RU2756924C2 - Method for early pcr diagnostics of the aleutian mink disease virus carnivore amdoparvovirus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular, to molecular biotechnology, and constitutes a method based on a fundamentally new approach to the method for conducting a polymerase chain reaction, excluding the stage of DNA extraction. The method for conducting a polymerase chain reaction eliminates the stage of DNA extraction and allows for PCR analysis using native blood without additional processing.

EFFECT: method provides a possibility to avoid the loss of genomic DNA of the Aleutian mink disease virus, occurring at the stage of DNA extraction, as well as the probability of achieving false positive and false negative results.

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Description

The method relates to the field of biotechnology, in particular to molecular biotechnology, and is a method based on a fundamentally new approach to the method of carrying out the polymerase chain reaction, excluding the stage of DNA extraction.

Today, the Aleutian disease caused by the parvovirus Carnivore amdoparvovirus of the genus Amdovirus is a serious threat to the health of the mink. Aleutian disease was originally observed in the United States in the late 1940s in the Aleutian mink (a gray-brown variety similar in color to Aleutian foxes). This disease belongs to plasmacytosis, since it detects a large number of plasma cells in tissues or exudates (Canuti et al, 2015). The classic form of this disease occurs in adult minks and is characterized by hypergammaglobulinemia, which causes glomerulonephritis, leading to acute renal failure, arthritis and decreased reproductive function (Prieto et al, 2018). The acute form of the disease is usually characterized by lethal interstitial pneumonia in mink pups. There is no effective treatment or vaccine against Aleutian mink disease.

Aleutian disease may be temporarily asymptomatic, but the course of the disease is mainly determined by the virulence of the viral strain. Known strains vary in pathogenicity, from the non-pathogenic AMDV G strain, the low-virulence SL-3 strain isolated in Germany, the Pullman strain, which is lethal to the Aleutian mink variety, and the highly lethal Utah strain. Aleutian disease affects the American mink (Neovison visori), the European ferret (Mustela putorius), the European marten (Martes martes), the common genetta (Genetta genetta), the raccoon dog (Nyctereutes procyonoides), and the skunk (Mephitis mephitis), which can be vectors.

The genome of the virus consists of a single DNA strand up to 4.8 tn long, containing information about three non-structural proteins (NS1, NS2, and NS3) and two structural proteins (VP1 and VP2). Figure 1 shows a diagram of the Aleutian mink disease virus genome. Non-structural proteins serve the expression of viral material, while structural proteins determine antigenic properties. It has been shown that caspase inhibitors in adult minks can limit viral multiplication, since caspases are involved in the regulation of NS1 virus replication. Of particular importance is the VP2 protein, which contains a region with a high polymorphism of amino acids, the combination of which is specific for certain strains. The high polymorphism of this region is due to its participation in the formation and localization of capsid proteins in the tertiary structure and makes it possible to differentiate strains.

In laboratory conditions, disease control is carried out based on the analysis of pathological and cytomorphological data, the results of laboratory studies of the blood serum of animals using an iodine test according to the Melloni method, which is not a specific method for identifying the pathogen and allows to identify no more than 40% of infected animals, and the reaction of immunoelectroosmophoresis (RIOEF ), enzyme-linked immunosorbent assay (ELISA). Animals with a confirmed diagnosis are discarded.

It should be noted that at a certain stage of the course of the disease, the level of specific antibodies to virus proteins is extremely low, therefore, ELISA methods used in diagnostics in most cases give false negative results.

However, there has been regular reinfection of farms despite significant efforts to eradicate the disease. This happens due to the resistance of viral particles to physical and chemical agents, the rapid spread of the virus by insects and humans.

With the help of PCR methods, which require the stage of DNA extraction, it is impossible to detect the virus in the chronic course of Aleutian mink disease, since in such animals the virus does not replicate and the amount of pathogen DNA is very low in biological samples.

PCR is generally not used on farms for examining the main herd due to the high cost of analysis, which is partly due to the need for DNA extraction. DNA extraction techniques involve the use of commercial kits that do not always meet quality standards, which leads to incomplete elimination of some inhibitors and, consequently, DNA loss, while the likelihood of cross-contamination of samples increases.

A known method of PCR diagnostics of the Aleutian mink disease virus "ABN" (manufacturing organization - FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow) for detecting DNA of the Aleutian mink disease virus (AMDV) in biological material by the polymerase chain reaction (PCR) ) with electrophoretic detection of amplification products in agarose gel. The method is based on the amplification of a specific DNA region from a biological material due to multiple repetitions of DNA denaturation cycles in the test sample, annealing of specific oligonucleotide primers (primers), and the synthesis of complementary DNA strands using the Taq polymerase enzyme.

The disadvantage of this method is that the developers propose the detection of AMDV, based on the amplification of viral DNA fragments homologous to one synthetic oligonucleotide, while, in the event of mutations in the DNA of the virus, amplification is impossible, which, according to the results of electrophoretic separation of amplicons, reveals a false negative analysis result. Another disadvantage of this method is that for PCR it is necessary to use a DNA extraction stage, in which the probability of the loss of viral DNA due to the short length of the AMDV genome is extremely high, and the possibility of contamination of the samples also increases. In addition to the listed disadvantages, the existing method is characterized by the complexity of the PCR setup, including the need to use a special wax plate that prevents mixing of reagents before exposure to certain temperatures. The analysis time without taking into account the special preparation of samples, DNA extraction and its processing is at least 2 hours 30 minutes.

The known method of PCR diagnostics of the Aleutian mink disease virus, proposed by M.V. Martynenko (Martynenko M.V. The use of polymerase chain reaction for tetection of the Aleutian mink disease virus // Agricultural biology. - 2004. - No. 6. - P. 120-122). The method involves carrying out PCR according to the standard technique with the stage of DNA extraction, the use of Taq polymerase and one pair of primers to the AMDV genome. The advantage of the method is that in comparison with RIEOF, this method has a sensitivity 5.5 times higher. The authors confirm the need for further improvement of diagnostic methods, including to exclude the stage of DNA extraction from the procedure for staging the reaction, but the authors failed to achieve this. Moreover, the authors propose to use one pair of primers. Thus, the presented method does not exclude the possibility of loss of the AMDV genome at the stage of DNA isolation, the possibility of overlaps of the desired nucleotide sequences of the short-length virus of the mink DNA, which indicates a high risk of obtaining false-negative results. Also, the use of one pair of primers increases the likelihood of complementarity of the oligonucleotide sequences with the mink genome and, consequently, obtaining false positive analysis results.

The patented method involves the identification of the causative agent of Aleutian mink disease by PCR using recombinant polymerase, in the N-terminus of which there are fewer amino acids, and glutamic acid is replaced by lysine in codon 708, which provides resistance to several types of PCR inhibitors, and, accordingly, an increase in the yield target DNA from whole blood, plasma and serum samples by at least 25%, which eliminates the stage of DNA extraction. Also, the patented method is based on the amplification of DNA fragments with three pairs of specific primers homologous to the sequences of conserved DNA regions of the AMDV genome, which excludes the possibility of obtaining false negative and false positive results based on the results of electrophoretic separation of DNA fragments. Primers used to diagnose AMDV according to the patented method: RP2 F TST AGA AGC AAC GCT TGG GGT GTA TG RGT TGT GTC ACT CCA CTG TST RP3 F TST AGA TTG GGC STA CCT CCT CTC TG R ATA CAG GAC CAA CGT TGT CT NS F TTG GTT GCT TTA CTC C R CTA CTT TTA CAT CAC CAC

The claimed method does not require special preparation of the sample, DNA extraction and any additional tools and involves the use of native tissues for direct amplification during PCR.

To confirm the effectiveness of the claimed method, a comparative analysis of the results of using the patented method and the standard PCR method (with the step of DNA extraction and PFU polymerase) in 50 samples of mink whole blood was carried out to identify AMDV. Of the 50 minks from which bioassays were taken, 10 animals were previously identified by other methods as free from AMDV infection, 19 - as carriers, 21 animals - were not tested.

PCR using PFU polymerase with DNA isolation from burrow biosamples with all three pairs of primers to different regions of the AMDV pathogen genome did not lead to amplification products and, thus, indicated the absence of the pathogen in the analyzed material. At the same time, in biosamples of the same minks using direct amplification methods without DNA isolation using recombinant polymerase, the corresponding fragments of the pathogen genome were simultaneously detected in the amplification products obtained using each of the three primers in PCR, which were absent in the control samples.

According to the results of the analysis of electropherograms of PCR amplification products, carried out without isolation of total DNA using three pairs of primers to regions of the AMDV pathogen genome, it was confirmed that 10 conditionally negative samples did not produce amplification products using all three primers to the pathogen genome, in 19 conditionally positive samples. the presence of amplification products was confirmed; in 21 blood samples of previously unexplored minks, amplification products of AMDV genomic DNA were also detected when all three primer pairs were used in PCR.

Example 1. Were investigated blood samples of mink, which were taken, including from animals previously identified by other methods as carriers of AMDV infection.

For PCR analysis, a PCR mixture with a total volume of 25 μL was used, obtained using a commercial kit of reagents for PCR (Synthol) and thermostable recombinant polymerase:

Intact. blood - 2.5 μl

dNTP - 2.5 μl

Buffer - 2.5 μl

MgCl2 - 2.5 μl

ddH2O -9.75 μl

Pr. F. - 2.5 μl

Pr. R. - 2.5 μl

Pol. rec - 0.25 μl

where primers: RP2 F TST AGA AGC AAC GCT TGG GGT GTA TG R GTT GTG TCA CTC CAC TGT CT RP3 F TST AGA TTG GGC CTA CCT CCT CTC TG R ATA CAG GAC CAA CGT TGT CT NS F TTG GTT GCT TTA CTC C R TST ACT TTT ACA TCA CCA C

The amplification program included the following stages:

1 cycle - primary denaturation 95 ° С 5 min

40 cycles - denaturation 94 ° С 30 sec

- annealing 55 ° С 1 min

- elongation 68 ° С 30 sec

1 cycle - final elongation 68 ° С 2 min

As control samples, we used whole blood from minks that later died from a confirmed diagnosis of Aleutian disease of minks (positive control), as well as a PCR mixture without a blood sample or with a rabbit blood sample.

According to the results of the analysis of electrophoregrams of PCR amplification products, carried out without isolation of total DNA using three pairs of primers to the genome regions of the AMDV pathogen, positive in terms of the content of the Aleutian mink disease virus genome was revealed, and it was also confirmed that the presence of amplification products was detected in all conditionally positive samples. Figure 2 shows an electrophoregram of amplicons obtained by PCR with recombinant polymerase without DNA isolation. In samples 1.1-4.3 in the lane number, the first digit is the animal's blood sample number. Samples 01.1-01.3 - negative control (rabbit blood), 02.1-02.3 - negative control (without the content of the blood sample in the PCR mixture). The second digit in the lane number means the primer number (# 1 - RP2, # 2 - RP3, # 3 - NS).

Example 2. According to the patented method, blood samples from minks were examined, which were taken from animals previously identified by other methods as free from AMDV infection.

For PCR analysis, a PCR mixture with a total volume of 25 μL was used, obtained using a commercial kit of reagents for PCR (Synthol) and thermostable recombinant polymerase:

Whole Blood - 2.5 μL

dNTP -2.5 μl

Buffer - 2.5 μl

MgCl2 - 2.5 μl

ddH2O - 9.75 μl

Pr. F. - 2.5 μl

Pr. R. - 2.5 μl

Pol. rec - 0.25 μl

where primers: RP2 F TST AGA AGC AAC GCT TGG GGT GTA TG R GTT GTG TCA CTC CAC TGT CT RP3 F TST AGA TTG GGC CTA CCT CCT CTC TG R ATA CAG GAC CAA CGT TGT CT NS F TTG GTT GCT TTA CTC C R TST ACT TTT ACA TCA CCA C

The amplification program included the following stages:

1 cycle - primary denaturation 95 ° С 5 min

40 cycles - denaturation 94 ° С 30 sec

- annealing 55 ° С 1 min

- elongation 68 ° С 30 sec

1 cycle - final elongation 68 ° С 2 min

The control samples were whole blood from minks that later died from a confirmed diagnosis of Aleutian disease of minks (positive control), as well as a PCR mixture without a blood sample or with a rabbit blood sample (negative control).

Based on the results of the analysis of electrophoregrams of PCR amplification products, carried out without isolating total DNA using three pairs of primers to the genome regions of the AMDV pathogen, it was confirmed that the presence of amplification products was not detected in all conditionally negative samples. Figure 3 shows the results of electropherogram of amplicons obtained as a result of PCR with recombinant polymerase without DNA isolation using three pairs of primers to regions of the AMDV genome in mink samples conditionally free from infection. The first track is a molecular weight marker.

Example 3. PCR using PFU polymerase with DNA isolation from mink biosamples, the research data of which are given in examples 1 and 2, with all three pairs of primers to different regions of the AMDV pathogen genome, did not lead to the production of amplification products and, thus, indicated about the absence of a pathogen in the analyzed material. Figure 4 shows the results of an electrophoregram of amplicons obtained as a result of PCR with DNA from mink blood using PFU polymerase, where in the lane number the first digit denotes the number of the animal's blood sample, and the second is the primer number (primer No. 1 RP2, No. 2 -RP3 , No. 3 - NS). The first two lanes are molecular weight markers.

For PCR analysis, DNA was first extracted using a commercial kit "M-Sorb" according to the manufacturer's recommendations.

PCR mixture with a total volume of 25 μl was obtained using a commercial kit of reagents for PCR (Synthol) and PFU polymerase 5 units / μl (Silex):

DNA -2 μl

dNTP - 2 μl

Buffer - 2 μl

MgCl2 - 2 μl

ddH2O -14.8 μl

Pr. F. - 2 μl

Pr. R. -2 μl

PFU-Pol - 0.2 μl

The amplification program included the following stages:

1 cycle - primary Denaturation 95 ° C 2 min

40 cycles - denaturation 94 ° С 15 sec

- annealing 55 ° С 15 sec

- elongation 72 ° С 2 min

The technical result. The patented method excludes the stage of DNA extraction, allows PCR with samples of native tissues, reduces the risks of cross-contamination of samples and the time of the study, eliminates the possibility of overlap with the host genome during amplification, eliminates the possibility of obtaining both false positive and false negative results, includes diagnostics for three markers, suggests diagnostics based on the identification of conservative regions of the pathogenic genome. The principle of the method proposed for patenting can be used to detect other viruses with small genomes, both in veterinary medicine and in medicine.

Claims (1)

A method for PCR diagnostics of the Aleutian mink disease virus Carnivore amdoparvovirus, characterized by the use in the PCR mixture of a sample of native mink blood, a thermostable recombinant polymerase, in the N-terminus of which there are fewer amino acids, and glutamic acid is replaced by lysine at codon 708, as well as three pairs primers RP2 F TCT AGA AGC AAC GCT TGG GGT GTA TG RGT TGT GTC ACT CCA CTG TCT RP3 F TCT AGA TTG GGC CTA CCT CCT CTC TG R ATA CAG GAC CAA CGT TGT CT NS F TTG GTT GCT TTA CTA C R CTA CTT CAT CAC CAC, carrying out amplification, including "hot start" and 40 cycles of denaturation, annealing and elongation - the presence or absence of the Aleutian mink disease virus is confirmed by the results of electrophoregram: the presence or absence of bands, respectively.

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