RU2409673C1 - Synthetic oligonucleotide primers for detecting 1a and 1b virus subgenotypes of viral diarrhoea - bovine mucosal disease and method of applying thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to synthetic oligonucleotide primers for detecting 1a and 1b virus subgenotypes of viral diarrhoea - bovine mucosal disease (VD - BMD) and to a method of applying said synthetic oligonucleotide primers for detecting 1a and 1b virus subgenotypes of viral diarrhoea - bovine mucosal disease. The method can be used in veterinary virology for subgenotype differentiation of strains and isolates of genotype 1 virus. The offered method involves a PCR with pairs of primers SEQ ID NO:1 5'-tcgacgccttaacatgaaggt-3' and SEQ ID N0:3 5'-ccatgtgccatgtacag-3' for subgenotype la, SEQ ID NO:2 5'-tcgacgctttggaggacaagc-3' and SEQ ID NO:3 5'-ccatgtgccatgtacag-3' for subgenotype lb. It is followed with detecting a DNA fragment sized 186 bp in each reaction. Further, the presence in an analysed material of 1a and lb subgenotype virus of viral diarrhoea - bovine mucosal disease is concluded.

EFFECT: invention allows express subgenotype differentiation of genotype one VD - BMD virus strains.

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Description

The invention relates to biotechnology, namely to genetic engineering, and can be used in veterinary virology for genotyping strains and isolates of the virus of viral diarrhea - a disease of the mucous membrane of cattle (VD-BS cattle).

Until now, the main method for genotyping strains and isolates of viral diarrhea - diseases of the mucous membranes of cattle is a method based on the accumulation of viruses in cell culture, RNA isolation, reverse transcription and polymerase chain reaction, purification of PCR products, amplicon sequencing and comparison with known sequences of the genome of reference strains (Shulpin M.I., Ayanot P.K., Mishchenko V.A. Indication of the virus of cattle viral diarrhea, genotyping and phylogenetic analysis of isolates detected in the Russian Federation. Issues of Virology. - 2003. - No. 6. - P.41-46.).

The disadvantages of this method include the need to obtain a large number of highly purified amplicons, duration and high cost.

The closest solution to this invention are primers and a method for genotyping VD-BS virus of cattle, including the synthesis of oligonucleotide primers for the NS5B gene and nested PCR (Gilbert A., Burton KM, Prins SE, Deregt D. Typing of Bovine Viral Diarrhea Viruses Directly from Blood of Persistently Infected Cattle by Multiplex PCR. Journal of clinical microbiology. - Vol. 37, No.6. - p.2020-2023).

External primers for the first round of amplification:

5'-aagatccacccttatga (a / g) gc-3 '

5'-aagaagccatcatc (a / c) ccaca-3 '.

Multiple internal primers for the second round of PCR:

5'-tggagatctttcacacaatagc-3 ',

5'-gggaacctaagaactaaatc-3 ',

5'-gctgtttcacccagtt (a / g) tacat-3 '

The disadvantages of this method include the fact that it does not allow to separate strains and isolates of the virus of the first genotype into subgenotypes (1a and 1b).

The objective of the invention is the development of synthetic oligonucleotide primers and a method for identifying subgenotypes 1a and 1b of the virus of viral diarrhea - a disease of the mucous membranes of cattle using synthetic oligonucleotide primers.

The problem is solved in that synthetic oligonucleotide primers are selected and synthesized to detect subgenotypes 1a and 1b of the virus of viral diarrhea, a disease of the mucous membranes of cattle, according to the invention, the primers have the nucleotide sequences: SEQ ID NO: 1 5'-tcgacgccttaacatgaaggt-3 ID NO: 2 5'-tcgacgctttggaggacaagc-3 ', SEQ ID NO: 3 5'-ccatgtgccatgtacag-3'.

The problem is also solved by the fact that in the method of using synthetic oligonucleotide primers to detect subgenotypes 1a and 1b of the virus of viral diarrhea - a disease of the mucous membranes of cattle, including PCR, characterized in that separate reactions are carried out with pairs of primers SEQ ID NO: 1 5 ' -tcgacgccttaacatgaaggt-3 'and SEQ ID NO: 3 5'-ccatgtgccatgtacag-3' for subgenotype 1a, SEQ ID NO: 2 5'-tcgacgctttggaggacaagc-3 'and SEQ ID NO: 3 5'-ccatgtgccatgtagg and if a DNA fragment of 186 n.p. in each reaction, a conclusion is drawn about the presence of viral diarrhea virus in the test material, a disease of the mucous membranes of cattle subgenotypes 1a and 1b, respectively.

The technical result of the invention is the ability to quickly differentiate strains and isolates of the virus VD-BS cattle of the first genotype into subgenotypes.

The invention is illustrated by the following examples.

Example 1. The method of selection and preparation of synthetic oligonucleotide primers.

The search for new specific primers is carried out on the basis of the complete genomes of strains of the VD-BS virus of cattle of genotype 1 presented in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html). All sequences are aligned using the Blast method, genomic regions are selected that show at least 80% homology with sequences of the 5'-untranslated region of reference strains of VD-BS cattle Oregon C24V (subgenotype 1a) and ILLC (subgenotype 1b). Using the Lasergen software package, primers are searched for selected genome regions. The obtained sequences of several pairs of primers are additionally tested for specificity by PCR modeling in the Vector NTI Suite program with the genome sequences of all investigated strains of VD-BS cattle virus of genotype 1, classical swine fever viruses, border sheep disease and VD-BS cattle virus of genotype 2 . The presence or absence of homology with these pathogens is the basis for the selection of primers. The final selection of primers is based on the following criteria: high similarity index of the fragment and RNA of different virus strains, high annealing temperature (GC method), long consensus length. The chemical synthesis of primers is carried out by the amidophosphite method on an ASM-102U automatic synthesizer. The concentration of specific oligonucleotide primers in the mother liquor is determined by spectrophotometric method.

To confirm the synthesis of primers of a given sequence, polyacrylamide gel electrophoresis is carried out by comparison with a molecular weight marker pBR322 / BsuRI.

Figure 1. Comparison of the nucleotide sequences of the 5'-untranslated region of strains of the virus VD-BS cattle 1 genotype. Bold italics indicate nucleotide regions for specific binding to primers.

Thus, this method allows to obtain synthetic oligonucleotide primers complementary to the highly conserved 5'-untranslated region of the VD-BS cattle virus genome specific for subgenotypes.

Example 2. A method of using synthetic oligonucleotide primers to detect subgenotypes 1a and 1b of the virus of viral diarrhea - a disease of the mucous membranes of cattle.

The method is carried out in several stages.

Stage 1. Isolation of RNA virus VD-BS cattle.

To isolate RNA, suspensions of strains and isolates of VD-BS cattle virus of the 1st genotype isolated in cell culture, as well as a suspension of biomaterial from animals in which genotype 1 virus RNA was detected by PCR, are taken.

100 μl of a clarified suspension of a virus or biomaterial is transferred to a test tube with 450 μl of a lyse solution, mix thoroughly and add 25 μl of a resuspended sorbent, mix and leave in a rack for 1 minute, mix again, leave for 5 minutes, and then centrifuge for 30 seconds at 10 thousand rpm The supernatant is removed, 400 μl of washing solution 1 is added, stirred and centrifuged for 30 seconds at 10 thousand rpm. The supernatant is removed, 500 μl of washing solution 3 is added and the procedure is repeated. 400 μl of washing solution 4 is added to the tubes, carefully resuspended, centrifuged for 30 sec at 10 thousand rpm. Remove supernatant. Open tubes are placed in a 60 ° C thermostat for 5-10 minutes to dry the sorbent. Then add 40 μl of RNA eluate, mix, incubate for 2-3 minutes at 60 ° C, centrifuged.

Step 2. Conducting a reverse transcription reaction to obtain cDNA.

In a test tube containing 10 μl of the reaction mixture: buffer for RT (50 mM Tris-HCl [pH 8.3], 3 mm MgCl2, 75 mm KCl, 10 mm DTT), 0.1 mm dNTP, 0.1 μg primer for RT, 80 e.a. M-MulV reverse transcriptase, add 10 μl of RNA sample, mix gently and place in a 37 ° C thermostat for 30 minutes. Then add 20 μl of TE buffer, mix thoroughly and use for PCR.

Stage 3. Amplification of the cDNA region of the virus VD-BS cattle.

Polymerase chain reaction. A separate PCR mixture is prepared for each pair of primers. Composition of the reaction mixture: PCR buffer (60 mM Tris-HCl [pH 8.5], 1.5 mM MgC12, 25 mM KCl, 10 mM 2-mercaptethanol, 0.1% Triton X-100), 0.2 mM dNTP, 0.1 μg of each primer, 1.25 ea Taq DNA polymerase, 5 μl reverse transcription product.

Temperature regime of PCR: The amplification program consists of the following temperature regimes: 95 ° С - 5 min - 1 cycle; 95 ° С - 45 sec, 55 ° С - 45 sec, 72 ° С - 1 min - 35 cycles; 72 ° C - 5 min.

Stage 4. Sizing of PCR products.

PCR products are analyzed by electrophoresis in a 2% agarose gel in standard Tris-borate buffer (pH 8.0).

10 μl of the PCR product is mixed with 2 μl of sample buffer (0.25% bromophenol blue, 40% sucrose) and added to an agarose gel well. Electrophoresis is carried out at a voltage of 10 V / cm of the length of the gel until the dye passes from the start of at least half of the gel (approximately 30 minutes). The results of electrophoresis are taken into account when viewing the gel in ultraviolet light with a wavelength of 254 nm on a Transilluminator instrument.

100bp molecular weight marker.

The PCR result is considered positive if the PCR product corresponds to a fragment size of 186 nucleotide pairs.

Figure 2 Electrophoregram of an amplified fragment of the genome of 2 strains of VD-BS cattle virus "Oregon C 24 V" and "VK-1" (Lanes 1 and 2 PCR with a pair of primers for subgenotype 1a, tracks 3 and 4 - PCR with a pair of primers for subgenotype 1b, lane 5 - molecular weight marker).

Example 3. Determination of the specificity of PCR.

The results of experiments to determine the specificity of the reaction with two pairs of primers are presented in table 1.

Table 1 Virus (strain) PCR Results Primers for subgenotype 1A Primers for subgenotype 1b VD-BS virus of cattle 1 genotype (NADL) + - VD-BS virus of cattle 1 genotype (Oregon C24V) + - VD-BS virus of cattle 1 genotype (TM-340) + - VD-BS virus of cattle 1 genotype (VK-1) - + VD-BS virus of cattle 2 genotype (BL isolate) - - Swine fever virus - - Type 1 cattle adenovirus (BV-10) - - Type 1 cattle rhinovirus (SD-1) - - KST cell culture - - Note: “+” is a positive result;
"-" - negative result.

Thus, the proposed synthetic oligonucleotide primers and the method of their use are highly specific for the detection of RNA strains and isolates of the virus VD-BS cattle.

Example 4. The results of testing strains and isolates of the virus VD-BS cattle related to the first genotype.

The results of testing isolates of the virus of viral diarrhea - diseases of the mucous membranes of cattle of the 1st genotype are presented in table 2.

table 2 No. p / p Name of isolate The clinical form of the disease, source of excretion Subgenotype one Buffalo Respiratory, calf / spleen 1a 2 Bongo Respiratory, Bongo / spleen antelope 1b 3 1140 Respiratory, giraffe / spleen 1b four T-l Sire / sperm 1a 5 AND Genital / vaginal discharge 1a 6 I4 Genital / vaginal discharge 1b 7 SV-4 Sire / sperm 1a 8 T-2 Sire / sperm 1a 9 RA Respiratory, calf / lungs 1b 10 Cream Abortion / spleen 1a eleven Irm 01/06 Respiratory, calf / lungs 1a 12 Boron PI cow / blood serum 1b

Thus, the developed primers and the method of their use allow efficient subgenotyping of strains and isolates of the virus of viral diarrhea, a disease of the mucous membranes of cattle of the 1st genotype, which can be used in the study of molecular epizootology of the disease and in the planning of antiepizootic measures.

Claims (2)

1. Synthetic oligonucleotide primers for detecting subgenotypes 1a and 1b of the virus of viral diarrhea of the disease of the mucous membranes of cattle, characterized in that the primers have nucleotide sequences: SEQ ID NO: 1 5'-tcgacgccttaacatgaaggt-3 ', SEQ ID NO: 2 5' -tcgacgctttggaggacaagc-3 ', SEQ ID NO: 3 5'-ccatgtgccatgtacag-3'. 2. The method of using synthetic oligonucleotide primers to detect subgenotypes 1a and 1b of the virus of viral diarrhea of the disease of the mucous membranes of cattle, including PCR, characterized in that they conduct separate reactions with pairs of primers SEQ ID NO: 1 5'-tcgacgccttaacatgaaggt-3 'and SEQ ID NO: 3 5'-ccatgtgccatgtacag-3 'for subgenotype 1a, SEQ ID NO: 2 5'-tcgacgctttggaggacaagc-3' and SEQ ID NO: 3 5'-ccatgtgccatgtacag-3 'for subgenotype 1b and if a DNA fragment is detected size 186 n.p. in each reaction, a conclusion is drawn about the presence in the test material of the virus of viral diarrhea of the disease of the mucous membranes of cattle subgenotypes 1a and 1b, respectively.

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