate sets with plastic bottles and test tubes:

Set 1 - for the isolation of nucleic acids, containing a nucleosorbent with washing buffer based on guanidine thiocyanate, a buffer for the reaction, a negative control;

Set 2 - for staging RT-PCR, including plastic tubes containing: oligonucleotide primers, thermostable Taq polymerase enzyme, M-MLV reverse transcriptase enzyme, PCR buffer and reverse transcription buffer, a mixture of nucleoside triphosphates, positive and negative controls;

Set 3 - for electrophoresis, containing agarose, electrophoresis buffer.

The first set allows you to quickly and accurately isolate RNA of HBV virus without preliminary preparation of the suspension from the organs and without the use of toxic substances.

When setting up RT-PCR using the second set, denaturation of viral RNA is performed at 75 ° C and the primers are hybridized at the same time. As a result of further amplification, the concentration of the synthesized fragment in the test sample increases millions of times, which allows you to visually take into account the results of the analysis using agarose gel electrophoresis (third set).

The technical result of the invention is the accessibility, safety, sensitivity, as well as reducing the time for conducting diagnostic work with samples of organs and blood from infected animals.

The essence of the invention lies in the fact that the identification of the genome of HBVC virus is carried out by the method of polymerase chain reaction, which involves amplification of the cDNA of the virus, electrophoretic separation of the amplification products and accounting for the reaction results. In the presence of HBVK virus in the studied samples, a DNA fragment of 398 bp is synthesized. A molecular weight marker is introduced to determine the fragment size.

A significant difference of the proposed method is that:

1. The analysis can be carried out directly from a sample of an organ (for example, liver, lung, heart, muscle) without first preparing a suspension. At the same time, a portion of the organ is homogenized in a lysis buffer. Thus, significantly reduces the time of setting the reaction.

2. Blood samples may be used for analysis. Thus, this method allows you to determine the presence of the pathogen in infected animals in the early stages after infection.

The invention is illustrated by the following examples.

The test system is equipped with plastic bottles and test tubes.

Example 1. Set No. 1 for the detection of nucleic acids is used to work with samples of organs and blood.

The test material (blood) in a volume of 200 microliters (hereinafter µl) or an organ sample weighing 0.2 g is added to a 1.5 cm ³ tube and a guanidine thiocyanate-based lysis buffer is added. Organ samples are homogenized in lysis buffer and incubated for 40 minutes at 56 ° C, then centrifuged for 5 minutes and the supernatant is collected in clean tubes. For 10 min, samples with blood and supernatant are incubated at room temperature with 40 μl of nucleosorbent, periodically shaking on a mixer. The tubes are centrifuged at 5000 rpm and the supernatant is removed. 300 μl wash buffer was added to the tubes, the tubes were shaken and centrifuged at 5000 rpm, and the supernatant was removed. The procedure is repeated twice. 400 μl of 70% alcohol are added to the tubes. The tubes are shaken and centrifuged at 12,000 rpm, the supernatant is removed. The procedure is repeated twice. The tubes are incubated for 8 min at 56 ° C, and 50 μl of double-distilled water are introduced into the tubes, thereby eluting the nucleic acids from the nucleosorbent into water. 5 μl of this solution is used for further synthesis of complementary DNA.

Example 2. Synthesis of cDNA and amplification of the cDNA region of HBV virus using kit No. 2 for RT-PCR with synthetic oligonucleotide primers.

Calculation of the primary structure of oligonucleotide primers.

To identify rabbit hemorrhagic disease virus

using the polymerase chain reaction, it was necessary to determine the portion of the genome, the nucleotide sequence of which would make it possible to differentiate this virus from other representatives of the Caliciviridae family and at the same time would be quite conservative for various strains and isolates of the GBK virus. After analyzing the literature data, we came to the conclusion that the VP60 gene encoding the capsid protein is the gene suitable for identifying HBVC.

The selection of primers was carried out on the basis of the analysis of the nucleotide sequences of strains and isolates of the causative agent of HBVC, available in the GeneBank database [http://www.ncbi.nlm.nih.gov/genbank]. Multiple alignment and sequence analysis was performed using the BioEdit 6.0 computer program. For the calculation of primers, a highly conserved region of the VP60 gene was selected. Calculation and analysis of oligonucleotides was carried out using the computer program "OLIGO 6.0". For selection and analysis of primers, the VP60 gene sequence of the rabbit hemorrhagic disease virus Triptis strain was used.

The test system has been tested on various strains and isolates of the HBVC virus. The strains of rabbit myxoma virus and pig vesicular exanthema virus were used as negative controls.

Primers are annealed, for which 5 μl of RNA, 0.2 μl of each primer and 5.6 μl of bidistilled water are added to pre-labeled 0.5 or 0.2 ml tubes.

RNA denaturation and annealing of the primers is carried out in a thermocycler at a temperature of 75 ° C for 5 minutes.

In a separate test tube, a general reaction mixture is prepared for N samples, which include a positive control (with an aqueous solution of HBV virus RNA) and a negative control (with bidistilled water). Reagents are added in the sequence shown in the table. The enzyme is added last.

Reagents Qty per sample (μl) Amount per N samples (μl) Reverse Transcription Buffer four 4 × (N + 1) dNTP one 1 × (N + 1) MMLV Enzyme Revertase 0.1 0.1 × (N + 1) Bidistilled water four 4 × (N + 1)

The mixture is applied on top of the oil. Samples are incubated at 37 ° C for 30 minutes in a thermocycler. After incubation, cDNA is used for further analysis or stored at -20 ° C for 2 weeks.

In a separate test tube with a volume of 0.5 or 0.2 ml, a total reaction mixture is prepared for N samples, which include positive and negative controls. Reagents are added in the sequence shown in the table. The enzyme is added last.

Reagents Qty per sample (μl) Amount per N samples (μl) Bidistilled water 14.6 14.6 × (N + 1) PCR buffer four 4 × (N + 1) Primer RHDF 0.2 0.2 × (N + 1) Primer RHDR 0.2 0.2 × (N + 1) dNTP one 1 × (N + 1) Taq polymerase enzyme 0.1 0.1 × (N + 1)

In the PCR reaction using 5 μl of cDNA obtained during the reverse transcription reaction.

The PCR reaction is carried out in a thermocycler under the following conditions:

94 ° C 30 sec 60 ° C 30 sec 42 cycles 72 ° C 30 sec

Example 3. Determining the size of PCR products using a kit for electrophoresis.

PCR products are analyzed by electrophoresis in a 2% agarose gel in standard Tris-boron buffer.

13 μl of the PCR product is added to a well of an agarose gel. Electrophoresis is carried out at a voltage of 10 V / cm of the gel length until the dye passes from the start of at least half of the gel (approximately 15 min). The result of electrophoresis is taken into account when viewing the gel in ultraviolet light with a wavelength of 230 nm on a Transilluminator instrument. The result is considered positive if the PCR product matches the fragment size of 398 base pairs.

Using the test system and calculated primers, all tested samples of the HBVC virus were detected. Negative controls were not amplified.

Example 4. Determination of the sensitivity and specificity of RT-PCR based on synthetic oligonucleotide primers.

The test system and synthetic oligonucleotide primers were tested for specificity and sensitivity. It was found that the test system does not amplify the cDNA of other bacteria and viruses. The test system showed diagnostic specificity and sensitivity at 100%.

Analytical sensitivity was determined relative to the activity of the virus, expressed in lg LD ₅₀ / cm ³ . For this, a series of 10-fold dilutions of the HBBC virus of strain “B-87” was carried out, with an initial titer of 4.5 lg LD ₅₀ / cm ³ and analyzed using the PNR method. The sensitivity limit was a 1:10 ³ dilution from the original sample with an infectious activity titer of 4.5 lg LD ₅₀ / cm ³ .

The analytical sensitivity of the proposed kit was also determined relative to the activity of the virus, expressed in GAE, by amplification of purified virus RNA isolated from serial four-fold dilutions of 10% suspension of samples of liver samples of rabbits infected with HBV. The calculated value of the analytical sensitivity exceeded the sensitivity limit of the RGA by 2 times.

Information sources

1. Vlasov N.A. Physico-chemical properties and antigenic structure of the rabbit hemorrhagic disease virus. // Doctoral dissertation. - Veil: VNIIVViM, 1998 .-- 351 p.

2. Shevchenko A.A., Vishnyakov I.O., Bakulov I.A., Vlasova T.A. Viral hemorrhagic disease of rabbits. // Veterinary medicine, 1994, No. 10. - S.22-23.

3. Tian L., Liao J., Li J., Zhou W., Zhang X., Wang H. Isolation and identification of a non-haemagglutinating strain of rabbit hemorrhagic disease virus from China and sequence analysis for the VP60 Gene. // Virus Genes, 2007, No. 35. P.745-752.

4. Ros Bascuñana S., Nowotny N. and Belák S. Detection and differentiation of rabbit hemorrhagic disease and European brown hare syndrome viruses by amplification of VP60 genomic sequences from fresh and fixed tissue specimens. // J. Clinical Microbiol., 1997, No. 35. - P.2492-2495.

Claims (3)

1. Synthetic oligonucleotide primers complementary to the highly conserved region of the VP60 gene of the rabbit viral hemorrhagic disease virus genome used to detect HBV virus RNA having the following nucleotide composition:
5'-caa cgt gct cca gtt ttg gta cg-3 '
5'-att ctg tct ggt tgg ggc gtg t-3 '. 2. A method for detecting HBV virus, including sample preparation, isolation of RNA from biological material, production of a polymerase chain reaction using synthesized primers, amplification of the RNA of the virus, evaluation of the reaction by gel electrophoresis in agarose, characterized in that the preliminary denaturation of viral RNA at 75 ° C for 5 minutes, cDNA synthesis was carried out at 37 ° C for 30 minutes, and two pairs of oligonucleotide primers according to claim 1 with 42 amplification cycles at temperatures denaturation 94 ° C 30 s, 60 ° C annealing 30 s, elongation 72 ° C 30 s, then subjected to evaluation results of the reaction without prior restriction of the resulting product, the result of the reaction is considered positive if the product corresponds to the size NDP 398 base pairs. 3. Test system for detecting RNA of the virus of viral hemorrhagic disease of rabbits, including plastic bottles and tubes, thermostable enzyme Taq polymerase, PCR mixture for the reaction, positive and negative controls, characterized in that it contains three sets: 1) set for detecting a nucleic acid containing a nucleosorbent with washing buffer based on guanidine thiocyanate, a reaction buffer, a negative control; 2) a kit for RT-PCR, including plastic tubes containing synthetic primers according to claim 1; 3) an electrophoresis kit containing agarose, electrophoresis buffer.