Synthetic oligonucleotide primers-probe and method to detect genomes of 1st, 4th, 16th serotypes of bluetongue disease virus by method of rt-pcr in real-time mode

FIELD: biotechnologies.

SUBSTANCE: synthetic oligonucleotide primers and probes are disclosed to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus. Also the method is described to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus using such synthetic oligonucleotide primers and probes.

EFFECT: method makes it possible to perform determination of a serotype of the bluetongue disease virus with the help of PCR with a stage of reverse transcription in real time mode, and also to increase specificity and sensitivity, to reduce time to perform work aimed at determination of a serotype of the bluetongue disease virus in samples of an investigated biological material.

4 cl, 4 tbl

 

The invention relates to veterinary Virology, namely by means of genetic typing of viruses.

Bluetongue is an infectious, non-communicable viral disease with a wide spectrum of domestic and wild ruminants, currently defined by the OIE as a major socio-economic problem, which creates great difficulties for international trade in animals and animal products [1].

A preliminary diagnosis on bluetongue is based on the clinical and pathological signs. For a definitive diagnosis requires laboratory confirmation. Currently, the laboratory diagnosis of bluetongue as a set of interconnected virological and serological methods based on isolation of the virus (use chicken embryos or cell culture, antigen detection of bluetongue by immunofluorescence, with subsequent typing of the selected virus neutralization reaction (PH) with typespecification serum, RNA detection of bluetongue virus RT-PCR or RT-PCR in real time; the detection of group-specific antibodies to bluetongue virus in the blood serum of the long reaction of complement fixation (RDSC)and competitive ELISA, indirect ELISA. Sensitivity tests can vary between laboratories, but in General VIR gene is sa can be detected in blood samples, starting from the 3rd day after infection onwards (data according to the results of RT-PCR in real time) and detection antibody ELISA can be expected, since 5-7 days after infection.

It is now known about the existence of 26 serotypes of the virus and molecular epidemiology data indicate a high genetic heterogeneity of its population, which allows you to split virus types, topotype, quanitity. Already known for more than 300 variants of the virus, genetically different from each other. In July 2011, the UK declared himself free from the disease, however, on the Mediterranean continent flash still continue to be logged. According to data posted on the website eubtnet.org from may 2010 to July 2011 were the following outbreaks in Algeria, France, Italy, Portugal and Spain 1 serotype - 108 outbreaks in Italy and Spain - 10 flashes 4th serotype, in Italy and Spain - 4 flash 8 and in Cyprus, Greece and Turkey - 23 flash 16th serotype. Molecular and epidemiological data of all isolates of bluetongue viruses belonged to the European lines/groups and corresponded to the 1st serotype - Western topotype, 4th serotype - Western topotype, 8th serotype - Western topotype and 16th serotype - East topotype.

The disease is caused by the bluetongue virus, which is one of the types of ro is and Orbivirus, family Reoviridae [2]. The viral genome consists of 10 segments of dsrnas Packed in dodecahedral nucleocapsid, consisting of seven structural proteins [3]. The outer layer of the capsid presents two proteins VP2 and VP5, which have a high degree of heterology amino acid sequence, equally both between and within each serotype [4]. The serotype of the virus is determined VP2 protein, which has a neutralizing epitopes of the virus [5].

The need to improve the means and methods of laboratory diagnostics with the new achievements of science and diagnosis in asymptomatic course of the disease has set us the task of developing a panorama in real-time to detect the genomes of the 1st, 4th, 16th serotypes of bluetongue virus. Difficulties in its development due to several factors: firstly, the structure of the genome, as it consists of 10 segments of dsrnas and as a result is currently known fact frequent reasontly viruses as field options field and with the vaccine, not only within the same serotype, but also between serotypes.

Known quantitative method RT-PCR real-time detection of vaccine and/or field strains WB. Since the detection of PCR products was based on the fluorescence intensity is significantly increased Pro-and the productivity of the test and reduces the risk of contamination. There were developed two universal test based on RT-PCR in real time, they allowed us to detect all serotypes WB [6, 7]. In addition, currently widely used tests based on RT-PCR real-time detection of individual serotypes that in the event of an outbreak quickly and in each case to differentiate virus serotype [8; 9]. All existing methods and based on these test systems have been developed abroad, therefore, a significant drawback is their high cost and long delivery time in our country.

The purpose of this invention is to develop a method of differentiation of the 1st, 4th, 16th serotypes of bluetongue virus using RT-PCR in real time, based on the use of specific for each serotype systems "oligonucleotide primers-probe.

This goal is achieved by specific systems, "oligonucleotide primers - probe to detect the 1st, 4th, 16th serotypes of bluetongue virus, which are then used in a polymerase chain reaction with the stage of reverse transcription real-time. This procedure includes the following steps.

1. Denaturation dsrnas of bluetongue virus.

2. Reverse transcription - synthesis of cDNA.

3. In fact PCR in real time.

the Denaturation of dsrnas is carried out at a temperature of 95°C for 5 minutes in the reaction mixture with serotype-specific pair of oligonucleotide primers. Then denatured RNA WB needs to be fixed by freezing. Synthesis of cDNA is carried out at a temperature of 42°C for 30 minutes in the reaction mixture for reverse transcription, including deoxyribonucleotides (dNTP), a buffer for reverse transcription and enzyme revertase (reverse transcriptase). Then the reaction terminorum at 95°C for 5 minutes. The obtained cDNA is used for polymerase chain reaction. Prepared standard mixture for PCR in real time, including a buffer for PCR dNTP, a pair of oligonucleotide serotype-specific primers and the corresponding probe and the enzyme DNA polymerase. The reaction profile is as follows: hold temperature 94°C - 3 min; Cycling: 94°C 10 s, 60°C - 20 s, 72°C - 15 C. the Cycle is repeated 45 times.

The basis of the developed method RT - PCR, real-time technology hybridization probes TaqMan controlling the kinetics of PCR directly during amplification using resonance fluorescence quenching. For detection used a probe carrying a fluorophore and cositel, complementary part amplifierarava with specific primers fragment. Selection and analysis of serotype-specific primers (table 1, 2, 3) and probes was performed using the software package "Bio Edit 7.0.8", "Oligo 6.0" and based on the analysis of nucleotide n is sledovatelnot 2nd segment 120 isolates 1, 4-th and 16-th serotypes, the main genetic representatives of European strains of bluetongue virus. Nucleotide sequences were taken from GenBank. As a source of fluorescence on the 5' end of the probe used dye FAM, and for suppression of fluorescence on the 3' end BHQ1.

Table 1
Serotype-specific primers and probe for detection of the genome of 1 serotype of bluetongue virus.
Name of the primer/probeThe nucleotide sequence, 5'-3'Position on the nucleotide sequence of a segment
l/2/qfCGA ATT TAC GGT GTA TAG With2756
l/2/probeTGG TAA CGA CGA GAT GCT GAC AA2864
1/2/qrATA CGT TGA GAA GTT TTG T2906

Table 2
Serotype-specific primers and probe for detection of the genome of the 4th serotype of bluetongue virus.
Name of the primer/probeThe nucleotide sequence, 5'-3'Position on the nucleotide sequence of a segment
4/2/qfATG CAT AAA GAG TAC GGY GT2429
4/2/probeGAA CAC GAA GAT ATC GCA GGG2470
4/2/qrACT TGG ACG TCA CAA CAG G2523

Table 3
Serotype-specific primers and probe for detection of genome 16th serotype of bluetongue virus.
Name of the primer/probeThe nucleotide sequence, 5'-3'Position on the nucleotide sequence of a segment
16/2/qfCGC GTA TAA TCA ATT CAT GAA708
16/2/probeGAT ACG AGA GGA AAT GGC CGC771
16/2/qrGAT GTG TGT TCG CTC GAA G846

Technical is the result of the invention is the ability to determine the serotype of bluetongue virus by PCR with the stage of reverse transcription real-time as well as increasing the specificity and sensitivity, the reduction of time in determining the serotype of bluetongue virus in the samples of the studied biological material.

The invention consists in that by means of these serotype-specific oligonucleotide primers and samples carried out RT-PCR in real time, allowing to detect the genomes of the 1st, 4th, 16th serotypes WB. The use of this technique allows to increase the specificity and sensitivity of the reaction.

Specific example

Detection of virus genomes bluetongue 1st, 4th and 16th serotypes in samples of biological material (blood from infected with bluetongue the animals, the cultural material of the virus, freeze-dried culture material of the virus from the Museum of strains of microorganisms wildebeest Vniivvim RAAS, as well as in samples of intact cell cultures, blood from intact animals and cultural material heterologous viruses).

1. From the presented samples using reagent Trizol (Invitrogen) and according to manufacturer's instructions were isolated nucleic acid. Further preparations of nucleic acids were used in the reaction: 8 μl of sample was added to the reaction mixture to denature. In a test tube with denatured NC was added to the mixture for reaction reverse tra is scriptie. Drugs received complementary DNA in the amount of 10 μl was used for setting the actual PCR in real time (results are presented in table 4). PCR can be carried out, for example, on the device Rotor gene 6000 (Corbett Research, Australia).

Table 4
The results of the use of the present invention to detect the genomes of the 1st, 4th and 16th serotypes of bluetongue virus.
No. of samplesCharacterization of biomaterialThe resulting PCR
1 serotype : 4th serotype16th serotype
1.freeze-dried culture, bluetongue virus BTV-1 reference strain+--
2.freeze-dried culture, bluetongue virus BTV-2 reference strain---
3.dried ulturally, the bluetongue virus BTV-4 reference strain-+-
4.freeze-dried culture, bluetongue virus BTV-8 reference strain---
5.freeze-dried culture, bluetongue virus BTV-10 reference strain---
6.freeze-dried culture, bluetongue virus BTV-11 reference strain---
7.freeze-dried culture, bluetongue virus BTV-13 reference strain---
8.freeze-dried culture, bluetongue virus BTV-16 reference strain--+
9.freeze-dried culture, Viru is bluetongue, BTV-17 reference strain---
10.freeze-dried culture, bluetongue virus BTV-18 reference strain---
11.freeze-dried culture, bluetongue virus BTV-20 reference strain---
12.freeze-dried culture, bluetongue virus BTV-23 reference strain---
13.freeze-dried culture, bluetongue virus BTV-24 reference strain---
14.the blood of the lamb No. 4, infected 4th bluetongue virus serotype (.BTV-4, Spain), 15th day after infection, infectious activity of 3.75 lg EDS50/0.1 ml-+-
the blood of the lamb # 20 of infected 1st bluetongue virus serotype (.BTV-1, Spain), 21st day after infection, infectious activity of 3.0 lg EDS50/0.1 ml+--
16.the blood of calf No. 50 infected with bluetongue virus 8 serotype (field isolate FCL/4-09, Russia), 4 days after infection, the infectious activity of 4.5 lg EDS50/0.1 ml---
17.the blood of calf No. 464, infected 1st bluetongue virus serotype (PCs-BTV-1, Spain), 7 days after infection, infectious activity of 5.0 lg EDS50/0.1 ml+--
18.the blood of the calf from 01.02.10 was infected with bluetongue virus 1 serotype (strain BTV-1 Spain) and 8th serotype (strain BTV-8 Denmark) at the same time+--
19.the blood of deer No. 2, infected 16th bluetongue virus serotype (strapper), 15 days after INF the Oia, infectious activity of 2.0 lg EDS50/0.1 ml--+
20.freeze-dried culture, virus AGBO strain new Jersey, 1st serotype---
21.freeze-dried culture, virus ACHL St/60-100, 9th serotype---
22.intact cell culture Vero, 32 pass---
23.intact cell culture KSS-21.1 pass---
24.blood from the intact animal (cattle - cows)---
25.blood from healthy animals (small ruminants - sheep)---

Carrying out the reaction of reverse transcription.

1. In the prepared tubes contribute 7 ál of the mixture to denature, then make 40 μl of mineral oil. Under the oil make 8 ál of the drug selected RNA. The reaction denaturation is carried out at a temperature of 95°C for 5 minutes. Next, you need to put tubes in conditions of negative temperatures, for this purpose use a tripod with refrigerant.

2. Cook the mixture for reverse transcription for this one sample in a separate test tube is mixed with 9.5 μl of the mixture for reverse transcription and 0.2 μl of MMLV-revertase, the mixture is stirred. In tubes with denatured RNA contribute to 9.7 ál of the mixture for reverse transcription. Reverse transcription is performed at a temperature of 42°C for 30 minutes. After the reaction cDNA used for PCR.

Carrying out multiplex PCR in real time.

1. In a separate tube, prepare the General reaction mixture for the required number of samples, one sample add 8 ál of the mixture for PCR, 7 μl of a mixture of primers and 0.15 µl Taq polymerase. The mixture is stirred, avoiding foam formation, and dig in 15 ál of tube a volume of 0.2 cm3on the surface of the mixture make the wax in the volume of 15 µl.

2. In a report prepared for PCR tubes on wax make 10 ál of cDNA.

The profile reaction:
1. Keeping temperature94°C - 3 min
2. The Cycling94°C - 10
60°C - 20
72°S - 15
The cycle is repeated 5 times
3. The Cycling 2
94°C - 10
60°C -20 ° C - Detection
72°C -15 ° C
Cycle to repeat 40 times

The fluorescence measured in the channel Fam/Green at 60°C.

The threshold cycles are determined automatically by the device.

Accounting and interpretation of results amplification.

The device Rotor gene 6000 set the parameters to optimize the detection of fluorescence for both channels. The minimum signal - 15F, the maximum signal - to 25 F.

The results are interpreted on the basis of the presence (or absence) of intersection of the graph of fluorescence installed on ACC is cstuuyxm threshold line.

When analyzing the results of amplification of specific plot cDNA bluetongue virus (channel Fam/Green) level threshold line R=0,01.

The sample is considered positive for the presence of RNA virus bluetongue, if the Ct value in the Fam channel is less 31. The sample is considered negative if the Ct value in the Fam channel is missing.

Thus, for the first time chosen a specific system "oligonucleotide primers - probe and a method that allows using RT-PCR in real time using data systems to detect the 1st, 4th and 16th serotypes of bluetongue virus.

The main advantages of the claimed invention are:

- high reliability of the method, thanks to selective detection of unique sequences of the three serotypes of bluetongue virus;

- the speed of analysis, as a number of procedures needed to register the amplification products in classical PCR;

- easy-to-follow techniques, eliminating the need to attract highly qualified personnel;

- the relative cheapness of the method, through the use of RT-PCR in real time to detect serotypes.

1. Synthetic oligonucleotide primers and probe to detect the genomes of 1 serotype of bluetongue virus, wherein they shall complementary areas variable 2nd segment 1 serotype of bluetongue virus used to identify genome of 1 serotype of bluetongue virus, having the following nucleotide composition (5'-3'):

1/2/qfCGA ATT TAC GGT GTA TAG With
1/2/probeTGG TAA CGA CGA GAT GCT GAC AA
1/2/qrATA CGT TGA GAA GTT TTG T

2. Synthetic oligonucleotide primers and probe for detection of genomes 4th serotypes of bluetongue virus, characterized in that they are complementary parts of variable 2-th segment of the 4th serotype of bluetongue virus used to identify genome 4th serotypes of bluetongue virus, having the following nucleotide composition (5'-3'):

4/2/qfATG CAT AAA GAG TAC GGY GT
4/2/probeGAA CAC GAA GAT ATC GCA GGG
4/2/qrACT TGG ACG TCA CAA CAG G

3. Synthetic oligonucleotide primers and probe for detection of genomes 16th serotype of bluetongue virus, characterized in that they are complementary parts of variable 2-th segment of the 16th serotype of bluetongue virus used to identify genome 16th serotype of bluetongue virus with the following nucleot the command structure (5'-3'):

16/2/qfCGC GTA TAA TCA ATT CAT GAA
16/2/probeGAT ACG AGA GGA AAT GGC CGC
16/2/qrGAT GTG TGT TCG CTC GAA G

4. How to detect the genomes of the 1st, 4th, 16th serotypes of bluetongue virus, including reverse transcription polymerase chain reaction, producing a polymerase chain reaction using synthetic primers according to claims 1 to 3, the amplification of RNA virus, evaluation of the reaction, characterized in that the carry out reaction amplification of PHK with stage cDNA synthesis, which in turn consists of denaturation dsrnas of bluetongue virus and the reaction of reverse transcription, and then interpret the results based on the presence/absence of crossing of fluorescence curve with the established threshold line.



 

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