RU2416647C1 - Oligonucleotide primers, method and test system for identifying genome of sheep nairobi disease virus by reverse transcription - polymerase chain reaction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, namely to synthetic oligonucleotide primers complementary to a conservative S-segment region of a genome of sheep Nairobi disease virus, to a method for identifying sheep Nairobi disease virus and to a test system for identifying DNA of sheep Nairobi disease virus. The offered invention can be used in veterinary virology. The method for identifying sheep Nairobi disease virus involves RNA recovery from the biological material. It is followed with conducting a polymerase chain reaction with using primers NSD F₁ 5'TATGCTTCTGCCTTGGTTG-3' NSD R₁ 5'-ATCCGATTGGC AGTGAAG-3', NSD F₂ 5'-AGAGCACATTGACTGGGC-3', NSD R₂ 5'-GCCTTCCAAAGCCAGTAG-3' Thereafter, virus RNA is amplified. Then, the reaction is assessed by agarose gel electrophoresis, with a reaction result considered as positive if the PCR product corresponds to the size of 360 base pairs.

EFFECT: offered invention allows identifying genome RNA of sheep Nairobi disease virus by means of a nidicolous version of RT-PCR with using two synthesized pairs of oligonucleotide primers complementary to conservative gene region of core protein N.

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Description

The invention relates to veterinary virology, and in particular to diagnostic tools.

Sheep Nairobi disease (hereinafter - BNO) is a zooanthroponous transmissible disease of sheep and goats, manifested by recurrent fever, hemorrhagic gastroenteritis, glomerulonephritis, mucopurulent discharge from the nose and diarrhea; characterized by a mortality rate that can vary between 40 and 90%.

The disease is constantly recorded in Kenya, Tanzania and Mozambique and in India. According to the OIE, outbreaks of BNO have also been reported in the Middle East (Kuwait, 1995) and in Europe (Greece, 2003). The source of infection is sick sheep and goats, as well as hidden virus carriers.

Clinical symptoms are similar in sheep and goats, although sheep are more susceptible to infection.

A person usually becomes infected with BNO when they are bitten by infected ixodid ticks, and their natural susceptibility is high. After the disease, long-term immunity remains, repeated diseases are rare. In African countries, men get sick more often due to the fact that in these areas they tend to breed animals. The incubation period lasts 4-5 days, the main clinical symptoms are: an increase in body temperature to 39-40 ° C with 2-3-day breaks, chills, pain in muscles and joints, bloody diarrhea. During the height of the disease, a rash appears on the trunk and limbs. The asymptomatic course of the disease in humans is described.

During an outbreak of BNO during antiepizootic measures, it is necessary to conduct quick and accurate expert studies that allow the pathogen to be identified as soon as possible.

Known methods for diagnosing infection, such as a neutralization reaction, enzyme-linked immunosorbent assay, which are characterized by high sensitivity and are widely used [1, 2]. The main drawback of these methods is the duration of the analysis, and the fact that the neutralization reaction gives cross-reactions with related bunyaviruses precludes the possibility of verifying the authenticity of the analysis results.

All studies of the BNO virus genome were carried out by employees of the Viral and Rickettsia Disease Division of the Center for Disease Control and Prevention (Atlanta, Georgia, USA) [3, 4]. They determined the nucleotide sequences of the S- and L-segments of the strains "NSD 708" and "Ganjam", carried out a phylogenetic analysis among related nairoviruses and developed methods for amplification of various parts of the genome of the BNO virus. However, one half of the oligonucleotide primers designed by these scientists is strain-specific, and the other is genus-specific. Thus, these primers do not allow us to solve the problem of identifying the genome of any strain of BNO virus from pathological material and its reliable differentiation from other viruses by PCR.

The aim of this invention is to develop a method for detecting genomic RNA of the BNO virus using the nested RT-PCR variant using two synthesized pairs of oligonucleotide primers complementary to the conserved region of the N nucleocapsid protein gene N.

Using the first (external) pair of primers, it is possible to amplify a DNA fragment with a size of 690 pairs of nucleotides (hereinafter referred to as bp), which is the target for annealing the second (internal) pair of primers, allowing to synthesize a 360 bp fragment. The use of two pairs of primers allows to achieve high specificity and sensitivity of the reaction.

The goal is achieved by a method for detecting BNO virus in blood samples, samples of organs of latently infected, sick or dead animals and / or in infected cell cultures, in which the complementary DNA is first synthesized on a viral RNA matrix and then amplified. This procedure is carried out using 3 sets that make up the test system:

1. a kit for the isolation of nucleic acids, including a nucleosorbent, a lysing buffer with guanidine thiocyanate and 2 solutions for sequential washing of the sorbent;

2. a kit for the production of PCR, including the reaction mixture for the production of PCR and 2 pairs of specific oligonucleotide primers;

3. kit for electrophoresis, including agarose and electrophoresis buffer with ethidium bromide.

The cDNA synthesis was carried out at a temperature of 50 ° C for 30 min with a primer NSD R ₁ , and then the reaction was terminated by 5-minute heating of the reaction mixture at a temperature of 94 ° C.

The obtained cDNA is used for the 1st round of PCR with the participation of an external pair of primers - NSD F ₁ and NSD R ₁ . The DNA fragments obtained in the 1st round were 930 bp in size. used as a matrix for the 2nd round of PCR using an internal pair of primers NSD F ₂ and NSD R ₂ . The nucleotide composition of the primers used is as follows:

NSD F ₁ 5 '-TAT GCT TCT GCC TTG GTT G - 3'

NSD R ₁ 5 '- PBX CGA TTG GCA GTG AAG - 3'

NSD F ₂ 5 '- AGA GCA CAT TGA CTG GGC - 3'

NSD R ₂ 5 '- GCC TTC CAA AGC CAG TAG - 3'

The temperature regimes for both PCR rounds are the same and include the following steps: 2 min preliminary denaturation at 94 ° C, 35 amplification cycles (94 ° C for 30 sec, 61 ° C for 30 sec, 72 ° C for 30 sec), 1 cycle chain completion (94 ° С - 2 min, 72 ° С - 5 min).

After staging RT-PCR, the products are separated by horizontal electrophoresis in a 2% agarose gel containing ethidium bromide. Analysis of the results of the reaction is carried out only on the 2nd round of PCR.

The technical result of the invention is to increase the degree of specificity and sensitivity, as well as reducing the time it takes to carry out diagnostic work to detect the virus in samples of organs and blood from sick animals.

The essence of the invention lies in the fact that using these primers (NSD F ₁ , NSD R ₁ , NSD F ₂ , NSD R ₂ ) carry out nested RT-PCR, allowing to identify the genome of the virus NNR. If there is a BNO virus in the RNA samples under study, a DNA fragment of 360 bp in size is synthesized in the second round of NDP. The use of two pairs of primers allows to increase the specificity and sensitivity of the method.

A significant difference between these primers is that they:

1) are complementary to the conservative region of the nucleocapsid protein gene N of the BNO virus and are not complementary to any parts of the genomes of other viruses;

2) flank on a cDNA obtained on a viral RNA template, a fragment of 970 bp (primers NSD F ₁ , NSD R ₁ ) and inside it is a fragment of 360 bp (primers NSD F ₂ , NSD R ₂ ).

The invention is illustrated by several examples.

Example 1. Set No. 1 is used to isolate RNO virus RNA from blood, organ samples, and culture-containing virus-containing material.

The test material (blood, a suspension of the virus, 10% suspension of organs) in a volume of 100-200 microliters (hereinafter referred to as μl) is introduced into 1.5 cm ³ tubes into which 600 μl of lysing buffer based on guanidine thiocyanate are previously added. The tubes are mixed and incubated at room temperature for 8-10 minutes. Then add 40 μl of nucleosorbent, incubated at room temperature for 10 minutes, periodically mixing the mixture. After incubation, the tubes are centrifuged for 30 sec at 8-13 thousand rpm, the supernatant is discarded. 300 μl of washing buffer is added, the sorbent is carefully resuspended, centrifuged for 30 sec at 8-13 thousand rpm, and the supernatant is removed. The sorbent is washed twice with 75% ethyl alcohol and then dried for 10 minutes at a temperature of 56 ° C. To the precipitate add 50 μl of elution buffer, mix and incubate for 10 min at a temperature of 56 ° C. Test tubes are centrifuged for 1 min at 13 thousand rpm and transfer the supernatant to new tubes. The supernatant is further used for the synthesis of cDNA.

Example 2. Synthesis and amplification of the cDNA region of the BNO virus using kit No. 2 for conducting nested PCR with synthetic oligonucleotide primers.

Calculation of the primary structure of oligonucleotide primers.

Using the program "BioEdit 7.0" the nucleotide sequences of the S-segment of the genome of strains "NSD 708" and "Ganjam G619" of the Nairobi sheep disease virus were aligned. After analyzing the constructed alignment, a conserved region located between 238 and 1169 nucleotides (5 '→ 3') was found inside the nucleocapsid protein N gene. Using the Oligo 4.0 program, 2 pairs of primers were selected - NSD F ₁ (238-256 nucleotide positions) and NSD R ₁ (1152-1169), flanking a fragment of 930 base pairs, and 2 pairs of primers - NSD F ₂ (642 -659) and NSD R ₂ (984-1001) flanking a fragment of 690 base pairs.

Using the program "Oligo 4.0" describes the main properties of the calculated primers, which determined the possibility of their use in PCR. These properties are shown in the table.

Table Properties of primers calculated using the program "Oligo 4.0" Primer Name Melting point, ¹ ° С ΔG primer hybridization per template, kcal / mol ΔG formation of hairpin structure, kcal / mol Primer annealing efficiency per matrix (Priming efficiency number) ² NSD F ₁ 61.5 -35.9 1.4 392 NSD R ₁ 60,4 -34.7 1.4 384 NSD F ₂ 60.1 -34 1.4 429 NSD R ₂ 60 -35.3 one 373 Notes. ¹ Melting point calculated using the Nearest neighbor method. ² Priming efficiency number is a value that determines the efficiency of annealing a given primer for a given section of the matrix, calculated according to a unique algorithm developed by the creators of the "Oligo" program. The minimum value of Priming efficiency number, at which the primer during PCR is effectively annealed to a specific area of the matrix, -210.

The test system has been tested on various strains of the BNO virus. Strains of related bunyaviruses were used as negative controls: rift valley fever (LDR), Congo-Crimean hemorrhagic fever (CCHF) and Akabane.

To obtain cDNA, 0.6 cm ³ tubes are prepared and labeled for the N-number of samples, including negative controls. 15 μl of the reaction mixture, including a buffer for revertase, 10 pmol of primer NSD R ₁ , 2.5 mmol of each dNTP, 20 units, are added to the tubes. MMLV revertase; 1 drop (40-45 μl) of mineral oil is layered on top of the mixture. Next, 5 μl of the test RNA is added under the oil and the tubes are placed in an amplifier with the following temperature conditions:

50 ° C 30 minutes 1 cycle 94 ° C 5 minutes

After incubation, the reaction mixture is used as a cDNA preparation. To carry out the 1st round of PCR, a 25 μl reaction mixture was prepared, including 10 pmol of NSD F ₁ and NSD R ₁ primers, 2.5 mmol of each dNTP, Taq polymerase buffer with 15 mM MgCl ₂ , 1 unit. Taq polymerase. This mixture is added to pre-labeled tubes, a drop of mineral oil is layered on top and 5 μl of cDNA is added under the oil. The tubes are placed in an amplifier with the following temperature conditions:

94 ° C 2 minutes 1 cycle 94 ° C 30 sec 35 cycles 61 ° C 30 sec 72 ° C 30 sec 94 ° C 2 minutes 1 cycle 72 ° C 5 minutes

After the completion of the program, they begin to carry out the 2nd round of PCR. To do this, transfer 5 μl of the mixture from the tubes in which the 1st round was carried out to new tubes with 20 μl of the reaction mixture, including 10 pmol of NSD F ₂ and NSD R ₂ primers, 2.5 mmol of each dNTP, Taq polymerase buffer with 15 mM MgCl ₂ , 1 unit Taq polymerase; 1 drop of mineral oil is layered on top. The tubes are loaded into an amplifier with a temperature regime similar to that for the 1st round of PCR.

Example 3. Determining the size of PCR products using kit No. 3 for electrophoresis.

The products of the 2nd round of PCR are analyzed by electrophoresis in a 2% agarose gel in standard Tris-borate buffer.

15 μl of the PCR product (already containing the gel buffer) is added to the agarose gel well. Electrophoresis is carried out at a voltage of 10 V / cm of the gel length until the dye passes from the start of at least half of the gel (approximately 15 min). The result of electrophoresis is taken into account when viewing the gel in ultraviolet light with a wavelength of 260 nm on a Transilluminator instrument. The result is considered positive if the PCR product matches the fragment size of 360 base pairs.

Using the test system and calculated primers, all test samples of the BNO virus were detected. Negative controls were not amplified.

Example 4. Determination of the sensitivity and specificity of PCR based on synthetic oligonucleotide primers for protein N.

The results of PCR with primers complementary to the gene of protein N of the virus BNO. Name of drugs The virus titer TCD50 / ml Cipher Expected Result Actual result BNO virus strain "MM" 6.0 one + + BNO virus strain "MM / K-05 5.8 2 + + BNO virus strain "MM / K-05 1.8 3 + + virus LDR strain "VNIIVViM1974" 6.5 four - - Akabane virus strain "B8935" 4.75 5 - - CCHF virus (positive control from the AmpliSens CCHF kit produced by the Federal State Budget Institution Central Research Institute of Epidemiology of Rospotrebnadzor to detect the CCHF virus genome by RT-PCR) 5,0 6

The test system and synthetic oligonucleotide primers were tested for specificity and sensitivity. It was found that the test system does not amplify the cDNA of other viruses.

Thus, the proposed test system allows you to achieve a high level of sensitivity using two pairs of primers, allowing you to get in the 2nd round of PCR fragment of 360 bp in size.

Information sources

1. Davies F.G. Nairobi sheep disease // Parassitologia. - 1997. - V.39 - No. 2 - C.95-98.

2. Davies F.G., Mungai J.N., Taylor M. The laboratory diagnosis of Nairobi sheep disease // Trop. Anim. Health Prod. - 1977. - V.9 - No. 2 - S.75-80.

3. Marczinke B.I., Nichol. S.T. Nairobi sheep disease virus, an important tick-borne pathogen of sheep and goats in Africa, is also present in Asia // Virology. - 2002. - V.303. - No. 1 - S.146-151.

4. Terpstra C. Physical and biological properties of Nairobi sheep disease virus // Vet. Microbiol. - 1983. - V.8 - No. 6 - S.531-541.

Claims (3)

1. Synthetic oligonucleotide primers complementary to the conserved region of the S-segment of the sheep Nairobi disease virus genome, used to identify sheep Nairobi disease virus DNA, having the following nucleotide composition:
NSD F ₁ 5'-TATGCTTCTGCCTTGGTTG-3 '
NSD R ₁ 5'-ATCCGATTGGCAGTGAAG-3 '
NSD F ₂ 5'-AGAGCACATTGACTGGGC-3 '
NSD R ₂ 5'-GCCTTCCAAAGCCAGTAG-3 '. 2. A method for detecting a sheep’s Nairobi disease virus, including isolation of RNA from biological material, production of a polymerase chain reaction using synthesized primers, amplification of virus RNA, evaluation of the reaction by gel electrophoresis in agarose, characterized in that DNA synthesis is carried out at a temperature of 50 ° C for 30 min with a primer NSD R ₁ , and then terminate the reaction with 5-minute heating of the reaction mixture at a temperature of 94 ° C, then the DNA obtained is used to carry out the 1st round of PCR with the participation of an external pa primers of NSD F ₁ and NSD R ₁ according to claim 1, and the DNA fragments obtained in the 1st round are used as a template for the 2nd round of PCR using an internal pair of primers NSD F ₂ and NSD R ₂ according to claim 1 the temperature regime of PCR is the same for the 1st and 2nd rounds and includes the following steps: 2 min preliminary denaturation at 94 ° С, 35 amplification cycles (94 ° С - 30 s, 61 ° С - 30 s, 72 ° C - 30 s), 1 cycle of chain completion (94 ° C - 2 min, 72 ° C - 5 min), then after the 2nd round of PCR the reaction results are taken into account, while the reaction result is considered positive if the size f the segment corresponds to 360 pairs of nucleotides. 3. A test system for the detection of DNA from the Nairobi disease virus of sheep, including plastic bottles and tubes, thermostable Taq polymerase enzyme, a PCR mixture for the reaction, positive and negative controls, characterized in that it contains three sets: 1) isolation kit nucleic acids, including a nucleosorbent with a lysis buffer based on guanidine thiocyanate and 2 solutions for sequential washing of the sorbent; 2) a PCR formulation kit comprising a PCR reaction mixture and synthetic oligonucleotide primers according to claim 1; 3) a kit for electrophoresis, including agarose and electrophoresis buffer with ethidium bromide.