RU2360971C1 - Method and test system to detect dna of african swine fever virus by means of specific oligonucleotide primers in polymerase chain reaction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there is offered method to detect DNA of African swine fever virus, and also test system to detect DNA of PAP virus.

EFFECT: invention can be used in veterinary science.

2 ex, 3 tbl

Description

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

African plague (ASF) is the most dangerous disease for pigs, which can lead to disastrous consequences for pig production in any country. Clinical signs and pathological changes have a significant similarity with the symptoms that are observed with classical swine fever, therefore, in laboratory studies, these diseases must always be differentiated.

A known method for detecting the virus of classical swine fever using specific oligonucleotide primers and a polymerase chain reaction, including the synthesis of synthetic oligonucleotides, primers in the conservative region of the greatest similarity of the genomes of different strains of classical swine fever virus, but with the greatest differences from the nucleotide sequences of the RNA strains of the large viral di virus cattle, then the synthesis of complementary DNA on a viral RNA matrix in the reaction of reverse transcription and amplification the synthesis of complementary synthesized DNA by polymerase chain reaction using the aforementioned primers [1].

The closest analogue (prototype) is a method and kit for detecting ASF virus DNA by PCR. In this case, a DNA fragment of 1144 nucleotides with primers N1 and N3 and a fragment of 562 nucleotides with primers N2 and N3 containing the site for Pst I restriction enzyme are amplified. The kit includes plastic tubes containing specific primers (N1, N2, N3); Tag polymerase enzyme; 10x reaction buffer, mixture of deoxynucleotide triphosphates; a positive control is a recombinant plasmid containing the Vp 72 protein gene. The disadvantage of this method and kit is the need for special preparation of biological samples and a phenol-detergent method for nucleic acid isolation, in which the concentration of nucleic acid yield is not constant and requires subsequent dilution [2].

The aim of the invention is to develop a more sensitive and specific method and an appropriate test system for detecting the ASF virus genome in organs and blood samples from sick animals.

This goal is achieved by a method for detecting ASF virus DNA in samples of organs and blood samples of chronically infected, sick and dead animals, and in an infected cell culture, in which, for PCR, DNA is denatured at 94 ° C, DNA hybridization with specific primers complementary a highly conserved region of the ASF virus genome of the P30 gene region, having the following nucleotide composition:

P30F-5 '- aga atg ggg atc cat tct gca tgt gct gtt tga - 3'

P30R - 5 '- cta aaa atc cgg atg tag gtg aga tta aag ctt a - 3', with an annealing temperature of 55 ° C for 10-30 sec with the number of amplification cycles from 30-35, then the results of the reaction are evaluated without prior restriction the resulting product and a test system that includes three sets with plastic bottles and tubes:

1) a kit for the isolation of nucleic acids containing a nucleosorbent with washing buffer based on guanidine thiocyanate;

2) a kit for staging PCR, including specific oligonucleotide primers complementary to the highly conserved region of the ASF virus genome of the P30 gene region, having the following nucleotide composition:

P30F-5 '- aga atg ggg atc cat tct gca tgt gct gtt tga - 3'

P30R-5 '- cta aaa atc cgg atg tag gtg aga tta aag ctt a - 3'

thermostable enzyme Tag-polymerase, PCR mixture for the formulation of the reaction, positive control;

3) an electrophoresis kit containing agarose and an electrophoresis buffer with ethidium bromide.

The first set allows you to quickly and accurately isolate ASF virus DNA without first preparing the suspension from the organs and isolating the formed elements from the blood.

When setting up PCR in the second set, repeated cycles of denaturation of the studied cDNA at a temperature of 94 ° C are repeated, DNA hybridization with specific primers at a temperature of 55 ° C and synthesis of complementary DNA chains from them using stable DNA polymerase at a temperature of 72 ° C and simultaneous landing primers, the use of mercury methyl hydroxide is excluded. As a result of 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 to increase the degree of specificity and sensitivity, as well as reducing the time of carrying out diagnostic work in samples of organs and blood from sick animals.

In the presence of the ASF virus genome in the test sample, a 374 bp fragment is synthesized.

It was found that in the process of isolation there occurs a “standard” DNA output, which does not require preliminary multiple dilution of the nucleic acid to perform PCR.

Significant differences of the proposed method from the prototype is that:

1. No preliminary sample preparation is required. Those. at the first stage of the isolation method, a lysis buffer (1:10) is added to the biomaterial without preliminary preparation of suspensions from organs. This affects the reduction of contamination and significantly reduces the time of the diagnostic examination.

2. When DNA virus is isolated, phenol is replaced by guanidine thiocyanate, which has less toxicity.

3. In the process of isolation, a “standard” DNA output occurs, which does not require its additional dilution before PCR.

The invention is illustrated by the following examples.

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

Example 1. Detection of ASF virus DNA grown in cell culture (13 virulent and avirulent strains) and samples of biological material obtained from sick animals.

The following were used as a negative control: classical swine fever virus, Aujeszky's disease virus, and samples of organs of healthy pigs.

When using the first set of test systems, a lysis buffer containing guanidine thiocyanate, 1 M TrisHCl, triton × 100, 0.5 EDTA is added to the sample of biological material (lymph nodes, spleen, or organ pool). The mixture is incubated for 0.5 h at room temperature, a nucleosorbent for sorption of DNA is added, then the sorbent is washed with a washing buffer consisting of guanidine thiocyanate, 1 M TrisHCl and water from the components of the destroyed virus, after the DNA is eluted from the sorbent with bidistilled water. Isolated DNA is used in PCR.

When using the 2nd set, amplification of a portion of DNA (P30) of the ASF virus is performed. The incubation mixture with a final volume of 25 μl contains: Tag polymerase buffer with 15 mM MgCl ₂ , 2.5 mM each dNTP, 10 mM each primer, 5 units. enzyme. Over the mixture layered 25 μl of mineral oil. The temperature regime of PCR is shown in table 1

Table 1. Process Temperature Time Cycles Denaturation 94 ° C 3 min one Denaturation 94 ° C 10 s Annealing 55 ° C 20 s thirty Elongation 72 ° C 15 s Elongation 72 ° C 1 min one

PCR products are analyzed by electrophoresis in a 2% agarose gel in standard Tris-boron buffer using the 3rd set.

10 μl of PCR product is mixed with 2 μl of sample application buffer and added to a well of 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 (15 min). The result of electrophoresis is taken into account when viewing the gel in ultraviolet light with a wavelength of 254 nm on a Transilluminator instrument. The result is considered positive if the PCR product matches the fragment size of 374 base pairs.

Using the proposed detection test system, ASF virus is detected in all studied ASF virus samples grown both in cell culture and from organ samples.

Example 2. Determination of the sensitivity and specificity of PCR based on synthetic primers synthesized for the P30 gene

The specificity of the PCR method is determined by the amplification of DNA isolated from control samples according to the size of the synthesized PCR product by agarose gel electrophoresis.

The high specificity of the proposed method is shown in the study of DNA preparations of strains of various serotypes that differ in virulence and haemadsorption ability.

The suitability of the developed method for detecting ASFV DNA in samples of organs of infected animals was evaluated. In experiments to detect the ASF virus genome, DNA preparations were used that were isolated from blood, spleen, liver, lymph nodes of fallen gilts infected with ASF virus (Madeira strain I serotype); gilts killed 21 days after immunization with the avirulent strain MK-220 of the III serotype (Table 2).

The high sensitivity of the method allowed us to conclude that using these primers, it is possible to identify animals with a chronic and asymptomatic course of the disease when the infectivity titer does not exceed 1.5-2.5 lg GADE50 / ml.

When using the test system, DNA-related and heterologous viruses were not detected. The requirements for the test system are presented in Table 3.

Table 3
Test System Requirements Name of indicator Characteristic and norm Sensitivity Should amplify the ASFV virus genome with a titer of 10 TCD ₅₀ / cm ³ Specificity Should not amplify DNA of other bacteria and viruses

A molecular weight marker is added to the gel, and the reaction is evaluated by the size of the PCR product, and the result of the reaction is considered positive if the PCR product corresponds to a size of 374 base pairs.

Thus, the proposed method and test system can achieve a high level of sensitivity using 2 primers instead of 3 used in standard PCR. This allows for 3 hours to detect 10 particles in 1 ml of the infectious virus in various materials, including samples of pig organs with chronic and latent forms of the disease.

The test system for detecting ASFV virus using PCR is designed for 50 reactions.

Information sources

1. Application RU 97103934, Puzyrev A.T., Donchenko A.S., Oreshkova S.F. et al., 1999

2. Patent RU 2125089, Tsybanova L.Ya., Vishnyakov I.O., Tsybanov S.Zh., 1999

Claims (2)

1. The method for detecting DNA of African swine fever virus, including the isolation of DNA from biological material, the polymerase chain reaction is carried out by denaturation of the studied DNA at a temperature of 94 ° C using synthesized primers, virus DNA amplification, evaluation of the reaction by gel electrophoresis in agarose, different the fact that DNA is produced by the method of sorption of nucleic acid using silica gel in the formulation of the polymerase chain reaction, the hybridization of DNA with specific primers, complementary to the highly conserved region of the ASF virus genome of the P30 gene region and not interacting with heterologous viruses having the following nucleotide composition:
P30F-5 '- aga atg ggg atc cat tct gca tgt gct gtt tga - 3'
P30R-5 '- cta aaa atc cgg atg tag gtg aga tta aag ctt a - 3'
with an annealing temperature of 55 ° C for 10-30 s with the number of amplification cycles from 30-35, then the reaction results are evaluated by agarose gel electrophoresis without preliminary restriction of the obtained product. 2. Test system for the detection of DNA from African swine fever virus, including plastic bottles and tubes, thermostable enzyme Tag polymerase, PCR mixture for the reaction, positive and negative controls, characterized in that it contains three sets: a kit for the isolation of nucleic acids containing a nucleosorbent with a guanidine thiocyanate lysis buffer; PCR kit with specific oligonucleotide primers complementary to the highly conserved region of the ASF virus genome of the P30 gene region according to claim 1, having the following nucleotide composition:
P30F-5 '- aga atg ggg atc cat tct gca tgt gct gtt tga - 3'
P30R-5 '- cta aaa atc cgg atg tag gtg aga tta aag ctt a - 3'; electrophoresis kit containing agarose and electrophoresis buffer with ethidium bromide.