Set of synthetic oligonucleotides for dna identification in blood and other biomaterials of infectious agent of latent viral infection virus torque teno virus of circoviridae family by polymerase chain reaction

FIELD: medicine.

SUBSTANCE: synthetic oligonucleotides for indentifying DNA of Torque teno virus of all known genotypes are disclosed. Primers are combined in a set for DNA identification in blood and other biomaterials of the infectious agent of the latent viral infection Torque teno virus of Circoviridae family by polymerase chain reaction.

EFFECT: invention allows reliable identification of said virus in a biological material.

 

1. The technical field

The invention relates to the field of molecular genetic diagnosis of human viruses. Torque teno virus (TTV) was first discovered in 1997. Population-based studies revealed that the viral genome has a very high variability is currently allocated to 5 groups containing more than 40 genotypes. Also it is shown that the virus is widespread, at least among the 50% of the population. The identified Association between the presence of TTV and acute respiratory diseases, liver disease, asthma, HIV/AIDS, however, the exact function and manifestation of the virus in humans has not been determined.

The need to test the system for TTV obvious, as the literature data indicate the possibility of its use as at least a diagnostic parameter in identifying the diseases listed above.

2. The level of technology

In recent years developed a variety of methods of molecular genetic diagnosis of viruses, as well as assessment of their viral load. These methods allow you to identify specific DNA sequences unique to a particular virus. Thus, the corresponding virus is found in the biomaterial, and the conclusion is made about the presence of infection. The prior art, the following analogs of this invention are:

2.1 Patent # WO 2008138619 "Defined the e new sequence oligonucleotides TTV for warnings, diagnosis and treatment of childhood leukemia"

The present invention describes specific rearrangement of nucleic acid of the virus TTV, and the sequence of the primers and probes, which allow you to amplify and detect a given nucleic acid. In addition, the present invention describes a polypeptide encoded by a nucleic acid, and describes antibodies that bind these polypeptides. In the end, this invention describes methods of application of the obtained nucleic acid for the diagnosis of the development of childhood leukemia, for the diagnosis of other malignant tumors, for the diagnosis of autoimmune diseases, also for the production of vaccines for the prevention and treatment of these diseases.

2.2 Patent # WO 0185771 "New TTV-related virus s-TTV"

According to the latest data related to the virus TTV virus s-TTV may influence the occurrence of such diseases as chronic hepatitis and detection of DNA of this virus you need. On this basis, was cloned DNA virus s-TTV, which made it possible to analyze s-TTV DNA, s-TTV antigen and s-TTV-antibody. In the result, it was possible to diagnose the s-TTV infection or to monitor the infection process. Thus, it can be studied the relationship between s-TTV and human diseases, and technique, you is proving this relationship, can be tested on animals.

2.3 Patent US2001041331 "Methods for the determination of virus TTV"

The present invention is directed to obtaining primers or samples used to determine virus TTV in biological samples. The invention extends to methods, allowing the use of these primers and probes as well as to receive a set of reagents containing these oligomers. In addition, this invention is directed to the use of nucleotide sequences of the virus TTV as vectors and as markers for determining the transmission of the virus between organisms, as well as routes of transmission. In addition, this invention is directed to methods for the determination of TTV infection before xenotransplantation of organs or tissues.

2.4 Patent No. JP2000175700 "Determination of virus TTV using the method of real-time PCR, primer and probe used in this"

The subject of this invention is to provide a new oligonucleotide primers, forward and reverse, is used to determine virus TTV using the method of real-time PCR.

3. Disclosure of inventions

The most promising is the use for these purposes, the method of polymerase chain reaction (PCR) is a highly specific and highly sensitive method for the detection of viral DNA. The principle of PCR is many times the copy (amplification) of a specific DNA fragment, which is the marker for this virus. The PCR reaction is carried out by the enzyme Taq polymerase, which is able to extend oligonucleotide priming (primers) to a specific product.

The main advantage of PCR as a molecular biology research is its extremely high sensitivity. Used in medical practice test-system based on the principle of DNA amplification, can detect a human pathogenic microorganisms, even in cases when other methods (immunological, bacteriological) their identification impossible. System detection of PCR results in "real time", along with the answer to the question about the presence or absence in the sample DNA infectious pathogen, you can evaluate it, which in some cases allows you to specify the diagnosis and to choose the most appropriate method of treatment.

Work on the creation of the oligonucleotides used in these sets, is constructed generally as follows.

1) using open source and commercial databases of nucleotide sequences of the genomes of viruses or by self-determination of the nucleotide sequence of the studied virus selected region of the genome that is unique to this virus (or species groups).

2) based in the alarm region of the genome with the help of special software selected sequence of oligonucleotides, used for PCR reactions (usually it's 2 primer and probe). At this stage the work is to create alignment of many genomic sequences and the selection of the sequence, where the number of mutations corresponds to the desired location of the primers.

Alignment of the genomic sequences means the comparison of sequences of the genomes of many organisms with each other, finding a unique interest for virus genome regions that are unmatched by any other viruses.

The match between the number of mutations and the location of primers means that the currently known mutations, may have this virus, should not affect the selected primers for a region of the genome replacement in the genomic sequence (mutations) may adversely affect the work of the primers.

3) the Manufacture of primers are ordered in a special service center.

4) using practical experiments proved the suitability of the selected sequences for specific purposes (for example, to determine the presence/absence of the virus in the biomaterial; counting the number of DNA of this virus in the biomaterial - viral load).

The present invention allows to detect DNA virus Torque teno virus all known genotypes causing the latent viruso the infection. Similar sets, as well as the reaction mixture, the primers and methods of detecting DNA of this virus is unknown.

The technical result, which is aimed by the invention, is the reliable detection of the indicated virus in a biological material (whole blood, blood plasma, tissue samples).

This result is achieved by the use in the formulation of PCR of a set of synthetic oligonucleotides for the detection of virus DNA (Torque teno virus that causes latent viral infection, comprising primers:

5'-CCT GCA CTT CCG AAT GGC TGA GTT-3'

5'-CTT GAC TGC GGT GTG TAA ACT CAC-3'

and probe: 5'-FAM-CCT CCG GCA CCC GCC CTC GGG ACG-BHQ1-3',

where BHQ1 means attached to the 3'-terminal nucleotide dark quencher of fluorescence, a FAM fluorescent dye FRAMES attached to the nucleotides at the 5'end of the sample.

The specified set of synthetic oligonucleotides is part of the set of the following substances:

1) the Tubes with the reaction mixture, sealed with paraffin. This blend contains the specified primers and probe specific for the DNA part Torque teno virus - the so-called conservative domain, a nucleotide sequence which among different genotypes of TTV same mixture deoxyribonucleotides four types of reaction buffer (80 mm Tris-HCl (pH 8.6 at 25°C), 20 mm (NH4)2 SO4, 3mm MgCl2).

And the primers and probe are synthetic oligonucleotides. Primers specific to the marker region of the genome of the virus is introduced into a reaction directly for amplification (operating time) of the product. Sample (too specific to a given region of DNA) is composed of a fluorescent label, a fluorescence intensity which indicates the amount of product formed, which, in turn, depends on the efficiency of the primers, the number of the starting material and other parameters of the reaction. The primers in the reaction act as components, ensuring the passage of the reaction, and the sample as a component for monitoring the course of the reaction.

2) Solution of the enzyme Taq polymerase.

3) a Positive control sample (PC) - DNA of the recombinant plasmid with the cloned fragment of the conservative section of the TTV genome. The positive control sample is deposited into a separate test tube with the reaction mixture. The result of amplification of the PC shows the reference positive completion of the reaction, which compares the results of the amplification of the samples.

4) a Negative control sample is a sample that is injected in the experiment to control for possible contamination of reagents products previously conducted reactions. Put the capacity result for this sample demonstrates the need to replace the reagents and to rearrange the experiment.

4. The implementation of the invention

1) Biological material (whole blood, blood plasma, tissue samples) before carrying out PCR using the proposed set of reagents is carried out through the procedure of sample preparation using a different set of reagents for sample preparation is not the subject of this patent); in this procedure, from the biological material excreted DNA, which, in turn, used for PCR;

2) the Required number of tubes with the prepared reaction mixture containing specific synthetic oligonucleotide primers and a probe, labeled according to the number of samples;

3) all labeled test tubes without damaging the layer of wax is added to a solution of Taq polymerase;

4) all labeled tubes (except tubes To the-(negative control sample)To+(positive control sample)) shall be allocated according to claim 1 DNA;

5) In a test tube, labeled K-recorded negative control sample;

6) To the tube labeled C+, making a positive control sample;

7) All tubes are installed in the block of thermal cycler, the amplification is carried out according to the modes prescribed in the instructions to the set. Detection results is carried out by detecting thermal cycler automatically at the time and is plification (device: detecting thermocycler DT-322, DT-96 (JSC "APF of DNA-Technology").

Analysis of results is carried out in accordance with the instructions to the device.

In the case of formation of a specific product DNA sample is destroyed, which leads to an increased level of fluorescence, which is fixed by special devices by detecting thermocycler.

A set of synthetic oligonucleotides for DNA detection in blood and other biological pathogen latent viral infection - virus Torque teno virus of the family Circoviridae by polymerase chain reaction, characterized in that it includes primers:
5'-CCT GCA CTT CCG AAT GGC TGA GTT-3'
5'-CTT GAC TGC GGT GTG TAA ACT CAC-3'
and probe: 5'-FAM-CCT CCG GCA CCC GCC CTC GGG ACG-BHQ1-3',
BHQ1 means attached to the 3'-terminal nucleotide dark quencher of fluorescence, a FAM fluorescent dye F attached to the nucleotide C.



 

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