RU2541773C2 - Set of oligodesoxyribonucleotide primers and fluorescence marked probes for identifying rna of human respiratory syncytial virus - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to genetic engineering, can be used to identify a genetic material (RNA) of human respiratory syncytial virus in samples, and can be used in microbiology and medicine. A set of oligodeoxyribonucleotide primers for detecting RNA of human respiratory syncytial virus and comprises two pairs of oligonucleotide polymers possessing an activity of upstream and downstream primers in polymerase chain reaction, as well as two fluorescence marked DNA probes having the follow structure: Outer upstream primer (nested forward primer) 5'→3' F: 5'-AGRCARCARAGTTAYTCYATYATGTC-3' (26 s.); outer downstream primer (nested reverse primer) 5'→3' R: 5'-AATTTATTATWGGTTYMCCYTTTACATA-3' (25 s.); upstream primer (forward primer) 5'→3' F: 5'-GACACHATGAAYAGYTTRACATTACC-3' (26 s.); downstream primer (reverse primer) 5'→3' R: 5'-AAATGTYTTTATDATYCCRCGATTT-3' (25 s.); fluorescence marked DNA probes (probe) 5'→3' Z: 5'-FAM-TCTCTAGGAGCCATTGTGTCATGCTATGG-BHQ1-3' (29 s.) Z: 5'-FAM-TCTCTWGGAGCYATAGTGTCATGYTATGG-BHQ1-3' (29 s.).

EFFECT: extended spectrum of import-substituting oligodeoxyribonucleotide primers and fluorescence marked probes enabling identifying RNA of human respiratory syncytial virus by real-time PCR.

1 cl, 5 tbl, 1 dwg

Description

The invention relates to sets of primers and a fluorescently labeled probe for detecting the genetic material (RNA) of the human respiratory syncytial virus in clinical samples, sectional samples, environmental samples, culture virus-containing fluids and other biological products for the purpose of diagnosis, treatment correction, epidemiological investigation, as well as to solve research problems on monitoring and studying the properties of the respiratory syncytial virus, creating diagnostic, prophylactic iCal and therapeutic drugs and can be used in medicine, biotechnology and epidemiology.

Using the developed diagnostic primers and fluorescently-labeled probes, it is possible to identify the genetic material (RNA) of the human respiratory syncytial virus.

The reverse transcription polymerase chain reaction (RT-PCR) is the most common method used in laboratory practice for the differential diagnosis of infectious diseases. The method is based on two reactions - reverse transcription and polymerase chain reaction. In the reverse transcription reaction, a complementary DNA (cDNA) is built on the viral RNA matrix. In the subsequent PCR reverse transcription reaction, multiple selective doubling of the target cDNA region occurs (amplification). The amplified cDNA region is marker, since it is strictly limited to the sequences of DNA seeds (primers), without which PCR cannot occur. In order to detect the accumulation of amplification products in real time, in addition to a pair of primers, a fluorescently-labeled DNA probe is also required. The complexity of the choice of primers and probe is due to the requirement of their strict species specificity. If the selected oligonucleotides do not have species specificity for the target object or do not have sufficient homology to the target nucleotide sequence, false negative results of the study are possible. If the selected primers and probes demonstrate an affinity for non-target DNA sequences, then false-positive test results are possible. The correct choice of a combination of a pair of primers and a fluorescently labeled DNA probe allows for strictly specific amplification of the target cDNA fragment and real-time detection of PCR products accumulation.

Foreign analogues of oligonucleotides are known that allow the detection of the respiratory syncytial human virus. In Chinese Patent No. CN 1544656, publ. 2004, presented oligonucleotide primers and fluorogenic probes that allow for quantitative PCR in real time. Canadian Patent No. CA 2236022, publ. 1997, and US Application No. 1182004253582, publ. 2004, oligonucleotides are presented that allow the detection of genetic material (RNA) of the human respiratory syncytial virus by real-time PCR. In US application No. 2010221711, publ. 2010, a set of reagents is provided that allows the detection of respiratory syncytial virus in clinical samples from humans and animals and the assessment of viral load, and in US application No. 200440253582, publ. 2004, a set of reagents was published that made it possible to detect genomic RNA of the human respiratory syncytial virus types A and B in clinical samples.

The closest analogue (prototype) is a set of primers for the detection of cDNA of the respiratory syncytial virus of the person by PCR method "AmpliSens®ORVI-screen-FL" (http://www.pacxodka39.ru/product_3056.html Central Research Institute of Epidemiology, Moscow). The reagent kit is designed to identify the causative agents of acute respiratory viral infections of humans (ARVI): RNA of the respiratory syncytial virus, RNA of metapneumovirus, parainfluenza viruses of types 1-4, coronaviruses, rhinoviruses, adenoviruses of groups B, C and E and bocavirus in the clinical material by polymerase chain reaction (PCR) with hybridization-fluorescence detection.

However, in the known analogues and prototype, the primers have an insufficient level of homology to all currently existing respiratory syncytial human viruses in nature, as well as to the human metapneumovirus genomes available in the database, which reduces the reliability and reliability of the diagnosis of this virus using the claimed primers.

The technical result of the claimed invention is the expansion of the range of import-substituting oligodeoxyribonucleotide primers and fluorescently-labeled probes, allowing the identification of RNA of the respiratory syncytial virus of a person by real-time PCR.

The indicated technical result is achieved by the development of a set of oligodeoxyribonucleotide primers and fluorescently-labeled probes for identification of human respiratory syncytial virus RNA containing two pairs of oligonucleotide polymers having direct and reverse primer activity in the polymerase chain reaction, as well as two fluorescence-DNA fluorescence.

In the framework of the claimed technical solution, the developed primers and DNA probes have a high degree of homology to all known nucleotide sequences of the genomes of isolates of the respiratory syncytial human virus, available in the GenBank international database of biotechnological information.

The inventive set of reagents has several significant advantages. Due to the introduction of “degenerate” nucleotides into the sequence, patented primers and fluorogenic probes have a significant level of homology to all genomes of the respiratory syncytial virus in the database. Since the kit is a set of four primers and two fluorogenic DNA probes, two-round PCR can be performed, which increases the sensitivity of the method by orders of magnitude, and also allows to obtain a long-lasting PCR product, the nucleotide sequence of which can be determined on an automatic genomic analyzer. This, in turn, allows you to establish many molecular biological patterns in the field of epidemiology, contagiosity and pathogenicity of the virus. In addition, the primers and probes we developed were used in PCR mixtures consisting of components of domestic production, which makes the development easily and widely available for use in laboratories of the Russian Federation, this ensures a quick and reliable implementation of the development into practice. Used as a positive control sample, the plasmid construct carrying the human respiratory syncytial virus genome fragment can be used in quantitative PCR. This makes it possible to assess the viral load in the test sample.

Testing of the oligonucleotides was carried out using nasopharyngeal swabs from patients with symptoms of acute respiratory viral infections (the research procedure was approved by the ethical committee of the Federal State Budgetary Scientific Center of the Scientific and Research Center “Vector” (IRB 00001360)). It was experimentally shown that the selected primers and DNA probe provide reliable synthesis of target DNA fragments. The specificity of amplification was further confirmed by sequencing.

Characterization of a set of primers and plot amplified genomic RNA. The primers proposed for patenting flank a portion of the fusion protein (F gene) gene encoding the fusion glycoprotein. In the first round of PCR, a fragment of the genome from 6509 to 7091 nucleotides (582 bp) is amplified, in the second - from 6767 to 6970 nucleotides (203 bp). The use of fluorescence-labeled DNA probes complete with diagnostic primers allows real-time detection of the accumulation of amplification products.

It is important to note that the optimization of PCR conditions was carried out using sets of commercially available reagents, devices and enzymes intended for mass use in laboratory practice, which allows fast and reliable application of this invention in medical and research laboratories.

List of graphic materials

In figure 1. shows the results of the analysis of clinical samples by real-time PCR using patented oligonucleotides.

Method for constructing a set of diagnostic primers and fluorescently-labeled probes

Based on a theoretical study of the genome structure of the human respiratory syncytial virus to the conserved region of the fusion protein (F gene), primers and fluorescently-labeled DNA probes for polymerase chain reaction were calculated and synthesized (table 1). In the course of the work, 1562 nucleotide sequences of the human respiratory syncytial virus genome contained in the database were analyzed.

Table 1 Primers and probes for the detection of human respiratory syncytial virus Virus GenBank Follower Localization The nucleotide sequence of 5 '→ 3' Tm, ° C PCR product, bp RSV NC_001781.1 6509-6534 AGRCARCARAGTTAYTCYATYATGTC 51.1 582 7091-7067 AATTTATTATWGGTTYMCCYTTTACATA 51.2 6767-6792 GACACHATGAAYAGYTTRACATTACC 51.8 203 6970-6946 AAATGTYTTTATDATYCCRCGATTT 50.8 6890-6914 FAM-TCTCTAGGAGCCATTGTGTCATGCTATGG-BHQ1 63.3 FAM-TCTCTWGGAGCYATAGTGTCATGYTATGG-BHQ1 59.2

The method of obtaining positive control samples

Positive control samples were obtained by TOPO-T / A cloning. After that, competent E. coli cells of the TOP 10 line (Invitrogen, USA) were transformed with the obtained plasmids pCR2.1 (Invitrogen, USA) carrying a virus-specific insert of the synthesized DNA fragments. In the course of the work, a recombinant plasmid was constructed: pRSV. The presence of a viral genome-specific DNA insert was confirmed by sequencing.

Analysis of the effectiveness of the transformation was carried out by real-time PCR in accordance with the protocol described below, where a 1 × TE buffer containing recombinant pRSV plasmid was added to the reaction mixture with the insertion of a virus-specific synthesized DNA duplex. As a negative control, 1 × TE buffer was added to the reaction mixture.

Extraction of viral RNA. Viral RNA was isolated from 100 μl of a nasopharyngeal wash using the RIBO-prep kit (Central Research Institute for Emergencies, Moscow) according to the manufacturer's instructions.

Reverse transcription reaction. The synthesis of complementary DNA (cDNA) was carried out on a total RNA matrix using the Revert-L reagent kit (Central Research Institute for Electronic Research, Moscow) according to the manufacturer's instructions.

table 2 Mixture for the first round of PCR (per tube) Component Volume μl PCR buffer × 10 3 dNTP (5 mm) one MgCl ₂ (50 mm) 1.5 Hot Start Taq DNA Polymerase 0.3 Direct Primer - F (10 nM) 2 Reverse Primer - R (10 nM) 2 Sample 3 diH ₂ O 17.2

Table 3 Mixture for the second round of PCR (per tube) Component Volume μl PCR buffer × 10 3 dNTP (5 mm) one MgCl ₂ (50 mm) 1.5 Hot Start Taq DNA Polymerase 0.3 Direct Primer - F (10 nM) 2 Reverse Primer - R (10 nM) 2 A mixture of DNA probes - Z (each 15 nm) 0.5 First round PCR product one diH ₂ O 18.7

Carrying out a polymerase chain reaction. The amplification conditions were optimized by the concentration of magnesium ions, the concentration of primers and probes in the reaction mixture, and the temperature of annealing of primers.

The amplification reaction was carried out in 30 μl of a PCR mixture (tables 2 and 3) containing 1 × Taq amplification buffer, 2.5 mM MgCl ₂ , 0.17 mM each of nucleoside triphosphates, 1.5 active units of Smart Taq DNA polymerase (all components of MEDIGEN Lab LLC "), 20 pM forward and reverse primers, 8 pM fluorescent DNA probe.

The first round of PCR was performed according to the amplification program (table 4) on a Mastercycler gradient instrument (BioRad, USA). Monitoring the effectiveness of the passage of the first round of PCR was carried out by separating the amplification products in a 2% agarose gel.

Table 4 Amplification program for PCR with electrophoretic detection No. Operation T, ° C T, min: s one Hold / Activate Smart Taq DNA Polymerase 95 05:00 2 Cycling / Cycle (30 cycles) 95 00:10 fifty 00:30 72 00:20 four Hold / Final Elongation 72 7 a.m.

Table 5 Amplification program for PCR-RV No. Operation T, ° C T, min: s one Hold / Activate Smart Taq DNA Polymerase 95 05:00 2 Cycling 1 / Cycling 1 (10 cycles) 95 00:10 fifty 00:30 72 00:20 3 Cycling 2 / Cycling 2 (30 cycles) 95 00:10 fifty 00:30 detection 72 00:20

Real-time PCR was performed according to the amplification program shown in Table 5. Fluorescence intensity was detected via the Green 470/510 nm channel (FAM dye). Fluorescence was measured using Rotor Gene 6000 instruments (Corbet Research, Australia) and iQ5 (BioRad, USA). The results were evaluated by the presence of fluorescence up to 30 cycles (Fig. 1). In FIG. 1 presents the results of the analysis of clinical samples by real-time PCR using patentable oligonucleotides, the graphs of which show that: in samples No. 8, 14, RNA of the human respiratory syncytial virus was detected; K + is a positive control sample (pRSV).

Testing of patentable oligonucleotides

The oligonucleotides proposed for patenting were tested by the authors in an epidemiological study to study the species diversity of agents that caused SARS in residents of Novosibirsk and the Novosibirsk Region in October-April 2011-2012. The authors studied 164 samples of nasopharyngeal swabs from patients with symptoms of acute respiratory viral infection. The etiological agent of the disease was identified by the authors in 43% of cases (69 samples). Respiratory syncytial virus in the studied samples was detected in 4% of cases (7 samples) (Fig. 1). In the structure of combined infections, the respiratory syncytial virus was detected by the authors in 17.4% of cases (4 samples out of 11). The specificity of PCR studies was further confirmed by sequencing.

Thus, the claimed set of oligonucleotide primers and fluorescently-labeled probes provides, along with well-known analogues, reliable and reliable detection of the human respiratory syncytial virus in clinical samples and other biological materials.

Claims (1)

A set of oligodeoxyribonucleotide primers and fluorescently-labeled probes for identification of RNA of the respiratory syncytial human virus, containing two pairs of oligonucleotide polymers having direct and reverse primer activity in the polymerase chain reaction, as well as two fluorescently labeled DNA probes:
- external forward primer (nested forward primer) 5 '→ 3'
F: 5'-AGRCARCARAGTTAYTCYATYATGTC-3 ' (26 n.)
- external reverse primer (nested reverse primer) 5 '→ 3'
R: 5'-AATTTATTATWGGTTYMCCYTTTACATA-3 ' (25 n.)
- forward primer 5 '→ 3'
F: 5'-GACACHATGAAYAGYTTRACATTACC-3 ' (26 n.)
- reverse primer 5 '→ 3'
R: 5'-AAATGTYTTTATDATYCCRCGATTT-3 ' (25 n.)
- fluorescently-labeled DNA probes (probe) 5 '→ 3'
Z: 5'-FAM-TCTCTAGGAGCCATTGTGTCATGCTATGG-BHQ1-3 ' (29 n.) Z: 5'-FAM-TCTCTWGGAGCYATAGTGTCATGYTATGG-BHQ1-3 ' (29 n.)

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