RU2511440C2 - Method of quantitative determination of fixed rabies virus "moskva 3253" - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology and deals with method of quantitative determination of fixed rabies virus strain "Moskva 3253". Method includes decontamination and separation of RNA from virus-containing material, carrying out reaction of reverse transcription and polymerase chain reaction with hybridization-fluorescence account of results in "real time" mode with application of specific primers RV5-5'-GTTGGGCACTGAAACTGCTA-3', RV6-5'-GAATCTCCGGGTTCAAGAGT-3' and probe RV7-5'-ROX-AATCCTCCTTGAACTCCATGCGACAGA-BHQ2. Quantitative assessment of virus is determined on the basis of registration of signal of analysed sample fluorescence and its comparison with signal of fluorescence of PCR-standards, which contain different quantities of DNA-targets. Claimed method makes it possible to determine quantitative content of virus in rabies antigen of organ-tissue and culture origin.

EFFECT: application of invention contributes to standardisation of stage of rabies antigen preparation in production of heterological anti-rabies immunoglobulin.

2 tbl, 3 dwg, 2 ex

Description

The invention relates to biotechnology and virology and can be used by specialists working in the field of production of medical immunobiological preparations (MIBP).

The invention was carried out as part of the implementation of the federal target program “National System of Chemical and Biological Safety of the Russian Federation (2009-2013)” and in accordance with State Contract No. 54-D of 04.06.2012

To date, rabies remains one of the most dangerous infectious diseases, from which annually about 50,000 people and 1 million animals die in the world [1]. The unfavorable epidemiological situation of rabies, which has developed in almost all regions of the Russian Federation due to an increase in the incidence of wild and domestic animals, is responsible for the sharply increased level of the number of people who seek rabies care annually - over 450,000 people. Half of this number receives a course of anti-rabies treatment [3], which necessitates the intensification of research to improve the quality of anti-rabies drugs.

For post-exposure prophylaxis of rabies in humans in the Russian Federation, a concentrated culture-based rabies vaccine is used in combination with rabies immunoglobulin.

To date, heterologous anti-rabies immunoglobulin can be considered an acceptable and safe alternative to a homologous drug obtained from human immune serum. The use of modern methods of purification of immunoglobulin preparations has significantly reduced the frequency of side effects to 1-2%, resulting from the use of rabies immunoglobulin of animal origin [15].

To immunize producers upon receipt of equine hyperimmune serum, a rabic antigen based on the fixed rabies virus strain Moscow 3253 is used. Upon receipt of the antigen, standardization of the quantitative assessment of the content of rabies virus "Moscow 3253" in the material for immunization is necessary.

Currently, the amount of virus in the test material can be determined in various ways.

Known methods for determining the amount of virus in the test material by local damage in infected cell cultures and on the chorion-allantoic membrane (HAO) of chicken embryos, in which the amount of virus is taken into account in plaque forming units (PFU), smallpox units (OOE). However, the use of methods is difficult due to the fact that at a high concentration of the virus in the test material it is impossible to avoid plaque fusion and, accordingly, it is impossible to conduct a quantitative assessment [4]. The method is performed with a live virus; it is laborious and time consuming to execute (72-96 hours).

A known method for determining the titer of the virus in units of 50% of the infectious effect. According to this method, a unit dose of the virus is taken to be its dose that can cause an infectious effect in 50% of infected animals. The method for determining the titer of the virus is to infect animals with the virus, followed by the clinical features of the manifestation of the infectious process in each animal (the occurrence of signs of paralysis on days 4-7) and the determination of 50% of the effective dose. However, this method of titration of viruses is rather laborious, time consuming and requires a large number of animals. The disadvantages of this method is that a higher dose of the virus does not always cause a higher infectious effect, which is associated with the individual sensitivity of each animal species [4].

A known method based on the ability to agglutinate red blood cells of certain animal species under certain conditions. The titer of such viruses is expressed in hemagglutinating units (GAE). The erythrocyte suspension and virus-containing material in dilutions are mixed in equal volumes, incubated at 37 ° C and the agglutination reaction is taken into account. However, not all viruses are able to agglutinate red blood cells; therefore, the disadvantage of this method is the possibility of setting the reaction only with hemagglutinating viruses [4].

All the above methods for determining the content of rabies virus in the material are based on the action of a living pathogen on various biological cells and tissues and are not suitable for the quantitative assessment of inactivated rabies virus.

One of the promising approaches to the quantitative assessment of viruses in inactivated antigen material is the use of molecular genetic methods.

In classical PCR with electrophoretic consideration of the results, it is not possible to quantify the DNA / RNA of infectious agents in the test material. In addition, the presence of an electrophoresis stage makes more stringent requirements for the organization of a PCR laboratory and the risk of contamination with PCR products increases. When using PCR with hybridization-fluorescence real-time results, it is possible to quantify the DNA / RNA of infectious agents in the test material, with automatic recording and interpretation of the results. The absence of an electrophoresis stage allows minimizing the number of false positive results. Since the registration of the results is carried out directly in the PCR process, the entire analysis can be performed in one or two rooms of the laboratory and there is no need for a separate room for the detection of reaction products [2].

This approach has been successfully used to determine the concentration of RNA of yellow hemorrhagic fever, human herpes virus-6 and cytomegalovirus [8, 9]. The effectiveness of using PCR with electrophoretic, hybridization-fluorescence, and sequencing results has been proven to detect rabies virus RNA in biological material from animals and humans [5, 6, 7, 10, 11, 12, 13, 14]. In all studies, the determination of the number of viral particles in the research material was not carried out.

The objective of the invention is the creation of a highly specific, sensitive, simple and quick method for the quantitative determination of fixed rabies virus strain "Moscow 3253" using the polymerase chain reaction in real time in the production of rabies immunoglobulin.

The technical result of the invention is to reduce the execution time, to obtain quantitative characteristics of the antigen used, which helps to standardize the stage of preparation of the rabid antigen in the production of rabies immunoglobulin.

The technical result is achieved by a method including disinfecting and isolating RNA from a virus-containing material, staging a reverse transcription reaction and polymerase chain reaction with hybridization-fluorescence taking into account the results in real time using specific primers PV5-5'-GTTGGGCACTGAAACTGCTA-3 ',

RV6-5'-GAATCTCCGGGTTCAAGAGT-3 '

and probe RV7-5'-ROX-AATCCTCCTTGAACTCCATGCGACAGA-BHQ2, providing amplification of the GL fragment of the genome of the fixed rabies virus strain Moscow 3253, annealing the primers is carried out at a temperature of 60 ° C for 10 cycles, then at 56 ° C for 35 cycles and a quantitative assessment of the virus is determined based on the registration of the fluorescence signal of the test sample and comparing it with the fluorescence signal of PCR standards containing various amounts of the target DNA.

As a target DNA for the development of a method for the quantitative determination of the fixed rabies virus of the strain "Moscow 3253", a fragment of the G-L region of the rabies virus of 881 bp was selected. When studying this nucleotide sequence, it was found that the G-L sequence of the rabies virus strain "Moscow 3253" is unique and differs from other sequences of this region from 12 nucleotides in strain RV-97 to 152 nucleotides for the NNV-RAB-H isolate. The results of the analysis are presented in table 1. The analysis of the nucleotide sequence was carried out using the AlignX algorithm and the Mega 3.1 program, as well as the BLAST algorithm and the GenBank genetic database. When studying the nucleotide sequence of this locus in the strain of rabies virus "Moscow 3253" its high stability was established in all passages of the virus used in the production of rabies immunoglobulin. Based on the results obtained, the consensus sequence of the G-L region fragment of the fixed strain of rabies virus “Moscow 3253” was derived. The homology analysis of the studied region of the G-L fragment of the region of derivatives of the SAD B19 strain showed 99.8% and 99.7% identity of the strains after passages on chicken embryos and Vero cells, respectively, which also confirms the high specificity of this fragment.

Based on the consensus sequence of the selected region of the GL region using the Primer Premier-V5 program (Premier Bio Soft International), the website www.genscript.com and the BLAST algorithm, the oligonucleotide primers RV5 and RV6 were selected to ensure fragment amplification within 436-585 nucleotides of the complete locus sequences and TaqMan RV7 format probe with a ROX fluorescent label and a BHQ2 quencher at position 542-565 of the nucleotide of the complete locus sequence, allowing real-time reaction results to be taken into account:

RV5-5'-GTTGGGCACTGAAACTGCTA-3 ',

RV6-5'-GAATCTCCGGGTTCAAGAGT-3 '

RV7-5'-ROX-AATCCTCCTTGAACTCCATGCGACAGA-BHQ2,

An analysis of the correspondence of the selected primers and probe according to the number of variable nucleotides is presented in Table 2. The results indicate a high specificity of the primers RV-5, RV-6 and probe RV-7 with respect to the above fragment of the G-L sequence of the region of the rabies virus strain "Moscow 3253". To increase the efficiency of hydrolysis of the RV7 probe due to the 5'-exonuclease activity of the Taq polymerase enzyme, three “AAT” nucleotides, non-complementary to the locus sequence, were added to the 5'-end of the probe. The arrangement of primers and probes is presented in figure 1.

The optimal composition of the reaction mixture for performing PCR with hybridization-fluorescence results was established experimentally. The optimal parameters of the amplification reaction with oligonucleotide primers and probe were selected. In particular, to perform PCR with hybridization-fluorescence results, the PCR mixture 1 contains primers RV5 and RV6, probe RV7, four deoxynucleotide triphosphates, sodium azide, nuclease-free water; reaction PCR mixture 2 contains 10 × Taq buffer with ammonium sulfate, magnesium chloride, nuclease-free water.

The mode of primer annealing and the effective number of amplification cycles were experimentally selected. Annealing of the primers is carried out at a temperature of 60 ° C for 10 cycles, then at 56 ° C for 35 cycles. The fluorescence analysis of the ROX channel is carried out at a temperature of 56 ° C by comparison with the PCR standards RV1, RV2, RV3, RV4, containing various amounts of target DNA and providing quantitative PCR.

The implementation of the proposed method is presented in the examples.

Example 1. Quantitative determination of the concentration of rabies virus strain "Moscow 3253" in the rabies antigen of organo-tissue origin.

Rabic antigen of organ-tissue origin is obtained from the brain suspension of rabbits, which previously introduced rabies virus strain "Moscow 3253". For analysis, 12 rabbit brain suspension samples were taken.

Determination of rabies virus concentration was carried out in several stages.

Disinfected in accordance with MU 1.3.2569-09 samples of a rabies antigen of organo-tissue origin were taken in 100 μl and transferred into 12 1.5 ml microcentrifuge tubes. Additionally, 100 μl of TE buffer (OKB) was added to a separate 1.5 ml microcentrifuge tube.

RNA isolation. For RNA isolation, we used a “kit of reagents for RNA / DNA isolation from the clinical material“ RIBO-sorb ”manufactured by the company (LLC“ InterLabService ”, Russia). The set of reagents for RNA isolation consists of a lyse solution, a solution for washing 1, a solution for washing 3, a solution for washing 4, a sorbent and an RNA buffer. In 12 microcentrifuge tubes with 100 μl of disinfected rabbit antigen of organo-tissue origin and 1 OKV tube, 450 μl of pre-heated lysing solution was heated at a temperature of (62 ± 2) ° C for 5-10 min and mixed thoroughly. Left in a tripod for 5-8 minutes, stirring 3-4 times. Centrifuged for 5 s at 3000-5000 rpm. The sorbent was carefully resuspended and 30 μl was added to each tube. Stirred, left in a rack for 1 minute, mixed again and left for 5 minutes. Tubes were centrifuged at 10,000 rpm for 45 s. The supernatant was removed, and 400 μl of washing solution 1 was added to the precipitate. The mixture was stirred until the sorbent was completely resuspended, centrifuged for 45 s at 10,000 rpm. After removal of the supernatant, 500 μl of washing solution 3 was added to the tubes. The sorbent was carefully resuspended and the tubes were centrifuged for 45 s at 10,000 rpm. After removal of the supernatant, the operation was repeated again. Then, 400 μl of washing solution 4 was added to the tubes, carefully resuspended, and centrifuged for 45 s at 10,000 rpm. The supernatant was removed and the tubes were placed in a thermostat at a temperature of 60 ° C for 10 min, while the lids were left open.

After drying, 50 μl of RNA buffer was added to each tube, mixed and placed in a solid state thermostat at a temperature of 60 ° С for 3 min. Stirred and centrifuged for 1 min at 13,000 rpm. The supernatant contains purified virus RNA.

Conducting a reverse transcription reaction.

To set up the reverse transcription reaction, a set of reagents was used to obtain cDNA on the Reverta-L RNA template (InterLabService LLC, Russia). The reagent kit for producing cDNA on an RNA template contains RT-G-mix-1, RT-mix-0.125 ml, revertase (MMLv) and DNA buffer.

Before starting work, tubes with RT-mix and RT-G-mix-1 (RT-G-mix-2) were removed from the freezer. After thawing the contents of the tubes at room temperature, they were thoroughly mixed and centrifuged for 5 s at 3000-5000 rpm to precipitate drops from the walls. To prepare the reaction mixture, 5 μl of RT-G-mix-1 (RT-G-mix-2) was added to the RT-mix tube, mixed thoroughly, centrifuged for 5 s at 3000-5000 rpm to precipitate drops the walls. To the resulting solution was added 6 μl of MMLv revertase, gently mixed and centrifuged for 5 s at 3000-5000 rpm to precipitate drops from the walls. 12 microcentrifuge tubes with a volume of 0.6 ml were selected, corresponding to the number of test samples and OKB. 10 μl of the resulting reaction mixture was added to each tube, then 10 μl of the RNA preparation and 10 μl of TE buffer in OKB were added to the tubes. The tubes were incubated in a thermocycler or solid state thermostat for 30 min at a temperature of 37 ° C. 20 μl of DNA buffer was added to all tubes and gently mixed 8-10 times. The obtained cDNA was used for PCR with hybridization-fluorescent results.

Arrangement of PCR.

0.2 ml microtubes containing PCR mixture-1, PCR mixture-2, TE buffer, Taq enzyme DNA polymerase with an activity of 5 units / μl, a set of PCR standards RV1, RV2, RV3 of various concentrations to determine high and low virus concentrations. The contents of the tubes were completely thawed.

PCR mixture-1 in a volume of 5 μl contains a solution of primers RV5, RV6 and probe RV7 in an amount of 8 and 4 pMol, respectively, 1 mMol of each of the four dNTPs, 0.025% sodium azide solution and nuclease-free water. PCR mix-1 was transferred 5 μl into 17 0.2 ml microcentrifuge tubes. In each tube, a molten 20% solution of paraffin in mineral oil was layered on the surface of the PCR mixture-1.

PCR mixture-2 in a volume of 10 μl, containing 2.5 × Taq buffer with ammonium sulfate, 6.25 mmol of magnesium chloride, Taq enzyme DNA polymerase, nuclease-free water.

The resulting PCR mixture-2 was mixed on a microcentrifuge / shaker and transferred to 10 μl in 17 microtubes with PCR mixture-1 without damaging the paraffin layer.

In 12 microtubes, 10 μl of a cDNA sample was added, in 3 microtubes 10 μl of each PCR standard RV1, RV2, RV3 was added, and 10 μl from an OKV sample containing TE buffer, the study of which is carried out with the analyzed samples, was introduced into 1 microtube starting at the stage of DNA extraction. This allows us to evaluate the absence of contamination at the stages of DNA isolation and reverse transcription. 10 μl of TE buffer was added to a 1 microtube with a negative amplification control (K-). It allows to evaluate the absence of contamination at the stage of amplification: control of the purity of the reagents and the absence of operator errors.

The prepared microtubes were placed in a RotorGene 6000 thermal cycler and PCR was carried out with hybridization-fluorescence results taking into account when the primers were annealed at a temperature of 60 ° C for 10 cycles, then at 56 ° C for 35 cycles. Accounting for fluorescence via the ROX channel was carried out at a temperature of 56 ° C.

PCR analysis is performed with disinfected material and takes 3.5-4.0 hours.

Accounting for results.

The rabies virus cDNA content in the test material was quantitatively assessed in the presence of a negative result in K- and OKV samples by the value of the signal fluorescence intensity by comparing the test sample with PCR standards RV1, RV2, RV3, designed to detect high concentrations. Quantification was determined using RotorGene 6000 thermal cycler software.

The concentration of fixed rabies virus strain Moscow 3253 in 12 test samples of the brain suspension of rabbits ranged from 2 × 10 ⁹ copies / ml to 2.81 × 10 ⁹ copies / ml (figure 2). The correlation coefficient amounted to 0.99708, the reaction efficiency - 0.89667.

Example 2. Quantitative determination of the concentration of rabies virus strain "Moscow 3253" in the rabbit antigen of cultural origin with a stationary method of growing.

For the study, 12 samples of rabies antigen obtained using the stationary method of growing Virus fixe on a Vero cell culture were taken. The stages of disinfection of an antigen of cultural origin, RNA isolation, reverse transcription reaction and PCR were carried out similarly to example 1, only when performing PCR, PCR standards RV2, RV3, RV4 were used, designed to detect low concentrations.

The concentration of fixed rabies virus strain Moscow 3253 in the studied samples ranged from 4.99 × 10 ² copies / ml to 7.59 × 10 ⁶ copies / ml (figure 3). The correlation coefficient amounted to 0.99893, the reaction efficiency - 0.96566.

Thus, the claimed method for the quantitative determination of the fixed rabies virus of the strain "Moscow 3253" by the method of polymerase chain reaction with hybridization-fluorescence taking into account the results in real time allows you to determine the quantitative content of the virus in the rabid antigen of organo-tissue and cultural origin and ensures simplicity and safety of work. Significantly reduces analysis execution time. The use of this method helps to standardize the stage of preparation of the rabbit antigen in the production of heterologous rabies immunoglobulin.

Literature

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10. Sato G. et al. 2005 Sato G., Tanabe H., Shoji Y. et al. Rapid discrimination of rabies viruses isolated from various host species in Brazil by multiplex reverse transcription-polymerase chain reaction // J. Clin. Virol. - 2005 .-- Vol.33 (4). - P.267-273.

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15. WHO Expert Consultation on Rabies 931, Geneva, 2004.

table 2 The nucleotide sequence of GenBankNCBI The number of polymorphic nucleotides in the genome of the virus compared to the sequence The total number of polymorphic nucleotides primer RV5 primer RV6 RV7 probe EU293115 one 2 four 7 AB645847 one 3 four 8 HQ450386 one four 3 8 JN234411 2 2 four 8 AB044824, AB128149 3 2 3 8 DQ875050 one four four 9 AB517659, EF437215 2 3 four 9 DQ875051 3 2 four 9 GQ918139 one 5 four 10 M31046, EF206717, EF206718, EF206720, EF206709, EF206708, EF206719, EF206707, FG913470, EU182346, EU 182347 2 6 3 eleven EF206710, EF206711 3 6 3 12 AF499686 2 6 four 12 EF542830 0 0 0 0

Claims (1)

A method for the quantitative determination of fixed rabies virus of the strain "Moscow 3253", including disinfection, isolation of RNA from virus-containing material, formulation of the reverse transcription reaction and polymerase chain reaction with hybridization-fluorescence real-time results using specific RV5-5'- primers GTTGGGCACTGAAACTGCTA-3 ', RV6-5'-GAATCTCCGGGTTAAAGAGT-3' and probe RV7-5'-ROX-AATCCTCCTTGAACTCCATGCGACAGA-BHQ2, providing amplification of the GL fragment of the genome of the fixed rabies virus from strain 32 from Moscow 32 aimers are carried out at a temperature of 60 ° C for 10 cycles, then at 56 ° C for 35 cycles, and the quantitative assessment of the virus is determined by recording the fluorescence signal of the test sample and comparing it with the fluorescence signal of PCR standards containing different amounts of target DNA .

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