RU2361924C1 - Way of detection of virus of flu a of h5n1 subtype - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: way provides carrying out of reaction of return transcription with RNA of the virus, amplification combined with reaction to two genes, analysis of a reactionary admixture by means of an electrophoresis in an agarous gel and revealing RNA of strains of viruses of flu A by results of the analysis. The invention can be used for detection of virus of flu of A type H5N1 subtype based on detection of genetic material of the originator, and is intended for mass analyses with possible automation of process.

EFFECT: way allows carrying out one-phase express identification of virus of flu A, subtype H5N1.

2 dwg, 2 tbl, 4 ex

Description

The invention relates to molecular biology, virology and can be used to detect influenza virus type A subtype H5N1, based on the detection of the genetic material of the pathogen, and is intended for mass analysis with possible automation of the process.

Type A influenza virus belongs to the family Orthomyxoviridae, is widespread in nature, affects many species of mammals and birds, mainly the aquatic and near-water complex, and can cause epidemics, pandemics, and epizootics (Brown I.H. at al 1993, 1995, 1998). Influenza A virus has a high degree of variability, especially for the surface glycoproteins of the virion: hemagglutinin (HA) and neuraminidase (NA). There are 16 known antigenic subtypes of hemagglutinin (HI-HI5) and 9 subtypes of neuraminidase (N1-N9).

H5N1 subtype influenza viruses were periodically isolated from chickens and other bird species (Castrucci, M.R., et al 1993, Cauthen AN et al 2000). In 1997, an outbreak of the disease was observed in Hong Kong due to direct transmission of the H5N1 avian influenza virus from chickens to humans (Capua I. et al 2002). The spread of avian influenza caused by the H5N1 virus is observed in Asia and Europe (Li, Wang J. et al 2004). During 2004, WHO constantly informed of cases of human exposure to avian influenza, with the disease having a mortality rate of more than 70%. In 2005, epizootics were recorded among birds in Siberia, the Far East, Kalmykia, Astrakhan, and other regions of the Russian Federation. It has been proven that the H5N1 subtype of influenza A virus is able to mutate at high speed and form highly pathogenic strains of the influenza virus, which may pose a serious epidemic danger in the future (Li, Wang J. et al 2004).

The classic diagnostic methods for this pathogen are the hemagglutination inhibition reaction, the complement binding reaction, the neutralization reaction, which determine the presence of viral antigens, and the enzyme-linked immunosorbent assay, which determines the presence of antibodies to viral antigens in blood serum. In addition, there are a number of kits for detecting the genetic material of influenza A virus in biological samples by polymerase chain reaction (WHO 2005), (Payungporn S. Et al, 2004). A scientific publication describing a PCR test system for determining influenza virus RNA describes a diagnostic method that differs from the present invention in specific components and methodological approaches.

An object of the invention is the creation of a method of one-step rapid identification of influenza A virus subtype H5N1. In this case, the technique can be carried out both in two PCRs and using multiplex PCR (identification of two gene regions in one PCR reaction). Multiplex PCR will allow the detection of viral material in one reaction mixture, reducing the time of diagnosis, the percentage of financial costs and technological errors.

The problem is solved by selecting pairs of oligonucleotide primers specific for influenza A virus subtype H5N1.

For the selection of primers used more than 10,000 sequences of HA and NA genes of influenza A virus of various subtypes isolated before 2007. Sequences were analyzed using the Alignment Service and Lasergene programs (version 6.0). The homology level of the selected primers is not less than 95%.

The essence of the invention lies in the fact that a method for detecting a virus based on the amplification of the cDNA region of the influenza A virus using the combined reverse transcription reaction and polymerase chain reaction (RT-PCR) of the viral gene of the hemagglutinin and neuraminidase to increase the number of copies of the DNA of influenza A virus subtype H5N1 . Moreover, the homology level of the selected primers is at least 95% for both genes. A virus is detected if, after separation of part of the reaction mixture in an agarose gel, 184 nucleotide pairs for primers specific for H5 and 213 nucleotide pairs for primers specific for N1 are detected in the analyzed sample.

Primers were selected and analyzed using the Oligo software (version 3.3.) (Breslauer et al 1986).

The lack of homology with other viruses of the Orthomyxoviridae family and with other subtypes of influenza A virus was the main criterion for the selection of oligonucleotides.

Thus, for each gene (HA and NA) of influenza A virus subtype H5N1, a pair of primers was selected that is highly conserved and specific to its subtype and does not have homology with other subtypes of influenza A virus and other related viruses.

Schemes and calculated fragments of PCR are presented in the drawing.

The calculated oligonucleotides were synthesized on a 349 DNA / RNA synthesizer (Applied Biosystems (USA)) using the standard method.

The proposed method includes two main reactions. The first one is the first RT-PCR with primers specific for the region of the influenza A virus hemagglutinin gene. The matrix for this reaction is the single-stranded RNA of the influenza virus, and the primers are primers F-H5, consisting of 20 units: ACAAGGTCCGACTACAGCTT, and R-H5, consisting of of 22 links: ATTGATTCCAATTTTACTCCAC. The second reaction is the second RT-PCR with primers specific for the region of the neuraminidase gene. The matrix for this reaction is single-stranded RNA of the influenza virus, and the primers are primers F-N1, consisting of 22 units: CAGAACATTAATGAGTTGTCCT, and R-N1, consisting of 22 units: TCTCAGTATGTTGTTCCTCCAA. The use of a polymerase chain reaction, combined with a reverse transcription reaction, reduces the reaction time.

The reaction mixture was analyzed by electrophoresis on a 2% agarose gel. The presence of 184 nucleotide pairs of DNA fragments in the analyzed sample indicates the presence of influenza A virus subtype H5 in the RNA starting material, and 213 nucleotide pairs of DNA fragments of influenza A virus of subtype N1.

The proposed method for the diagnosis of influenza A virus by PCR is highly sensitive and highly specific to the influenza A virus subtype H5N1 and can reliably determine the presence of RNA of the virus of this subtype in biological samples without first accumulating the test virus, which is especially important when working with highly pathogenic strains.

The invention is illustrated by the following examples.

Example 1. The choice of the sequence of primers.

Given the large variability of the genome of the influenza A virus type, when detecting it, it is necessary to use primers that correspond to the most conserved parts of the viral genome. The basis for the search for primer annealing sites was a comparison of the nucleotide sequences of the hemagglutinin and neuraminidase gene of all influenza viruses presented in the international database. In total, more than 10,000 nucleotide sequences of the genomes of influenza viruses were used.

When choosing primer sites, the following requirements were taken into account: the specificity of primers for all sequences of viruses of the H5N1 subtype, the absence of primer annealing sites on the sequences of viruses of other subtypes. This work was performed using the programs Lasergene (version 6.0), BioEdit (version 7.00) and Amplify (version 1.06 Univers. Of Wisconsin, Genetics, Madison). As additional criteria for determining the suitability of the primers, the following were taken into account: the absence of primer duplexes and their annealing on each other, the absence of false annealing sites on the genome matrices of various strains of influenza A virus and related viruses. All selected primers satisfy these criteria.

Example 2. The selection of RNA from samples.

To isolate RNA from samples is to obtain it in its pure form, i.e. separate from proteins and other cellular molecules that may interfere with molecular analysis. To avoid cross-contamination, clean 1.5 ml tubes were labeled and 600 µl of a solution containing guanidine thiocyanate were added to them before work. Samples of the test material (in a volume of 200 μl) were used for analysis. The method of separation from the inorganic sorbent "SiO ₂ " was used. RNA was suirable in 30 μl of deionized water, free of RNAase at 56 ° C in closed tubes.

Example 3. Conducting RT-PCR with primers specific for regions of the H5 and N1 genes.

In a separate tube, a total reaction mixture is prepared for N samples, which include positive and negative controls. Reagents are added in the sequence shown in the table. Enzymes are added last.

Reaction RT-PCR-1 for the H5 subtype of influenza A virus. Reagent Qty per sample Number of N samples Buffer for PCR-1 14.625 14.625 × (N + 1) Primers for PCR-N5 4.5 4.5 × (N + 1) Taq polymerase enzyme 0.25 0.25 × (N + 1) Enzyme MMLV revertase + RNase inhibitor 0.625 0.625 × (N + 1) Reaction RT-PCR-1 for subtype N1 of influenza A. Reagent Qty per sample Number of N samples Buffer for PCR-1 14.625 14.625 × (N + 1) Primers for PCR-N1 4.5 4.5 × (N + 1) Taq polymerase enzyme 0.25 0.25 × (N + 1) Enzyme MMLV revertase + RNase inhibitor 0.625 0.625 × (N + 1)

The mixture is stirred and 20 μl are instilled into tubes, 2-3 drops of oil (approximately 40 μl) are added to each tube.

Then, 5 μl of RNA of the corresponding analyzed or control samples (K ⁺ and K ^- ) are introduced into them with a separate tip with an oil filter. Samples are introduced into the amplifier with the following temperature conditions:

50 ° C  - 40 min 95 ° C - 3 min 1 cycle 95 ° C  - 20 sec 57 ° C - 20 sec 40 cycles 72 ° C  - 20 sec 72 ° C - 5 min 1 cycle

Example 4. Detection of amplification products.

Detection of amplification products is carried out by electrophoresis in a 2% agarose gel containing ethidium bromide.

An aliquot is examined (7 μl of the aqueous phase of the amplification mixture).

Electrophoresis is carried out at a voltage of 8 volts / cm length of the gel, for 60 minutes During this time, the reaction product manages to pass at least half of the gel. The recommended gel size is 5-10 cm. The direction of movement of the samples in the gel from "-" to

"+".

The results of electrophoresis are viewed in ultraviolet light with a wavelength of 254 nm on a Transilluminator instrument. The reaction results are detected in the form of luminous reddish stripes.

Samples are considered positive, the bands in which are located in the gel at the level of the positive control band.

The test samples are considered negative if they do not reveal any bands or the bands do not correspond to the size of the fragment in the control sample (i.e., are located at a different distance from the start).

In the negative control, no bands should be detected. The presence of colored fragments in the negative control at the level of the positive control bands indicates cross-contamination in the analysis process. Results are void. The analysis must be repeated.

The proposed one-stage method for the detection of influenza A virus is highly sensitive to influenza A virus subtype H5N1 and can be used for rapid identification. It is carried out both in two PCR and in multiplex PCR. Multiplex PCR reduces the percentage of financial costs and the number of technological errors.

LITERATURE

1. Breslauer et al // Proc. Natl. Acad. Sci USA. - 1986. - V.83. - P.3746.

2. Brown I.E., Harris P.A., Alexander D.J. Serological studies of influenza virus in pigs in Great Britain 1991-2 // Epidemiol. Infect. - 1995 .-- Vol. 114. - P.511-520.

3. Brown LH., Done S.H., Spencer Y.I., Cooley W.A., Harris P.A., Alexander D.J. Pathogenicity of swine influenza H1N1 virus antigenically distinguishable from classical and European strains. // Vet. Rec. - 1993. - Vol. 132. - P.598-602.

4. Brown, I.H., Harris P.A., McCauley J.W., Alexander D.J. Multiple genetic reassortment of avian and human influenza A viruses in European pigs, resulting in emergence of an H1N2 virus of novel genotype. // J. Gen. Virol. - 1998. - Vol. 79. - P.2947-2955.

5. Capua I, Alexander D.J. Avian influenza and human health. // Acta Trop. - 2002 - Vol. 83. No. 1. - P.1-6. Review

6. Castrucci M.R., Donatelli I., Sidoli L, Barigazzi G., Kawaoka Y., Webster R.G. Genetic reassortment between avian and human influenza viruses in Italian pigs. // Virology. - 1993 .-- Vol.l93. - P.503-506.

7. Cauthen A.N., Swayne D.E., Schultz-Cherry S., Perdue M.L., Suarez D.L. Continued circulation in China of highly pathogenic avian influenza viruses encoding the hemagglutinin gene associated with the 1997 H5N1 outbreak in poultry and humans. // J. Virol. - 2000. - Vol. 74. - N 14. - P.6592-6599.

8. Li, Wang J., Smith G.J.D. Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia // Nature. - 2004 .-- Vol. 430. - P.209-213.

9. Payungporn S, Phakdeewirot P, Chutinimitkul S, Theamboonlers A, Keawcharoen J, Oraveerakul K, Amonsin A, Poovorawan Y. Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection // Viral Immunol. 2004; 17 (4): 588-93.

10. Recommended laboratory tests to identify avian influenza A virus in specimens from humans • WHO Geneva • June 2005.

Claims (1)

A method for detecting influenza A virus of subtype H5N1, comprising conducting a reverse transcription reaction with virus RNA, combined with an amplification reaction for two genes, analyzing the reaction mixture by agarose gel electrophoresis and detecting RNA of influenza A virus strains according to analysis results, characterized in that As primers for carrying out a combined reverse transcription and amplification reaction in both PCR and multiplex PCR, oligonucleotides of 20 and 22 units of F-H5 ACAAGGTCCGACTACAGCTT and R-H5 ATTGATTCCAATTTTACTCCAC, complete at least 95% hereditary hemagglutinin gene, and oligonucleotides from 22 F-N1 units CAGAACATTAATGAGTTGTCCT and R-N1 TCTCAGTATGTTGTTCCTCCAA, complementary to at least 95% neuraminidase gene, identification of DNA samples in 18 samples the use of primers specific for the hemagglutinin gene and 213 nucleotide pairs when using primers specific for the neuraminidase gene indicates the presence of virus RNA in the starting material.

Images