RU2270252C2 - Differentive oligonucleotide kit for species identification of genus orthopoxvirus viruses, biological microarray, kit and method for species identification of genus orthopoxvirus viruses - Google Patents

Abstract

FIELD: virology, medicine, veterinary, molecular biology.

SUBSTANCE: claimed method includes two-step PCR to produce single strain fluorescent labeled DNA fragment; hybridization on microarray, containing differentive oligonucleotide kit; date registration and interpretation. Results are evaluated by comparison of obtained data with set of standard, unique for each species templates, wherein said templates are selected in such a manner that fluorescent signal of perfect duplex allows unambiguous identification of genus Orthopoxvirus viruses. Differentive oligonucleotide kit, biological microarray, and kit useful in carrying out the method also are disclosed.

EFFECT: accelerated analysis method of increased accuracy.

8 cl, 1 dwg, 1 tbl, 1 ex

Description

Technical field

The invention relates to molecular biology, virology, and specifically to species identification of viruses of the genus Orthopoxvirus, including the step of hybridizing a specially prepared viral DNA on a biological microchip. The invention also discloses the construction of highly specific DNA probes that differentiate types of orthopoxviruses according to the sequence of the crmB gene.

State of the art

Currently, the following methods are used for species identification of orthopoxviruses:

I. Detection of smallpox antigen by immunofluorescence [1, 2];

II. Immune electron microscopy [3, 4];

III. Isolation on chicken embryos [5, 6];

IV. Enzyme-linked immunosorbent assay as applied to the species identification of orthopoxviruses [2, 7];

V. Indirect hemagglutination reaction [8];

VI. PCR based methods:

a) followed by analysis of the length of the amplified fragment [9];

b) followed by restriction analysis [9, 10].

The effectiveness of the immunofluorescence method depends on the type of test material. The reliability of the results of studies of materials from pustules and crusts is small due to the admixture of leukocytes with autofluorescence.

Methods (II, IV) require the presence of species-specific monoclonal antibodies for each type of orthopoxviruses. The possibility of applying the methods at the moment is shown only on a very limited number of orthopoxviruses. In addition, the use of methods (I, II, IV) for species diagnosis is limited due to the close antigenic relationship of representatives of Orthopoxvirus.

Isolation on chicken embryos (III), being essentially a cultural method, requires special equipment and highly qualified personnel. The analysis takes several days and is associated with increased biological hazard. In addition, the method is very sensitive to the quality and age of the used chicken embryos and often causes difficulties in interpreting the results.

Method (V) requires not only the presence of monoclinal antibodies, but also the use of specially prepared ram sheep erythrocytes. The method has the same disadvantages as for methods I, II, IV, in addition, it does not have a sufficiently high sensitivity.

The above disadvantages are eliminated in the present invention.

SUMMARY OF THE INVENTION

The object of protection of the present invention is an accelerated method for species identification of six species (and two subspecies) of the viruses of the genus Orthopoxvirus. The crmB gene encoding the viral analogue of the tumor receptor factor cell receptor was selected as a target for species identification. The method is based on two-stage PCR to obtain a single-stranded fluorescently labeled DNA fragment, followed by hybridization on a biochip containing a set of differentiating oligonucleotides, as well as registration and interpretation of the results.

To implement the method, biological microchips are used, made of microarrays, which are a glass substrate with gel cells deposited on it. Each gel cell contains an individual differentiating oligonucleotide.

The preparation of a sample for hybridization with oligonucleotides immobilized on a chip consists in obtaining a single-stranded fluorescently labeled product by two-stage PCR. The first stage of PCR is used to amplify the studied fragment of the crmB gene, the second stage is called asymmetric PCR and is aimed at preferentially amplifying only one of the DNA strands using an amplicon from the first round of PCR and a primer labeled with a fluorescent dye, for example TexasRed®.

Hybridization of the studied DNA with microchip oligonucleotides is carried out by incubating the hybridization mixture in a sealed reaction chamber over the chip for several hours at a given temperature. Oligonucleotides immobilized on a microchip are characterized by species specificity for various species of the orthopoxvirus genus.

The fluorescent signal is detected from a dry microchip after washing it using a detecting device, for example, an experimental setup consisting of a fluorescence microscope, a CCD camera and a computer, or a portable setup that uses laser excitation light and the fluorescence signal is detected using photographic film. The interpretation of the obtained hybridization picture is carried out by comparing it with a set of standard, individual for each type of orthopoxviruses, templates.

The main advantages of the method for identifying orthopoxviruses by hybridization of amplified fluorescently labeled DNA on a microchip are:

- high reliability of the method, based on the simultaneous analysis of several species-specific sections of the gene crmB viruses;

- speed of analysis;

- ease of implementation of the method, eliminating the need to attract highly qualified personnel;

- the relative cheapness of the method.

Another object of protection proposed by the present invention is a kit for implementing the method, including:

a) a microchip containing a set of differentiating oligonucleotides and used for the specific diagnosis of orthopoxviruses, and

b) if necessary, reagents for DNA isolation, primers for PCR (for amplification of the studied fragment of the viral gene crmB), reagents for hybridization, as well as a device for recording analysis results (for example, a portable chip analyzer that uses light as an exciting source lasers and equipped with a camera or CCD camera).

Another object of the invention is a biological microchip used in the claimed method for carrying out species identification of viruses of the genus Orthopoxvirus and which is a gel microarray on a glass substrate on which differentiating oligonucleotides are immobilized.

Another object of the invention is a set of differentiating oligonucleotides intended for immobilization on a biological microchip and characterized by species-specificity (complementarity) to species of the genus orthopoxviruses.

In addition, the invention is illustrated by the drawing, which shows hybridization patterns and their comparison with patterns (control).

DETAILED DESCRIPTION OF THE INVENTION

The terms and definitions used in the description of the invention mean the following.

Biological microchip - (biochip, DNA chip), a carrier plate with an area of about 1 cm ² , on which in a certain order there are cells containing immobilized single-stranded oligonucleotides with a unique base sequence. The number of cells can be up to 1 million per 1 cm ² .

Antigen - (from anti ... and ... gene) substances that are perceived by the body as foreign and cause a specific immune response.

Antibodies - proteins (immunoglobulins) of human blood plasma and warm-blooded animals, which have the ability to specifically bind to antigens. Interacting with microorganisms, they prevent their reproduction or neutralize the toxic substances released by them.

PCR (polymerase chain reaction) is an enzymatic reaction that utilizes the ability of DNA polymerase to synthesize new DNA chains using existing DNA molecules as a template according to the principle of complementarity. The use of the PCR method allows a thousand-fold increase in the number of the studied fragment in the sample, respectively, increasing the sensitivity of methods based on PCR diagnostics.

Amplification - (from lat. Amplificatio - extension) - is used in molecular biology to indicate an increase in the amount of DNA in the PCR process.

The objective of the present invention is to create an express method of species identification of orthopoxviruses on biochips. The method suggests typing according to species-specific regions of the crmB gene, which is convenient in that it allows reliable identification of six species of this genus. A set of species-specific oligonucleotide probes (a set of differentiating oligonucleotides) immobilized on a chip is proposed. Using standard methods for the isolation of viral DNA allows the analysis of natural samples, including animal and human tissues, for the presence of a particular virus of the genus Orthopoxvirus. In the presence of special equipment, the method can be used for diagnostics in the field.

The schematic diagram of the differential diagnosis of orthopoxviruses on a biochip includes the following items:

I. The principle of creating a biochip for species identification of orthopoxviruses.

1) Target selection.

The product of the crmB gene is a viral analogue of the cell receptor of the tumor necrosis factor of humans and some mammals, one of the main factors of virulence of this virus group. Secreting an infected cell, it binds the cell factor of tumor necrosis, thereby inhibiting the cytokine-mediated immune response. Species specificity of the above mammalian cytokine, reflecting on the structure of the viral analogue of the receptor, determines the narrow host specificity of most orthopoxviruses. Thus, the crmB gene contains conserved species-specific regions suitable for species identification.

2) DNA isolation.

DNA is isolated from a natural sample (animal and human tissue) using the methods described in the literature for the isolation of DNA from animal cells.

3) Selection of primers and differentiating oligonucleotides.

The primers were selected taking into account the requirements of high specificity for conserved regions of the crmB gene of orthopoxviruses, which allows one to selectively amplify the studied fragment directly from natural samples, as well as the requirements that are standardly applied to primers - the absence of highly stable secondary structures and not exceeding 3-4 ° С difference in melting temperatures. Differentiating oligonucleotides are characterized by species specificity for various species of the genus orthopoxviruses.

II. The manufacture of microarrays for the immobilization of oligonucleotides in order to obtain a biochip.

A microarray containing cells 100 × 100 × 20 μm in size was prepared by photopolymerization of a 5% polyacrylamide gel, as described previously [11]. The gel was activated, after which the deposition of solutions of oligonucleotides and their covalent binding to the active groups of the gel were carried out.

III. Sample preparation for analysis.

The crmB gene fragment is amplified using highly specific primers. The resulting amplification is used to generate fluorescently labeled single-stranded DNA for subsequent hybridization to a microchip. For this, the method of the so-called asymmetric PCR is used.

IV. Hybridization.

The fluorescently labeled PCR product, the preparation of which is described in paragraph III, then hybridizes with differentiating microarray oligonucleotides in a specially selected buffer for 4-6 hours.

V. A method for recording and interpreting hybridization results.

Differentiating oligonucleotides are divided into five groups depending on the species-specific region of the crmB gene. The fluorescence signal intensity of the resulting perfect duplexes within each group is significantly higher than the signal intensity for imperfect duplexes, which allows reliable species identification of orthoproxviruses. Interpretation of the results is carried out based on the hybridization picture, which is obtained using an experimental setup, including, for example, a fluorescence microscope, a CCD camera and a computer.

Information confirming the possibility of carrying out the invention

Oligonucleotides

Biochip immobilizing oligonucleotides were synthesized on a 394 DNA / RNA synthesizer (Applied Biosystems) using a standard phosphoamidite procedure. For the synthesis of oligonucleotides for the hybridization microchip, a 3'-Amino-Modifier C7 CPG 500 was used, so that the synthesized oligonucleotide contained a free amino group spacer at its 3'-end. PCR primers were modified at the 5'-end using a 5'-Ammo-Modifier C6 (or C12) (Glen Research, VA).

Preparation of micro-chips for microchip.

A microarray containing cells 100 × 100 × 20 μm in size was prepared by photopolymerization of a 5% polyacrylamide gel, as described previously [11].

The gel was activated with 2% trifluoroacetic acid at room temperature for 10 minutes, washed with water and dried. Next, the gel was sequentially processed in Repel-Silane (2% solution (weight / volume) of dimethyldichrolosilane in 1,1,1-trichloroethane, LKB-Produkter AB, Bromma, Sweden) for 30 s; dichloromethane - 10 s; ethanol (95 vol.%) - 10 s; washed with water for 3 min and dried.

A 1 mM oligonucleotide solution was applied twice with a robot needle in a volume of 1 nl. To stabilize the covalent bonds between the amino groups of the spacers flanking one of the ends of the oligonucleotides and the aldehyde groups of the gel, the matrix was placed in a 0.1 M solution of the pyridine-borane complex (Aldrich Chemical Co., Inc., Milwaukee: WI) in chloroform, the upper aqueous phase was layered and kept 12-16 hours at room temperature. Next, the biochip was washed twice with ethanol (95 vol.%) And water. Unreacted aldehyde groups were reduced with a freshly prepared solution of 0.1 M NaBH ₄ (Aldnch) for 20 minutes at room temperature. The biochip was washed with water, dried and stored at room temperature.

Preparation of material for amplification.

Isolation of DNA from a natural sample (animal and human tissue) is performed using methods described in the literature for the isolation of DNA from animal cells.

Amplification of a fragment of the crmB gene and obtaining a single-chain labeled product by PCR.

The preparation of viral DNA for hybridization is carried out using two-stage PCR on a GeneAmp PCR-system 2400 amplifier (Perkin Elmer, Foster City, CA, USA). For amplification, TNFR1f and TNFR3r primers are used, flanking a 267 bp fragment. for human smallpox virus and varying in length in other species.

The first stage of PCR is performed to obtain the selected viral DNA fragment. The reaction buffer contains 16.6 mM (MH ₄ SO ₄ , 67 mM Tris-HCl, pH 8.6 at 25 ° С, 1.75 mM MgCl ₂ , 120 μM dNTP, 0.1% Triton X-100, 1.5 Unit Taq DNA polymerase activity (Silex, Moscow, Russia) and 5 pMol of each of the primers TNFR1f and TNFR3r in a volume of 30 μl.

Primers

TNFR1f 5'-GCTTCCAGATTATGTGATAGCAAGACTA-3 '

TNFR3r 5'-TexasRed®-NH-TCCGGATACTCCGTATCCTATTCC-3 '

Temperature: denaturation at 95 ° C for 5 minutes, then 30 amplification cycles (95 ° C for 35 sec, 64 ° C for 45 sec, 72 ° C for 45 sec), final incubation at 72 ° C for 5 min. Two microliters of the reaction mixture after the first stage of PCR is used as a matrix for the second stage.

The second stage is carried out to obtain a predominantly single-stranded product necessary for hybridization with differentiating oligonucleotides. For this, the TNFR1f primer and the TNFR3r fluorescently labeled primer are used in a ratio of 1/10, respectively. The temperature regime was the same as for the first stage, the number of cycles was increased to 35.

Hybridization of the amplified labeled product on a biochip 12 μl of a sample obtained in asymmetric PCR (~ 1.2 μg ssDNA) is used for hybridization on a biochip in a buffer of the following composition: 1 M NaCl, 50 mM HEPES, pH 7.5, 5 mM Na ₂ EDTA. Hybridization is carried out in a commercially available hybridization chamber with a volume of 30 μl (Sigma) for 4-6 hours at 37 ° C. Washing is carried out three times in a buffer: 0.8 M NaCl, 50 mM HEPES, pH 7.0, 6 mM EDTA, 0.5% Twin 20 at 37 ° C, after which the chip is dried.

Registration and interpretation of hybridization results

Registration of the hybridization pattern is carried out in an experimental setup consisting of a fluorescence microscope, a CCD camera and a computer. For digital image processing, WinView (Princetone Instruments, USA) is used. The measurement of the intensity of fluorescent signals is carried out using the original software.

The interpretation of the results is as follows. Inside each column of gel cells that contain oligonucleotides for one of the species-specific positions of the crmB gene, the intensities of fluorescent signals are visually (or using software). A hybridizable DNA sample is capable of forming a perfect duplex with only one species-specific oligonucleotide in each row, and imperfect ones with all the others. The fluorescence signal of a perfect duplex in each row is several times greater than the signals of imperfect duplexes. As a result, a hybridization pattern is formed on the chip as a whole, which is different for six types of orthopoxviruses, which allows us to unambiguously interpret the obtained data.

Biochip structure

The biological microchip contains 15 immobilized oligonucleotides, a list of which is presented in Table 1. Each of the five vertical rows of the biochip contains oligonucleotides for any one species-specific position of the crmB gene. Thus, oligonucleotides for five species-specific positions of the indicated gene are presented on the chip, which allows for reliable species identification. The resulting hybridization picture is compared with the patterns (Fig. 1), based on the correspondence to one of them, the results are interpreted.

Table 1.
Differentiating oligonucleotides for species identification of orthopoxviruses. No. / No. Amino Acid Position Strain The nucleotide sequence, 5'-3 ', 3'-NH ₂ Length, nucle. T pl. duplex (1 M NaCl), ° С ΔG duplex, Kcal / mol one 63, ins. cow STAA (C / T) ACAAACACACA-NH ₂ 16 61.8 -24.3 2 63 All the rest STAA (C / T) ACACAATGTAC-NH ₂ 16 61.8 -23.0 3 63, subst. rabbit + some v.v. STAA (C / T) ACACGATGTAC-NH ₂ 16 64,4 -24.4 four 81 All the rest TTACCCGCTTGTC-NH ₂ 13 61.3 -25.4 5 81, subst. monkey TTACAGGCTTGTCT-NH ₂ fourteen 60.5 -23.8 6 81, subst. tatera TTACCCACTTGTCT-NH ₂ fourteen 60.5 -23.5

7 90 variola AAGATGCAATAGTAAT-NH ₂ 16 60.7 -25.2 8 90, subst. camel + cow AAGATGCGATAGTAAT-NH ₂ 16 61.2 -26.6 9 90, subst. monkey + tatera AAGATGTGATAGTAAT-NH ₂ 16 60.5 -23.1 10 90, del. Buff. + Vv + rab. GGAAGACGCGAT-NH ₂ 12 60.9 -24.5 eleven 116 Variola GTCTTCTTAAAGGA-NH ₂ fourteen 60.6 -22.6 12 116, subst. camel GTATTCTCAAAGGA-NH ₂ fourteen 60.6 -22.5 13 116, subst. All the rest GTCTTCTCAAAGGA-NH ₂ fourteen 61.5 -23.2 fourteen 126 All the rest ATTTCCCAAACAAAA-NH ₂ fourteen 60.7 -25.8 fifteen 126 monkey ATTTCTAAAACAAAA-NH ₂ fourteen 60.8 -22.2

Example 1. Differential diagnosis of orthopoxviruses on a biochip.

Theoretical patterns and their corresponding real hybridization patterns.

The drawing shows a hybridization pattern of DNA samples of various orthopoxviruses and patterns corresponding to each case. It is seen that for each of the given types of orthopoxviruses a unique hybridization pattern is characteristic. Inside each column, the cell with the highest fluorescent signal intensity corresponding to perfect duplex is visually distinguishable.

The advantages of the method

The method of species identification of viruses of the genus Orthopoxvirus on a miniature biological chip compares favorably with the speed of analysis, the simplicity of the method used to prepare a sample of viral DNA for hybridization, the possibility of analysis in the field, and also relative cheapness.

Bibliography

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Claims (8)

1. A set of differentiating oligonucleotides for species identification of viruses of the genus Orthopoxvirus, the nucleotide sequence of which is given in table 1. 2. A biological microchip for species identification of viruses of the genus Orthopoxvirus, which is a carrier plate on which a gel microarray is located, in the cells of which differentiating oligonucleotides described in paragraph 1 are immobilized. 3. Set for species identification of viruses of the genus Orthopoxvirus, including a) the biological microchip described in claim 2; b) reagents for sample preparation and identification; c) device (s) for interpreting identification results. 4. The kit according to claim 3, in which the reagents for sample preparation and identification include reagents for DNA isolation, reagents for PCR and reagents for hybridization. 5. The kit according to claim 3, in which a portable chip analyzer is used as a device for interpreting the identification results, using laser light as an exciting source and equipped with a camera or a CCD camera. 6. A method for species identification of viruses of the genus Orthopoxvirus, comprising the steps of: a) the preparation of the biological microchip described in claim 2, which consists in applying a gel microarray to a glass substrate and subsequent immobilization of the differentiating oligonucleotides described in claim 1; b) amplification of a fragment of the cpv gene by two-stage asymmetric PCR using a fluorescently labeled primer to obtain amplified single-stranded viral DNA; c) hybridization of single-stranded DNA obtained from stage b) with the indicated differentiating oligonucleotides on a biological microchip obtained according to stage a) by incubating the specified microchip in a sealed reaction chamber to form duplexes of amplified viral DNA fragments with differentiating microchip oligonucleotides; d) recording the result obtained by detecting a fluorescent signal of a labeled sample, and e) the interpretation of the result obtained by comparing the obtained hybridization pattern with patterns. 7. The method according to claim 6, according to which the registration of the fluorescent signal is carried out visually or using software. 8. The method according to claim 6, according to which the interpretation of the obtained hybridization pattern is carried out by comparing it with a set of standard, individual for each type of patterns, selected in such a way that the fluorescence signal of a perfect duplex allows for unambiguous species identification of viruses of the genus Orthopoxvirus.