RU2552795C1 - Set of oligonucleotide primers and fluorescence-tagged probe for identification of rna of rift valley fever virus by pcr real time process - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to fabrication of oligonucleotide primers and fluorescence-tagged probe for identification of inverse transcriptase polymerase chain reaction in real time.

EFFECT: higher efficiency of identification.

2 dwg, 2 tbl, 2 ex

Description

The invention relates to the field of biotechnology and can be used in veterinary medicine and to detect the genetic material (RNA) of the Rift Valley fever virus (VLDR) in clinical or biological samples, as well as in environmental objects.

Rift Valley Fever (LDR) is an acute zoonotic disease common in many countries in Africa and the Middle East. The causative agent belonging to the genus Phlebovirus of the Bunyaviridae family is characterized by high virulence against many species of wild and domestic ungulates and humans.

The VLDR genome is represented by single-stranded negative RNA, divided into three segments (S, M and L). The bipolar S segment contains the genes for nucleoprotein and non-structural proteins, the M segment encodes envelope glycoproteins, and the L segment encodes an RNA-dependent RNA polymerase.

The transmission of the virus is through the bites of mosquitoes Culex spp. and Aëdes spp., its natural reservoir is unknown. Human infection can also occur by contact with tissues and blood of diseased animals or by inhalation of virus-containing aerosols.

Rift Valley Fever is currently included in the Quarantine Infections Group controlled by the 2005 International Health Regulations. Epizootics of LDR cause enormous economic damage to animal husbandry, causing 80-100% death of young animals and intrauterine death of the fetus at any stage of pregnancy. The clinical picture is characterized by severe fever, necrotic hepatitis, gastroenteritis, heavy bleeding from the mucous membranes.

In humans, the manifestations of the disease range from a mild flu-like form to a severe one, accompanied by hemorrhagic syndrome, liver necrosis and meningoencephalitis. In the complicated course of LDR, mortality rates can reach 50%, residual neurological phenomena and a persistent decrease in visual acuity, leading to disability, are noted in surviving patients. Most often, the development of severe forms of LDR is observed during outbreaks preceded by epizootics among domestic animals.

The timely conduct of diagnostic measures, the assessment of the epidemic and epizootic situation according to LDR, the development of a comprehensive program for the prevention of infection require quick, highly sensitive and reliable identification of the pathogen.

One of the methods for the operative detection of VLDR RNA is the reverse transcription polymerase chain reaction (RT-PCR).

Known synthetic oligonucleotide primers used to identify the genetic material of VLDR, based on the amplification of conserved sequences of S- or M-segments of RNA [1-4].

However, these primer sets do not involve the use of fluorescently-labeled probes to detect results, which does not allow real-time accumulation of specific PCR products. In addition, in some works, the specificity of detection was achieved due to the so-called “Nesting”, or two-round, NDP using an additional pair of internal primers [2, 5]. Known synthetic oligonucleotide primers, a method for detecting and differentiating strains of rift valley fever virus based on polymerase chain reaction and restriction analysis (RF patent No. 2457255, IPC C12Q 1/68, published on July 27, 2012). The hydrolysis of PCR products is carried out by restriction endonucleases, and the detection of the results of the analysis by agarose gel electrophoresis.

The disadvantages of this invention include the following:

- an increase in the total time of analysis of samples up to 5-6 hours (instead of 2.5-3 hours), since the absence of a fluorescently-labeled probe does not allow to register the accumulation of specific PCR products in real time;

- the impossibility of automatic recording and processing of experimental data;

- additional manipulations with amplicons (enzymatic hydrolysis and gel electrophoresis) greatly increase the likelihood of false positive results associated with contamination of rooms and laboratory equipment with PCR products.

Known "Primers and probe for detecting fragment S of rift valley fever virus" (Chinese patent No. CN 102433392 (A), IPC C12Q 1/68, publ. 2012-05-02). The invention provides oligonucleotide primers and a fluorescently-labeled probe designed to identify Rift Valley fever virus RNA by real-time reverse transcriptase polymerase chain reaction. The hybridization region of the primers and probe (plot of the NS gene of the S segment, position No. 37-128).

However, since a more variable VLDR RNA fragment was selected for analysis in the invention, the primers and probe in some cases may not be specific enough for reliable identification of the virus.

The well-known "PRIMERS AND ITS APPLICATION IN MULTIPLEX PCR TO IDENTIFY RINDERPEST, PESTE-DES-PETITS-RUMINANTS VIRUS, BLUETONGUE VIRUS AND RIFT VALLEY FEVER" (Patent of the Republic of Korea No. KR 20110017706 (A), IPC Cp1Q 1/68. -02-22). The oligonucleotide primers presented in the patent are specific for Rift Valley Fever virus RNA. They are used as part of multiplex PCR with electrophoretic detection of analysis results.

However, competition between different oligonucleotide pairs with respect to the components of the reaction mixture (Taq polymerase, deoxynucleoside triphosphates, magnesium ions) can lead to a decrease in the analytical sensitivity of the system. In addition, the increase in the total time of analysis of samples is up to 5-6 hours (instead of 2.5-3 hours), since the absence of a fluorescently labeled probe does not allow real-time accumulation of specific PCR products to be recorded. The invention does not intend to automatically record and process experimental data. Additional manipulations with amplicons (enzymatic hydrolysis and gel electrophoresis) greatly increase the likelihood of false positive results associated with contamination of rooms and laboratory equipment with PCR products.

Known "New fluorescence quantitative polymerase chain reaction (PCR) detection method for rift valley fever virus and rift valley fever virus detection PCR System" (Chinese patent No. CN 102140528 (A), IPC C12Q 1/68, publ. 2011-08-03 ) The oligonucleotide primers and fluorescently-labeled probe of the invention are intended to identify Rift Valley fever virus RNA by real-time reverse transcriptase polymerase chain reaction. The hybridization region of the primers and probe differs from the claimed technical solution.

However, since a more variable VLDR RNA fragment was selected for analysis in this invention, the primers and probe in some cases may not be specific enough for reliable identification of the virus.

The closest analogue (prototype) are oligonucleotide primers and a fluorescently-labeled probe, allowing to detect VLDR RNA in the test sample by amplification of the S-segment (gene of the nucleocapsid protein N) with the detection of results in real time [6].

However, given the data on the genomic diversity of VLDR presented in GenBank, the indicated primers and probe selected from the six nucleotide sequences of reference strains may in some cases not provide reliable identification of the virus due to insufficient specificity for VLDR genovariants identified later.

The technical result of the claimed invention is the creation of a more specific set of oligonucleotide primers and fluorescently-labeled probe for identifying VLDR genetic material in clinical and biological samples and environmental objects by real-time RT-PCR.

The task is achieved by selecting oligonucleotide primers and a fluorescently-labeled probe having the following nucleotide composition:

Primer RVFV-2s: 5′-AGGAGCATCTGAAATAGG-3 ′

Primer RVFV-2a: 5′-GGCTCAAATGATCAAMCC-3 ′

Probe RVFV-2: 5′-FAM-TCATCATGGGAAACTCACGCA-BHQI-3 ′.

At the initial stage, the search and determination of the most conservative sections of the VLDR RNA L-segment was carried out using 94 known nucleotide sequences presented in the NCBI MegaBLAST database [http://www.ncbi.nlm.nih.gov/]. As a target for diagnostic primers and a probe, we selected a region that included 167 nucleotide pairs (positions No. 180-346).

Reverse transcription of viral RNA, combined with subsequent amplification of the cDNA fragment in the polymerase chain reaction, was performed using a CFX96 amplifier (BioRad, USA). The amplification products were detected directly during the NDP due to the use of a fluorescently-labeled oligonucleotide probe that hybridizes with the reaction product (amplicon) and provides additional specificity of the method. The fluorescence intensity increased during the reaction in proportion to the accumulation of amplicons.

The defining hallmark of the proposed primers and probe in comparison with the prototype is hybridization to more specific sequences of 94 known VLDR genomes.

The invention is illustrated by the following examples of specific performance.

Example 1. Verification of the analytical sensitivity of a set of primers and probes

To control the analytical sensitivity of the proposed set of primers and probes, a recombinant plasmid was constructed containing a VLDR cDNA insert: plasmid pUC19 hydrolyzed with the restriction enzyme SmaI (position No. 415) was ligated with a cDNA fragment of the VLDR RNA L-segment (position No. 180-346). The concentration of DNA of the recombinant plasmid (μg / ml) was determined using a NanoView Plus spectrophotometer (GE Healthcare, USA). The concentration of genomic equivalents (HE) of the LDR virus was calculated taking into account the molecular weight of the recombinant plasmid according to the formula (1):

where M is the concentration of plasmid DNA [GE / ml];

C is the concentration of the recombinant plasmid [μg / ml];

10 ^-6 is the conversion coefficient [μg / ml] to [g / ml];

6.02 · 10 ²³ - the number of Avogadro;

2853 — plasmid length [base pairs, bp];

3.26 · 10 ² - the average molecular weight of one link of the nucleotide chain [Yes].

From a concentrated solution containing an average of 10 ⁷ GE / ml, sequential 10-fold dilutions of plasmid DNA were prepared, which were used as samples for RT-PCR in real time (10 μl per reaction). Accordingly, the samples contained from 10 ⁵ or less GE per reaction. To control possible contamination, 10 μl of PCR water was added to a separate tube.

The reaction mixture with a total volume of 25 μl (including the volume of the test sample) contained 1 × AS polymerase buffer (67 mM Tris HCl, 16.6 mM (NH ₄ ) ₂ SO ₄ , 0.01% Tween-20), 0.4 mM each deoxynucleoside triphosphate, 3 mM MgCl ₂ , 1-5 ea reverse transcriptase M-MuLV, 1 EA Hot Start Taq DNA polymerase (Sibenzim LLC, Novosibirsk, Russia), 0.8 μM of each primer and probe (RVFV-2s: 5′-AGGAGCATCTGAAATAGG-3 ′, RVFV-2a: 5′-GGCTCAAA TGATCAAMCC-3 ′, RVFV-2: 5′-FAM-TCATCATGGGAAACTCACGCA-BHQI-3 ′) (FBSI SSC WB “Vector”).

RT-PCR with hybridization-fluorescence detection of reaction products in real time via the FAM / Green channel was performed on a CFX96 amplifier (BioRad, USA) according to the protocol shown in Table 1.

Table 1 Temperature ° C Time Fluorescence detection The number of cycles 45 30 minutes - one 94 3 min - one 94 10 s - 55 30 s - 5 72 20 s - 94 10 s - 55 30 s Fam / green 40 72 20 s -

The reaction results were interpreted on the basis of the presence (or absence) of intersection of the fluorescence curves with the threshold lines, which corresponds to the presence (or absence) of the threshold cycle value “Ct” in the corresponding column of the result table of the amplifier program. The result was considered positive (the sample contains the declared number of GE VLDR) if the fluorescence accumulation curve for the corresponding sample had a characteristic exponential growth phase and crossed the threshold line. In this case, the “Ct” value for this sample, increasing in proportion to its dilution, should not exceed 35.

The analytical sensitivity of the proposed set of primers and probes, determined as a result of the experiments (Fig. 1), was 100 GE VLDR (copies of plasmid DNA containing a virus-specific insert) per 25 μl of the reaction mixture.

Example 2. Determination of the genetic material of VLDR in a virus-containing sample

VLDR RNA detection efficiency was evaluated using an inactivated sample of the museum strain RVF 2002/09-PS provided by the GNU VNIIVViM of the Russian Agricultural Academy (Pokrov, Moscow Region, Russia).

RNA was isolated from the test material using the AmpliPrim RIBO-prep reagent kit (NextBIO LLC, Moscow, Russia) in accordance with the instructions for use in the presence of an internal control sample (CTP).

RT-PCR with hybridization-fluorescence detection of amplicons was carried out in the reaction mixture of the following composition: 1 × AS-polymerase buffer (67 mM Tris HCl, 16.6 mM (NH ₄ ) ₂ SO ₄ , 0.01% Tween-20), 0 , 4 mM of each deoxynucleotide triphosphate, 3 mM MgCl ₂ , 1-5 ea reverse transcriptase M-MuLV, 1 e.a. Hot Start Taq DNA polymerase (Sibenzim LLC, Novosibirsk, Russia), 0.8 μΜ of each primer and probe (RVFV-2s: 5′-AGGAGCATCTGAAATAGG-3 ′, RVFV-2a: 5′-GGCTCAAA TGATCAAMCC-3 ′, RVFV-2: 5′-FAM-TCATCATGGGAAACTCACGCA-BHQI-3 ′) (FBSI SSC WB “Vector”).

The total volume of the reaction mixture (per study) was 25 μl, including 10 μl of the test sample. To control possible contamination, 10 μl of PCR water was added to a separate tube.

Combined RT-GTZR and registration of results in real time was carried out on a CFX96 amplifier (BioRad, USA) according to the protocol shown in table 2.

Fluorescence was detected on the FAM / Green channel for amplification of VLDR cDNA and on the HEX / Yellow channel for amplification of CKD.

table 2 Temperature ° C Time Fluorescence detection The number of cycles 45 30 minutes - one 94 3 min - one 94 10 s - 55 30 s - 5 72 20 s - 94 10 s - 55 30 s FAM / Green, HEX / Yellow 40 72 20 s -

The reaction results were interpreted on the basis of the presence (or absence) of intersection of the fluorescence curves with the threshold lines, which corresponds to the presence (or absence) of the threshold cycle value “Ct” in the corresponding column of the result table of the amplifier program. The result was considered positive if the fluorescence accumulation curve for the corresponding sample had a characteristic exponential growth phase and crossed the threshold line. The value of "Ct" for this sample should not exceed 35 (figure 2).

Thus, from the above examples 1 and 2 shows the achievement of the claimed technical result: created oligonucleotide primers and probe to identify the genetic material VLDR in clinical and biological samples and environmental objects by RT-PCR in real time. The analytical sensitivity of the VLDR detection kit was also determined, which amounted to 100 genomic equivalents per 25 μl of the reaction mixture.

Information sources

1. Jupp P.G., Grobbelaar A.A., Leman P.A. et al. Experimental detection of Rift Valley fever virus by reverse transcription-polymerase chain reaction assay in large samples of mosquitoes // J. Med. Entomol., 2000, 37: 467-471;

2. Sail A.A., Macondo E.A., Sène O.K. Use of reverse transcriptase PCR in early diagnosis of Rift Valley fever // Clin. Diagn. Lab. Immunol., 2002, 9: 713-715;

3. Espach Α., Romito M., Nel L.H. Development of a diagnostic one-tube RT-PCR for the detection of Rift Valley fever virus // Onderstepoort J. Vet. Res., 2002, 69 (3): 247-52; Yeh J.-Y., Lee J.-H., Seo H.-J. Simultaneous detection of Rift Valley fever, bluetongue, rinderpest and peste des petits ruminants viruses by a single-tube multiplex reverse transcriptase-PCR assay using a dual-priming oligonucleotide system // J. Clin. Microbiol., 2011, 49: 1389-1394;

4. RF patent No. 2457255, IPC C12N 15/33, C12Q 1/68, publ. 07/27/2012

5. Yeh J.-Y., Lee J.-H., Seo H.-J. Simultaneous detection of Rift Valley fever, bluetongue, rinderpest and peste des petits ruminants viruses by a single-tube multiplex reverse transcriptase-PCR assay using a dual-priming oligonucleotide system // J. Clin. Microbiol., 2011, 49: 1389-1394;

6. Belov A.B., Grebennikova T.B., Zaberezhny A.D. et al. A real-time PNR-based test system for detecting the LDR virus // Veterinary Medicine, 2007, 6: 53-55.

Claims (1)

A set of oligonucleotide primers and a fluorescently labeled probe for the identification of Rift Valley fever virus by the real-time reverse transcriptase polymerase chain reaction, including a pair of oligodeoxyribonucleotides having specific direct and reverse primer activity, and a fluorescently labeled structure:

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