RU2441917C2 - Method for identification of 5т-htp genome of enteroviruses of evi and evii gene group implementing polymerase chain reaction - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the area of molecular biology and medicine, in particular - to the method of enterovirus infection diagnostics. The method is based on polymerase chain reaction of cDNA of human enteroviruses genome. The original differentiating gene group-specific primers corresponding to the conservative regions of 5'-HTP genome of enteroviruses of EVI and EVII gene group. The oligonucleotide sequences provide for high specificity of the method that may be implemented for 3-5 hours. The method may be implemented for gene diagnostics of the enterovirus infection to detect enteroviruses in the clinical material; for enterovirus infection epidemiological surveillance in the environmental facilities; in scientific research for specific amplification of fragments of enteroviruses genome of various gene groups, including mixed microbes.

EFFECT: enhanced and fast enterovirus detection.

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Description

The invention relates to molecular biology, medicine and industrial microbiology, in particular to the differential indication of human enteroviruses based on the specific amplification of a fragment of the 5'-untranslated region of the enterovirus genome.

For a long time, the differentiation of enteroviruses was carried out using classical virological methods based on the reaction of neutralizing the virus with specific hyperimmune sera in tissue culture. The cell culture method is time-consuming, quite laborious, economically expensive and does not have sensitivity against certain types of enteroviruses. As new knowledge about the structure of the genome and phylogenetic relationships of enteroviruses of different types was gained, it became possible to use highly sensitive and fast-performing molecular genetic methods for the differential analysis of these viruses.

The following methods are known for typing enteroviruses by analyzing cDNA fragments obtained by RT-PCR directly from the studied samples (samples of clinical material and from environmental objects).

1. Genotyping of enteroviruses by sequencing a fragment of the VP1 region of the genome obtained using oligonucleotide primers universal for human enteroviruses (Nix WA, Sensitive, seminested PCR amplification of VP1 seqences for direct identification of all enterovirus serotypes from original clinical spesimens / Nix WA, MS Oberste and MA Pallansch / J. Clin. Microbiol - 2006. - Vol. 44, No. 8. - P.2698-2704).

2. Differentiation of human enteroviruses into two gene groups according to the 5'-NTR genome by PCR (Golitsyna LN / Assessment of the developed “nested” variant of PCR for the detection of enteroviruses in patients. Golitsyna LN, Novikova NA, Dombrovskaya L .K., Knyagina O.N., Novikov D.V., Arkova T.A. // Vopr. Virusol. - 2002. - No. 5. - P.41-43).

In the first of the methods, PCR allows only the indication of enteroviruses; genotyping of the detected viruses is carried out at the second stage by sequencing. In addition, this method does not allow differentiation of enteroviruses if enteroviruses of several genotypes are simultaneously present in the test sample.

In the second method, the analysis of the genome of enteroviruses is carried out in 2 rounds of PCR, which lengthens the study and increases the risk of contamination of the sample with the product of the 1st round of PCR. In addition, the primers presented in this method have less specificity, as they were developed without taking into account modern knowledge about new strains and types of human enteroviruses.

The purpose of the invention is to increase the efficiency of the method for identifying the 5'-HTP genome of enteroviruses of the EVI and EVII genogroups using the polymerase chain reaction.

The essence of the invention lies in the fact that the cDNA fragment of human enteroviruses is amplified in a single-round PCR using our oligonucleotide primers of the following composition: FEvI - agatcaggyc gatgagycac yg, in which y means c or t; FEvII - ttggaatctt ygaygcgttg c, in which y means c or t; REv - cacggwcacc caragtactc g, in which w means a or t; r means a or g.

Using a FEvI - REv primer pair, an EVI gene group cDNA fragment of 251 bp is obtained, and using an FEvII - REv primer pair, a 295 bp gene enterovirus cDNA fragment is obtained. The results obtained are analyzed by agarose gel electrophoresis.

Example 1. The choice of the sequence of primers.

The selection of primer sequences is carried out by comparative analysis of the complete nucleotide sequences of the 5'-HTR genome of viruses belonging to the genus Enterovirus of the Picornaviridae family: human enteroviruses, animal enteroviruses, rhinoviruses and picornaviruses of other genera: parechoviruses, cobuviruses, aphthoviruses, hepatoviruses A., cardiovirus viruses A. the base of the nucleotide sequences of GenBank / EMBL. For analysis, the nucleotide sequences of the genome of 152 strains of human enteroviruses, 5 strains of animal enteroviruses, 21 strains of other picornaviruses listed above, which are important in human infectious pathology, are used. Sequences are aligned using the ClustalW program, the regions of the nucleotide sequence of a given region of the genome are found that are conserved within the group of human enteroviruses or each of the EVI and EVII genogroups, but are variable with respect to other picornaviruses. When choosing a nucleotide sequence to use as group-specific differentiating primers and a universal primer, the following criteria are used: the sequence should be conserved in different strains of human enteroviruses belonging to the same 5'-NTR geno group, but different from the corresponding nucleotide sequences of the 5'-NTR enterovirus genome humans belonging to another gene group of enteroviruses and other picornaviruses. Select sections of the direct nucleotide sequence (F) of size 22, 21 and 21 N.O., having the following sequences:

in enteroviruses of the EVI genogroup - agatcaggyc gatgagycac yg;

in enteroviruses of the gene group EVII - ttggaatctt ygaygcgttg c;

in enteroviruses of both genogroups, 5'-cacggwcacc caragtactc g.

At the next stage, the selected sequences are analyzed from the point of view of their homogeneity in different strains of enteroviruses, in the presence of nucleotide substitutions (variability of a nucleotide in different strains), degenerate bases are introduced. The reverse primer sequence (R) is set by inverting the forward (F) sequence.

Next, a comparative analysis is carried out with the sequences of the 5'-NTR genome of 5 strains of animal enteroviruses, 5 strains of human rhinoviruses, 6 strains of parachoviruses, 2 strains of hepatitis A virus, 1 strain of cobuvirus Aichi, 5 strains of aftoviruses, 2 strains of cardioviruses. It is established that the sequence homology in the region of FEvI, FEvII, REv primers with the corresponding sequences of the above-mentioned representatives of the picornavirus family is 25-40%, which indicates the theoretical specificity of the selected oligonucleotides in relation to the human enterovirus genome.

Then, a comparative analysis of the sequence of FEvI primer with 52 sequences of 5'-HTP genome of enteroviruses of the EVI geno group is carried out, and sequences of primer FEvII with 100 sequences of 5'-HTP genome of enteroviruses of the EVI geno group. It is established that the sequence homology in the region of FEvI and FEvII primers with the corresponding sequences of representatives within the gene group is 90-100%, and among representatives of opposite enterovirus geno groups - 43-59%, which indicates the theoretical specificity of the selected oligonucleotides in relation to the geno group of human enteroviruses.

Example 2. Identification of the 5'-NTR genome of enteroviruses of the EVI geno group and the EVII geno group and assessment of the specificity of the designed primers.

Isolation of enterovirus RNA is carried out using the nucleosorption method (Boom R. Rapid and simple method for purification of nucleic acids / R. Boom, CJSol, MMSalimans et al. // J. Clin. Microbiol. - 1990. - Vol. 28, No. 3. - P.495-500). The prepared sample is used to set up the reverse transcription reaction, polymerase chain reaction (PCR).

Amplification of cDNA is carried out in a volume of 25 μl in a thermocycler with active regulation of “Tertsik-MS2” (“DNA-technology”, Moscow).

For PCR, a reaction mixture of the following composition is prepared: 10 μl of the PCR mixture, 2 μl of a 4-component mixture of dNTP, 2 μl of DEPC water, 1 μl of forward and reverse primers. 15 μl of the mixture is added to a test tube containing 10 μl of DNA. Amplification of the DNA fragment is carried out in the following mode [94 ° C - 5 minutes; (94 ° С - 10 sec, 55 ° С - 10 sec, 72 ° С - 10 sec) X 42 cycles; 72 ° C for 5 minutes]. The result of amplification is a specific fragment of 251 bp in size. for a pair of FEvI-REv primers and a fragment of 295 bp for a pair of primers FEvII-REv. The amplification product is visualized by electrophoresis on a 2.0% agarose gel in the presence of ethidium bromide using 0.089 M Tris-borate buffer solution, pH 8.0, at 110 V per gel for 20 minutes. To determine the size of the fragment, a DNA dimension marker of 100-1000 bp, manufactured by Sileks, is used.

To assess the specificity of the proposed primers, reference strains of enteroviruses of the EVI and EVII genogroups are used: vaccine poliovirus type 1 (EVII), ECHO30 virus (EVI), hepatitis A virus, type 1 human parachovirus. Perform RT-PCR with samples of the indicated reference strains. When analyzing amplification products by agarose gel electrophoresis, the presence of amplicons of 251 bp in size is determined. and 295 bp It is established that FEvI, FEvII, REv primers amplify fragments of the genome of the desired size only when working with human enteroviruses of the corresponding gene group EVI and EVII, and do not give a positive or nonspecific reaction in the analysis of human enterovirus RNAs of the opposite genogroup and other picornaviruses (Figure A, B).

The results obtained indicate the high specificity of the designed primers in relation to the geno group of human enteroviruses, which allows them to be used for the differential indication of 5'-HTP enteroviruses of the EVI gene group and the EVII gene group.

The invention can be used in scientific research to gain new knowledge about the role of enteroviruses in human infectious diseases, to study the nature of the distribution and patterns of circulation of these viruses among the population and is of practical interest for use in the monitoring system for EVI in institutions of Rospotrebnadzor.

The method was developed and tested on the basis of the laboratory of molecular epidemiology of viral infections of the Nizhny Novgorod NIIEM them. Academician I.N. Blokhina.

The list of oligonucleotide sequences presented in the application No. 201114528/10 (020449) from 04/12/2010

A method for identifying the 5'-NTR of the genome of enteroviruses of the EVI and EVII genogroups using the polymerase chain reaction.

FEvI - agatcaggyc gatgagycac yg,

in which y is c or t;

FEvII - ttggaatctt ygaygcgttg s,

in which y is c or t;

REv - cacggwcacc caragtactc g,

in which w means a or t; r is a or g.

Claims (1)

A method for identifying the 5'-NTR of the genome of enteroviruses of the EVI and EVII genogroups, including the isolation of genomic RNA, the reverse transcription reaction, polymerase chain reaction and analysis of the obtained DNA fragments in an agarose gel, characterized in that oligonucleotides having the following sequences are used in the polymerase chain reaction :
FEvI - agatcaggyc gatgagycac yg,
in which y is c or t;
FEvII - ttggaatctt ygaygcgttg s,
in which y is c or t;
REv - cacggwcacc caragtactc g,
in which w means a or t; r means a or g,
the presence of enteroviruses of the EVI gene group is judged by a specific DNA fragment of 251 bp when using a FEvI-REv pair, the presence of enteroviruses of the EVII gene group is judged by a specific fragment of 295 bp when using a pair of FEvII-REv.

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