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

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.

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The invention relates to veterinary Virology, namely diagnostics.

Viral haemorrhagic disease of rabbits (VGBC) - OSTROBRAMSKA highly contagious disease, characterized by phenomena of hemorrhagic diathesis in all organs, especially the lungs and liver [1].

High mortality of rabbits with viral haemorrhagic disease of rabbits necessitated the development of rapid diagnostic methods UGBC for fast and timely adoption of quarantine measures and preventive measures. One of such methods is the polymerase chain reaction (PCR), which has found wide application in diagnostic studies in veterinary medicine.

There are many known methods of laboratory diagnostics UGBC, such as the reaction of haemagglutination (DSA), long reaction of complement fixation (RDSC), response inhibition of haemagglutination (rcga), the reaction of the enzyme-linked immunosorbent assay (ELISA) [2]. However, in some cases, the application of these methods does not give clear results, for example in the study of decayed postmaterial or objects vesennaja. Also, before setting reaction requires the preliminary preparation of the suspension organs, which is quite time-consuming.

There is a method of virus detection UGBC method of Poland, in which to highlight the PH of the used method of phenol-chloroform extraction with the use of proteinase K [3, 4]. The need to work with toxic chemicals and the duration of the procedure limits its wide application. In addition, the method includes the step of preparation of the suspension organs, which increases the time of the reaction.

The aim of the invention is to develop more specific, sensitive, rapid method and the corresponding test systems for the detection of virus UGBC using the socket option RT-PCR using a pair of synthesized oligonucleotide primers complementary to the conservative area of the VP60 gene of the viral genome, allowing to amplify a fragment of the complementary DNA size 398 BP

To achieve these goals were chosen and synthesized specific primers for identification of the pathogen UGBC used for cDNA synthesis and PCR:

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

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

The method of detection of virus RNA WGBC in samples of organs, blood samples of infected rabbits includes a preliminary step of RNA denaturation and annealing of primers at 75°C for 5 min, followed by cDNA synthesis at 37°C for 30 minutes the Reaction NDP includes 42 cycles of amplification: 94°C - 30 sec, 60°C 30 sec, 72°C - 30 sec. Then conduct an assessment and analysis of the reaction without restriction analysis of the resulting product with a set d is I productions electrophoresis.

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

Set 1 for the selection of nucleic acids containing nucleosomes with a wash buffer based guanidinoacetate, the buffer for the reaction, negative control;

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

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

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

When setting RT-PCR using the second set is the denaturation of viral RNA at 75°C and simultaneous hybridization of primers. As a result of further amplification, the concentration of the synthesized fragment in the test sample increases millions of times, so it is easy to consider the results of the analysis using agarose gel electrophoresis (third set).

The technical result of the invention is the accessibility, safety, sensitivity, and takinogawa time of the diagnostic work with samples of organs and blood from infected animals.

The invention consists in that the detection of the viral genome UGBC carried out by polymerase chain reaction, providing for amplification of cDNA of the virus, electrophoretic separation of the amplification products and the results of the reaction. If the samples of the virus UGBC synthesized DNA fragment length of 398 BP To determine the size of the fragment to be paid the molecular weight marker.

A significant difference of the proposed method is that:

1. The assay can be performed directly from the sample body (e.g. liver, lung, heart, muscle) without prior preparation of the suspension. When the hinge body is being homogenized in lytic buffer. Thus, significantly reduces the time setting reaction.

2. For analysis you can use blood samples. Thus, this method allows 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 used to work with samples of organs and blood.

The analyzed material (blood) in the amount of 200 microlitres (hereinafter µl) or a sample of body weight of 0.2 g contribute 1.5 cm3a test tube and add serouse buffer based guanidinoacetate. Samples of organs homogenized lyse buffer and incubated for 40 min at 56°C, then centrifuged for 5 minutes and take the supernatant into a clean test tube. Within 10 min of the sample with blood and the supernatant incubated at room temperature with 40 μl of nucleolonema, shaking occasionally on the mixer. The tubes centrifuged at 5000 rpm and remove the supernatant. In tubes make 300 ál wash buffer, shake the test tube and centrifuged at 5000 rpm and remove the supernatant. The procedure is repeated twice. In tubes make 400 ál of 70% alcohol. The tube is shaken and centrifuged at 12000 rpm and remove the supernatant. The procedure is repeated twice. The tube is incubated for 8 min at 56°C, and contribute in a test tube with 50 ál of bidistilled water, thereby elwira nucleic acid nucleolonema in the water. 5 μl of this solution is used for further synthesis of complementary DNA.

Example 2. Synthesis of cDNA and amplification plot cDNA of the virus UGBC with set No. 2 for carrying out RT-PCR using synthetic oligonucleotide primers.

The calculation of the primary structure of the oligonucleotide primers.

To identify haemorrhagic disease of rabbits

the polymerase chain reaction, it was necessary to determine the genome nucleotide sequence which is would allow to distinguish this virus from other members of the family Caliciviridae and at the same time would be quite conservative for various strains and isolates of the virus GBq. After analyzing the literature data, we concluded that the gene that is suitable for identification UGBC is VP60 gene that encodes a protein capsid.

Selection of primers was performed on the basis of the analysis of the nucleotide sequences of strains and isolates of the pathogen UGBC available in the GeneBank database [http://www.ncbi.nlm.nih.gov/genbank]. Multiple alignment and sequence analysis was performed using the computer program BioEdit 6.0". For calculation of primers was selected area highly conserved region of the gene VP60. Calculation and analysis of oligonucleotides was performed using the computer program OLIGO 6.0". For the selection and analysis of the primers used VP60 gene sequences of strain Triptis haemorrhagic disease of rabbits.

The test system has been tested on various strains and isolates of the virus UGBC. As negative controls used strains of virus myxoma rabbits and virus vesicular exanthema of swine.

Carry out the annealing of the primers, which in pre-labeled tubes of 0.5 or 0.2 ml make 5 µl RNA, 0.2 µl of each primer and 5.6 μl bidistilled water.

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

In a separate tube, prepare a total reaction mixture of n is N samples, including the positive control (with a water solution of RNA virus WGBC) and negative control (with bidistilled water). The contributed reagents in the order given in the table. The enzyme was added last.

ReagentsQty 1 sample (ál)In N samples (ál)
Buffer for reverse transcription44×(N+1)
dNTP11×(N+1)
The enzyme MMLV revertase0.10.1×(N+1)
Bidistilled water44×(N+1)

The mixture contribute on top of the oil. Samples incubated at 37°C for 30 minutes in the amplifier. After the incubation, the cDNA 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 prepare the total reaction mixture by N samples, including positive and negative controls. The contributed reagents in the order given in the table. The enzyme is added to the settlement of enyy the queue.

ReagentsQty 1 sample (ál)In N samples (ál)
Bidistilled water14,6of 14.6×(N+1)
Buffer for PCR44×(N+1)
Primer RHDF0,20,2×(N+1)
Primer RHDR0,20,2×(N+1)
dNTP11×(N+1)
The enzyme Taq polymerase0.10.1×(N+1)

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

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

94°C30 sec
60°C30 sec42 cycle
72°C30 sec

Example 3. Determine the size of PCR products using set for electrophoresis.

The PCR products analyzed by electrophoresis in 2%agarose gel in standard Tris-born buffer.

13 μl of the PCR product by making the hole in the agarose gel. Electrophoresis is carried out at a voltage of 10 V/cm the length of the gel as long as the dye will not work from the start at least half of the gel (approximately 15 min). The result of electrophoresis consider viewing the gel under ultraviolet light with a wavelength of 230 nm on the device "Transilluminator". The result is considered positive if the PCR product corresponds to the fragment size 398 base pairs.

With the use of test systems and is designed primers revealed all the analyzed samples of the virus UGBC. Negative controls did not amplificadores.

Example 4. Determination of the sensitivity and specificity of RT-PCR by use of synthetic oligonucleotide primers.

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

The analytical sensitivity was the determined relative to the activity of the virus, expressed in lg LD50/cm3. Why have performed a series of 10-fold dilutions of virus UGBC strain "-87", with an initial titer of 4.5 lg LD50/cm3and analyzed using the method of Poland. The limit of detection was 1:10 dilution of3from the original sample titer infectious activity of 4.5 lg LD50/cm3.

The analytical sensitivity of the proposed set was also determined relative to the activity of the virus, which is expressed in the GUY, amplification of purified RNA virus isolated from serial fourfold dilutions of 10-percentage of suspensions of liver samples of rabbits infected with UGBC. The calculated analytical sensitivity exceeded the limit of sensitivity of DSA 2 times.

Sources of information

1. Vlasov N.A. Physico-chemical properties and antigenic structure of the virus haemorrhagic disease of rabbits. // Doctoral thesis. - Cover: Vniivvim, 1998. - 351 S.

2. Shevchenko A.A., Vishnyakov ACTING, Bakulev I.A., T.A. Vlasova Viral haemorrhagic disease of rabbits. // Veterinary medicine, 1994, No. 10. - S-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 C., 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 Environ., 1997, No. 35. - P.2492-2495.

1. Synthetic oligonucleotide primers complementary to highly conservative area of VP60 gene of the genome of the virus of viral haemorrhagic disease of rabbits used for detection of virus RNA UGBC, 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. The method of detecting the virus UGBC, including sample preparation, separation of RNA from biological material, the formulation of the polymerase chain reaction using synthetic primers, amplification RNA virus, evaluation of the reaction by gel-electrophoresis in agarose, characterized in that conduct preliminary denaturation of viral RNA at 75°C for 5 min, the cDNA synthesis was performed at 37°C for 30 min, and for setting the NDP are selected and synthesized two pairs of oligonucleotide primers according to claim 1 with 42 cycles of amplification with denaturation temperature of 94°C 30 s, annealing 60°C for 30 s, elongation 72°C 30 s, then evaluation of the reaction without the prior restriction of the obtained product, and the reaction is considered positive if the product of Poland corresponds to the size of 398 base pairs.

3. Test-system for detection of virus RNA viral haemorrhagic disease of rabbits, including plastic bottles and tubes, thermostable enzyme Taq polymerase, PCR with the feature for the given reaction, positive and negative controls, characterized in that it contains three sets: 1) a kit for detecting a nucleic acid that contains nucleosomes with a wash buffer based guanidinoacetate, the buffer for the reaction, negative control; 2) a kit for carrying out RT-PCR, including plastic tubes containing synthetic primers according to claim 1; (3) set for electrophoresis containing agarose, buffer for electrophoresis.



 

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