RU2645263C1 - Test system of detecting dna of african swine fever virus by real-time polymerase chain reaction - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: invention relates to the field of veterinary virology, in particular to a test system for detecting the DNA of a particularly dangerous agent of African swine fever (ASF). Test system for detecting DNA of the African swine fever virus using real-time polymerase chain reaction includes plastic vials and test tubes, a thermostable Tag-polymerase enzyme, a buffer for the reaction, a mixture of four deoxynucleotide triphosphates, a positive control – recombinant plasmid containing a fragment of the vp72 gene of the African swine fever virus, synthetic oligonucleotide primers and a probe. Synthetic oligonucleotide primers and a fluorescent probe complementary to the preserved region of the genome of the African swine fever virus gene vp72 region are used 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’ – a fluorescent probe and taken at a ratio of 1:1:0.5, wherein BHQ1 – a dark fluorescence quenching agent is attached to 3'-terminal nucleotide, and FAM – a fluorescent dye is attached to nucleotide C, where a 5-fold buffer is used to set up the reaction and distilled water is used as the negative control.

EFFECT: invention is intended to increase the degree of specificity and sensitivity of the test system, as well as to shorten the duration of diagnosis while reducing the likelihood of technological errors during laboratory manipulations.

1 cl, 1 tbl, 9 dwg

Description

The invention relates to the field of veterinary virology, in particular to a test system for detecting DNA of a particularly dangerous pathogen of African swine fever (ASF) in the culture of infected cells and samples of pathological materials from pigs, and can be used in the diagnosis of ASF in research institutions, regional , regional veterinary laboratories and enterprises of the biological industry.

A known test system (see RF patent No. 2360971, class C12Q 1/68, 2009), including plastic bottles and tubes, thermostable Tag polymerase enzyme, PCR mixture for the reaction with specific oligonucleotide primers, complementary highly conserved region genome of the ASF virus of the P30 gene region, positive and negative controls.

A test system is also known (see RF patent No. 2125089, class C12Q 1/68, 1999 - prototype), including plastic bottles and tubes, thermostable Tag polymerase enzyme, reaction buffer, a mixture of four deoxynucleotide triphosphates, positive control - a recombinant plasmid containing a fragment of the vp72 gene of the virus of African swine fever, synthetic oligonucleotide primers and probe.

The disadvantage of the prototype is the lack of specificity and sensitivity of the test system, as well as the duration of the diagnosis, which does not exclude the likelihood of technological errors during laboratory manipulations.

The technical result is to increase the degree of specificity and sensitivity of the test system, as well as reducing the time of diagnosis while reducing the likelihood of technological errors during laboratory manipulations.

The technical result is achieved by the fact that in a test system for detecting DNA from African swine fever virus using a real-time polymerase chain reaction, including plastic bottles and tubes, thermostable Tag polymerase enzyme, reaction formulation buffer, a mixture of four deoxynucleotide triphosphates, a positive control - a recombinant plasmid containing a fragment of the vp72 gene of the virus of African swine fever, synthetic oligonucleotide primers and probe according to the invention use synthetic o igonukleotidnye fluorescent primers and probes complementary to the conserved region of the genome of the virus of African swine fever district gene vp72, having the following composition nukleitidny:

- прямой праймер,

- direct primer

- обратный праймер,

- reverse primer

- флуоресцирующий зонд и взятые при соотношении 1:1:0,5, при этом BHQ1 - темновой гаситель флуоресценции присоединен к 3'-концевому нуклеотиду, а FAM - флуоресцентный краситель присоединен к нуклеотиду С, причем для постановка реакции используют 5-кратный буфер и в качестве отрицательного контроля используют дистиллированную воду.

- a fluorescent probe and taken at a ratio of 1: 1: 0.5, while BHQ1, a dark fluorescence quencher, is attached to the 3'-terminal nucleotide, and FAM is a fluorescent dye attached to nucleotide C, and a 5-fold buffer is used to set up the reaction and distilled water is used as a negative control.

The essential features that distinguish the claimed technical solution from the prototype are:

- nucleotide sequences of primers and probe;

- format of the amplification mixture;

- the composition of the amplification mixture, in which two internal controls for the polymerase chain reaction are used.

The novelty of the claimed test system is that it allows in biological samples, which are optionally used: plasma, blood serum, smears from mucous membranes from latently infected and sick animals; pathological material from dead animals, in infected cell cultures, as well as in pig products and products of pig origin, to identify the specific sequence of the conserved region of the African swine fever virus genome without the use of additional amplicon detection methods, thereby reducing the percentage of technological errors. The format and composition of the amplification mixture reduces the complexity and duration of the analysis, and also allows for efficient production of nucleic acid fragments of the African swine fever virus containing selected functionally significant conserved loci of the target gene of the virus.

The test system is also characterized in that it uses synthetic oligonucleotide constructs:

(прямой праймер),

(direct primer)

(обратный праймер),

(reverse primer)

(флуоресцирующий зонд), где BHQ1 означает присоединенный к 3'-концевому нуклеотиду темновой гаситель флуоресценции, a FAM - флуоресцентный краситель FAM, присоединенный к нуклеотиду С.

(fluorescent probe), where BHQ1 is a dark fluorescence quencher attached to the 3'-terminal nucleotide, and FAM is a FAM fluorescent dye attached to nucleotide C.

Based on the synthetic oligonucleotide constructs of SEQ NO 1-3, an amplification mixture is prepared for real-time PCR; the presence of the desired virus in the studied nucleic acid sample is determined by the growth of the dye fluorescence signal. Moreover, the structure of synthetic oligonucleotide constructs provides binding only to fully complementary DNA targets, which causes a bright fluorescent signal. In addition, the physicochemical properties of the oligonucleotide constructs of SEQ NO 1-3 prevent the formation of studs and duplexes (high-energy internal structures), contribute to the formation of the required amount of amplification product, and are also selected taking into account the same annealing temperature on the target DNA (60-62 ° C). For the stable operation of oligonucleotide structures in the solution of the test system, the concentration of MgCl ₂ and the structures themselves, as well as the ratio of forward, reverse primers and fluorescent probes, the number of amplification cycles and their time at each stage are optimized.

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. 9 is a graph of the HEX channel — internal control sample of isolation — ASF synthetic DNA — doubtful sample;

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. 9 is a graph of the HEX channel — internal control sample of isolation — ASF synthetic DNA — doubtful sample;

Examples of specific use of the test system

The test system is equipped with plastic bottles and tubes, thermostable Tag polymerase enzyme, a 5-fold reaction buffer, a mixture of four deoxynucleotide triphosphates, a recombinant plasmid containing a fragment of the African swine fever virus vp72 gene as a positive control, synthetic oligonucleotide and oligonucleotide regions of the African swine fever virus genome region of the vp72 gene region, having the following nucleitide composition:

- прямой праймер,

- direct primer

- обратный праймер,

- reverse primer

- флуоресцирующий зонд и взятые при соотношении 1:1:0,5 и отрицательным контролем в качестве которого используют дистиллированную воду.

- a fluorescent probe and taken at a ratio of 1: 1: 0.5 and a negative control, which use distilled water.

Example 1. Conducting PCR analysis in the format of "uniplex PCR-RV" for the detection of virus isolated from pig tissues.

For PCR analysis, viral DNA was isolated from pig spleen tissues. Isolation was carried out according to the following method using the commercial kit of the company "IDS" for rapid isolation of DNA Express.

A spleen tissue homogenizer is removed and placed in a 1.5 ml tube with 300 μl of DNA Express solution. Add to each tube 10 μl of "EVK" (exogenous internal control of the release, part of the commercial kit). Vortex is mixed for 5-10 seconds. Precipitate droplets from the tube cap by short centrifugation. Warm up at 98 ° C for 15 minutes. Centrifuged for 2 minutes at maximum speed (10-14.5 thousand rpm). Transfer the supernatant to a clean 1.5 ml tube. The transferred supernatant is used for PCR.

To conduct PCR itself using a test system, you need:

Prepare the required number of tubes with the reaction mixture, including three tubes for standard DNA samples and a negative control sample. When designing primers and a probe, the basic requirements are: 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%), primer annealing temperature of 55 ° C, and probe annealing temperature of up to 72 ° C.

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 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 -1.5 pmol / μl, probe - 0.75 pmol / μl and sample volume - 10 μl.

The tubes with the amplification mixture are placed in accordance with a pre-prepared protocol, which indicates the numbers of the analyzed samples, as well as the tubes of the positive and negative controls. If the reaction is carried out on a Rotor Gene 6000 / Q analyzer (Corbett Research), the tubes can be labeled.

Add to all tubes with individual tips with aerosol filters of 7 μl:

a) in an amplification tube for negative control

- negative control sample (OKO) from the test system kit;

b) in test tubes of test samples — test DNA samples;

c) in an amplification tube for positive control

- positive control sample (FFP) from the test system kit.

To reduce the risk of contamination, samples should be added in this order.

If possible, the tube into which the sample was introduced should be closed with a lid and centrifuged for 15 seconds in a centrifuge or microcentrifuge-vortex. The tubes are then transferred to the instrument and amplification is carried out.

Amplification is carried out according to the program for the Rotor-Gene Q or Rotor-Gene 6000 amplifier (Corbett Research), shown in Table 1.

The amplification products are detected automatically by the device in each amplification cycle. Based on these data, the control program constructs the fluorescence signal accumulation curves for each of the channels specified for the samples.

To work with the test system, channels are used: FAM (specific signal), HEX (internal control signal), CY5 (exogenous internal control signal).

Evaluation of the effectiveness of using the test system was carried out according to the curves of fluorescence signal accumulation 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 the ASF (blue color) - the fluorescent signal began to accumulate until the 36th cycle, it can be considered positive. A negative control sample did not come out - there is no control contamination.

FIG. 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 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 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 - an internal control sample of isolation - synthetic DNA of ASF - accumulation of a fluorescent signal is enough to consider the reaction 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.

Example 1. For analysis by PCR, 2 samples of spleen tissues 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 included in the test system.

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 test system allows you to quantify the early stages of the highly conserved region of the VP72 gene of the African swine fever virus. 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 test system 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, based on 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)

Real-time polymerase chain reaction test DNA system for detection of African swine fever virus DNA, including plastic bottles and tubes, thermostable Tag polymerase enzyme, reaction buffer, a mixture of four deoxynucleotide triphosphates, positive control - recombinant plasmid containing the gene fragment African swine fever virus vp72, synthetic oligonucleotide primers and probe, characterized in that synthetic oligonucleotide primers and fluorescent are used probe complementary to the conservative region of the African swine fever virus genome of the vp72 gene region, having the following nucleitide 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 and taken at a ratio of 1: 1: 0.5, while BHQ1 - a dark fluorescence quencher is attached to the 3'-terminal nucleotide, and PAM - a fluorescent dye is attached to nucleotide C, a 5-fold buffer is used for the reaction and distilled water is used as a negative control.

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