RU2645262C1 - Method of detecting dna of african swine fever virus by real-time polymerase chain reaction - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: invention relates to veterinary virology, specifically to means of diagnosing African swine fever (ASF). Method for detecting DNA of the African swine fever virus by real-time polymerase chain reaction comprises isolating DNA using control samples (quality control of virus isolation and control of the reaction parameters); amplification of DNA using synthetic primers and a fluorescent probe, including denaturation at 95 °C, cycling: denaturation – annealing – elongation and estimation of the result from the fluorescent signal accumulation curves for each of the given channels. Recombinant plasmid with a fragment of the vp72 gene of the African swine fever virus is used as the control of the quality of virus isolation. Recombinant plasmid with a fragment of the healthy pig tissue gene is used as the control of reaction parameters. Synthetic oligonucleotide primers and a fluorescent probe, complementary to the preserved genome region of the African swine fever virus gene vp72 region, are taken in the ratio 1:1:0.5 and have the following nucleotide composition: SEQ NO 1:5'-CTG-CTC-ATG-GTA-TCA-ATC-3' – forward primer, SEQ NO 2:5'-GAT-ACC-ACA-AGA-TCG-CCG-3' – reverse primer, SEQ NO 3:5'-FAM-CCA-CGG-GAG-GAA-TAC-CAA-CCC-AGT-G-BHQ1-3' – fluorescent probe, where BHQ1 denotes a dark fluorescence quenching agent attached to 3'-terminal nucleotide, and FAM is a fluorescent dye attached to nucleotide C. Amplification is carried out under the following conditions: denaturation at 95 °C for 90 s, then cycling with detection, comprising denaturation at 95 °C for 15 s, annealing – 60 °C – 30 s and elongation – 72 °C for 4 s. Cycle: denaturation – annealing – elongation is repeated 40 times. Then, accumulation of the fluorescent signal is measured on channels: FAM for specific signal; HEX for internal control signal; CY5 for the signal of exogenous internal control. If the accumulation curves of the fluorescent signal reaches up to 36 cycles, then the result of the reaction is considered positive, and if the curves do not cross the threshold line or cross it after 36 cycles, the result of the reaction is negative.

EFFECT: invention makes improves accuracy of detecting DNA of the African swine fever virus by real-time polymerase chain reaction.

1 cl, 1 tbl, 9 dwg

Description

The invention relates to veterinary virology, and in particular to means for diagnosing African swine fever (ASF) both in the practice of the veterinary service and for scientific research.

African swine fever (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.

It is known to use oligonucleotide primers for the diagnosis of African swine fever with electrophoretic consideration of amplification results. With this method of setting up the reaction, it is not possible to quantify the nucleic acid of infectious agents in the test material. Also, if there is an electrophoresis stage, the risk of contamination of the laboratory with PCR products increases. When using PCR with hybridization-fluorescent results, it is possible to quantify nucleic acids with registration and interpretation of the results in real time, which allows to increase the reliability of diagnosis (Ekimov A.N., Shipulin G.A., Bochkarev E.G., Ryumin DV The latest technology in gene diagnostics: real-time polymerase chain reaction (Real-Time PCR) // Postgraduate medical education. - 2001. - No. 3. - P. 7-10).

Scientific publications are known that describe the use of PCR to detect and identify the African swine fever virus in biological samples (J of Virol. Meth. - 2003 - No. 107. - P. 53-61; J of Virol. Meth. - 2006 - No. 136. - P. 200-209; J of Virol. Meth. - 2011 - No. 178. - P. 161-170; J of Trans-boundary and Emerg. Des. - 2013 - No. 60. - P. 48-58 ), and also a number of patents are known (RU No. 2125089 C1, RU No. 2360971 C1, US 20140004504 No. A1, CN No. 101921878 A, CN No. 10333536 A, CN 103757134 B). The listed publications and patents describe various PCR formats that are used to diagnose ASF, including with agarose gel detection and hybridization-fluorescence detection.

The closest to the execution technique and the PCR format is the technical solution (see RF patent No. 2595373, CL C12Q 1/68, 2016), in which the detection of virus DNA is carried out by polymerase chain reaction in real time, by DNA extraction using samples : to control the quality of virus isolation, control the specificity of the reaction and a set of synthetic primers and a fluorescent probe for detecting virus DNA taken in equal proportions, amplification, including denaturation at 95 ° C, cycling: denaturation - tzhig - elongation and evaluation results of the accumulation of the fluorescent signal curves for each of the samples specified for the channel.

The disadvantage of this method is that it cannot be used to detect a fragment of the vp72 gene of the African swine fever virus due to the non-specificity of the primers and probe for African plague, a fragment of the vp72 gene of the African plague virus.

The technical result is to increase the accuracy of detection of African swine fever virus DNA by real-time PNR by constructing a kit containing a pair of specific oligonucleotide primers and a DNA probe, as well as selecting the conditions for PCR with them, which ensures minimal risk of contamination in the laboratory during analysis , as well as excluding subjectivity in assessing the results of PCR.

The technical result is achieved by the fact that in the method for detecting DNA of African swine fever virus by real-time polymerase chain reaction containing DNA isolation using control samples: to control the quality of virus isolation, control the specificity of the reaction; DNA amplification using synthetic primers and a fluorescent probe, including denaturation at 95 ° C, cycling: denaturation - annealing - elongation; and evaluating the result of the fluorescence signal accumulation curves for each of the given channels, according to the invention, use is made of a recombinant plasmid with a fragment of the vp72 gene of the African swine fever virus as a quality control of virus isolation, a recombinant plasmid with a gene fragment of healthy pig tissue as a control for the reaction, and synthetic oligonucleotide primers and a fluorescent probe are taken in a ratio of 1: 1: 0.5, complementary to the conservative region of the African swine fever virus genome of the gene region and vp72 and have the following nucleotide composition:

SEQ NO 1: 5'-CTG-CTC-ATG-GTA-TCA-ATC-3 '- direct primer,

SEQ NO 2: 5'-GAT-ACC-ACA-AGA-TCG-CCG-3 '- reverse primer,

SEQ NO 3: 5'-FAM-CCA-CGG-GAG-GAA-TAC-CAA-CCC-AGT-G-BHQ1-3 'is a fluorescent probe, where BHQ1 is a dark fluorescence quencher attached to the 3'-terminal nucleotide, a FAM is a fluorescent dye attached to nucleotide C, the amplification is carried out in the following mode: denaturation at 95 ° C for 90 s, then cycling with detection, including denaturation at 95 ° C for 15 s, annealing at 60 ° C - 30 s and elongation - 72 ° C for 4 s, and the cycle: denaturation - annealing - elongation is repeated 40 times, then the accumulation of the fluorescent signal is measured by the channels: FAM for a specific signal; HEX for internal control signal; CY5 for the signal of exogenous internal control, and if the fluorescence signal accumulation curves go out before the 36th cycle, then the reaction result is considered positive, and if the curves do not cross the threshold line or cross it after the 36th cycle, the reaction result is negative.

The invention is illustrated in the drawing, which presents graphs from the report of the device Rotor-Gene 6000 when using Real-time PCR:

in FIG. 1 is a graph of the FAM channel — the desired ASF DNA for a negative sample;

in FIG. 2 shows a graph of the channel Su5 - an internal control sample of specificity - pig DNA - negative sample;

in FIG. 3 shows a graph of the HEX channel — internal control sample of isolation — ASF synthetic DNA — negative sample;

in FIG. 4 is a graph of the FAM channel — the desired ASF DNA for a positive sample;

in FIG. 5 shows a graph of the Su5 channel — an internal control specimen of specificity — pig DNA — positive sample;

in FIG. 6 is a graph of the HEX channel — internal control sample of isolation — ASF synthetic DNA — positive sample; in FIG. 7 is a graph of the FAM channel — the desired ASF DNA for a questionable sample;

in FIG. Figure 8 shows a graph of the Su5 channel — an internal control specimen of specificity — pig DNA — a dubious sample;

in FIG. Figure 9 shows a graph of the HEX channel — an internal control sample of isolation — ASF synthetic DNA — a dubious sample.

A method for detecting DNA of African swine fever virus by polymerase chain reaction in real time is as follows.

To control the stage of DNA isolation of African swine fever virus, a recombinant plasmid with a fragment of the African swine fever virus vp72 gene is used as a control for the quality of virus isolation, and a recombinant plasmid with a gene fragment of healthy pig tissue as a control for the specificity of the reaction. Specific primers and probes were constructed using computer programs based on analysis of the nucleotide sequences of reference strains and isolates published on the GenBank resource and selection of conditions for real-time PCR using the developed primers and probe carrying a fluorophore and quencher, and a complementary part of the amplified with specific fragment primers. A pair of synthetic oligonucleotide primers and a fluorescent probe complementary to the conservative region of the African swine fever virus genome of the region of the vp72 gene are taken in a ratio of 1: 1: 0.5 and have the following nucleotide composition:

SEQ NO 1: 5'-CTG-CTC-ATG-GTA-TCA-ATC-3 '(direct primer),

SEQ NO 2: 5'-GAT-ACC-ACA-AGA-TCG-CCG-3 '(reverse primer),

SEQ NO 3: 5'-FAM-CCA-CGG-GAG-GAA-TAC-CAA-CCC-AGT-G-BHQ1-3 '(fluorescent probe), where BHQ1 is a dark fluorescence quencher attached to the 3'-terminal nucleotide, a FAM is a fluorescent dye attached to nucleotide C.

The reaction is characterized in that the amplification mixture is ready for use in the following mode (for Rotor-Gene 6000):

1. Temperature retention 95 ° С - 90 s

2. Cycle with detection 95 ° С - 15 s

60 ° С - 30 s - Detection (annealing with reading)

72 ° С - 40 s - elongation

The cycle is repeated 40 times.

To develop a method for diagnosing the genome of the virus of African swine fever, pigs were carried out in several stages.

1. Analysis of the structure of the genome of the virus of African swine fever.

2. Design of primers and probe.

3. Selection of the optimal pair of primers and probe.

4. Modeling the composition of the reaction mixture of the diagnostic method using the developed pair of primers and probe.

5. Testing the conditions of the method for diagnosing ASF using the developed pair of primers and probe.

6. Determination of the sensitivity of the method for the diagnosis of ASF using the developed pair of primers and probe.

7. Determination of the specificity of the method for the diagnosis of ASF using the developed pair of primers and probe.

When designing the primers and probe, the main requirements were: the degree of homology (complementarity) with the selected gene region; the absence of self-complementary regions within the oligonucleotides and complementarity to each other in order to prevent the emergence of stable secondary structures (dimers); the proximity of the annealing temperature of the primers.

The developed oligonucleotides have the optimal size (24–25 pl.) And GC composition (45.8 and 48%), annealing temperature of primers 55 ° С, and probe annealing temperature up to 72 ° С.

The composition of the reaction mixture was selected so that the concentration of MgCl ₂ ions was in the range of 2.0-2.5 mm, which ensures the optimal speed and accuracy of the Taq polymerase enzyme, the concentration of dNTP is not more than 2.5 mm, the concentration of each primer is 1.5 pmol / μl, probe 0.75 pmol / μl and sample volume 10 μl.

Testing of PCR conditions using the developed oligonucleotides was carried out on a Rotor-Gene 6000 amplifier (Corbett Research Pty Ltd., Australia).

The temperature-time regime of PCR is presented in the table.

The result was estimated from the fluorescence signal accumulation curves for each of the channels specified for the samples was carried out according to the graphs from the report of the Rotor-Gene 6000 device using Real-time PCR.

For negative sample

FIG. 1. Channel FAM - the desired DNA ASF (purple color). From the studied samples did not come out. Only the control signal is recorded (red).

FIG. 2. Su5 channel - an internal control sample of specificity - ASF DNA (violet color) - come out later than the graph of the positive control sample (red color), which indicates a slight inhibition of the reaction.

FIG. 3. HEX channel - internal control sample of isolation - synthetic DNA ASF (purple color). The graph of exogenous internal control of the release and the graphs of the test samples are the same, which indicates that the reaction was successful.

For a positive sample

FIG. 4. FAM channel - the desired DNA of ASF (blue color) - the fluorescent signal began to accumulate until the 36th cycle, it can be considered positive, the Negative control sample did not come out - there is no control contamination.

Figure 5. Su5 channel - an internal control sample of specificity - ASF DNA (blue color) - the accumulation of a fluorescent signal is sufficient to consider the reaction as positive. The black graph is a negative control sample. Fluoresces due to the presence of an exogenous control sample introduced into the tube at the isolation stage. The red graph is a positive control.

FIG. 6. HEX channel - internal control sample of isolation - synthetic DNA of ASF - accumulation of a fluorescent signal is enough to consider the reaction positive. The black graph is a negative control sample. Fluoresces due to the presence of an exogenous control sample introduced into the tube at the isolation stage. The red chart is positive.

For a dubious sample

FIG. 7. FAM channel - the desired DNA of ASF (blue color) - the graph shows that both the negative control sample and the test sample came out before the 36th cycle, this indicates a possible contamination.

FIG. 8. Su5 channel - an internal control sample of specificity - ASF DNA (blue color) - the accumulation of a fluorescent signal is sufficient to consider the reaction as positive. The purple plot is a negative control. Fluoresces due to the presence of an exogenous control sample introduced into the tube at the isolation stage. The red graph is a positive control.

FIG. 9. HEX channel - internal control sample of isolation - synthetic DNA of ASF - accumulation of a fluorescent signal is sufficient to consider the reaction as positive. The purple plot is a negative control. Fluoresces due to the presence of an exogenous control sample introduced into the tube at the isolation stage. The red graph is a positive control.

When taking into account the result, the threshold (threshold) was set manually at 10% of the maximum fluorescence level in the last amplification cycle. The threshold level was not more than 0.02. The values of the indicator "Ct" were at the level of 24-25. In positive samples, the fluorescence curve crosses the threshold line and, depending on the signal intensity, rises above the line more or less. In negative samples and negative control, fluorescence is not observed, which is reflected in direct detection at or below the threshold line.

An example of a specific implementation of the method for detecting DNA of the virus of African swine fever by polymerase chain reaction in real time.

Example 1. For analysis by PCR, 2 samples of spleen tissue from pigs, recognized as patients with African swine fever, were taken on the basis of PCR studies using a kit manufactured by GNU VNIIVViM, Pokrov. During PCR in the samples, fluorescence curves crossing the threshold line were obtained, in the absence of such in the negative control. This indicates the reproducibility of the results of the experiments.

Example 2. The use of the amplification reaction to detect DNA of the virus of African swine fever, using developed specific oligonucleotide primers and probe.

For analysis by PCR, 6 samples of pig spleen tissue were taken. In the PCR process, fluorescence curves crossing the threshold line were obtained in all samples in the absence of such in the negative control. The values of the "Ct" indicator ranged from 32 to 36. This indicates the reproducibility of the results of the experiments.

The proposed diagnostic method allows you to quantitatively identify in the early stages of the highly conserved region of the VP72 gene of the virus of African swine fever. The use of an oligonucleotide probe can increase the sensitivity and specificity of the method, eliminate subjectivity in evaluating the results. Using the proposed modification of PCR can significantly reduce the possibility of contamination of samples, premises, equipment and reagents, as well as reduce the time of analysis, which is important for both pet owners and veterinarians.

The proposed diagnostic method was tested with positive results and regular reproducibility of these results in 2016 on 8 samples of pig spleen tissues recognized as patients with African swine fever on the basis of PCR studies using a kit produced by GNU VNIIVViM, Pokrov.

The work was carried out on the basis of the Krasnodar State Agrarian University.

Claims (4)

A real-time method for detecting African swine fever virus DNA by real-time polymerase chain reaction, the method comprising: DNA isolation using control samples: to control the quality of virus isolation, control the specificity of the reaction; DNA amplification using synthetic primers and a fluorescent probe, including denaturation at 95 ° C, cycling: denaturation - annealing - elongation and evaluating the result from the accumulation curves of the fluorescent signal for each of the given channels, characterized in that they use a recombinant plasmid with a fragment of the vp72 virus gene African swine fever as a control for the quality of virus isolation, a recombinant plasmid with a gene fragment of healthy pig tissues as a control for the specificity of the reaction, and synthetic ol onukleotidnye primers and fluorescent probe taken in the ratio 1: 1: 0.5, complementary to conserved region of the genome of African swine fever virus of pigs vp72 gene region and have the following base composition:

- direct primer

- reverse primer

- a fluorescent probe, where BHQ1 means a dark fluorescence quencher attached to the 3'-terminal nucleotide, a FAM is a fluorescent dye attached to nucleotide C, the amplification is carried out in the following mode: denaturation at 95 ° C for 90 s, then cycling with detection, including denaturation at 95 ° C for 15 s, annealing - 60 ° C - 30 s and elongation - 72 ° C for 4 s, and the cycle: denaturation - annealing - elongation is repeated 40 times, while the accumulation of the fluorescent signal is measured channels: FAM for a specific signal; HEX for internal control signal; CY5 for the signal of exogenous internal control, and if the fluorescence signal accumulation curves go out before the 36th cycle, then the reaction result is considered positive, and if the curves do not cross the threshold line or cross it after the 36th cycle, the reaction result is negative.

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