RU2744665C1 - Method for detecting lujo virus rna by reverse transcription and polymerase chain reaction, taking into account real-time results - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to medical diagnostics, specifically to a method for determining the RNA of the Lujo virus (a member of the family Arenaviridae) with the use of reverse transcription and a real-time polymerase chain reaction (RT-PCR) on the basis of biological material of people, animals and environmental objects and can be used in research and medical institutions, agencies of Rospotrebnadzor. The accumulation of the fluorescent signal along the JOE channel in the samples under study indicates the presence of the RNA of Lujo and the result is considered valid if the accumulation of the fluorescent signal of an internal control sample is observed in the same samples via the FAM channel.

EFFECT: invention constitutes a method for detecting the RNA of the Lujo virus which involves carrying out reverse transcription and PCR taking into account the results in real time using the appropriate primers and probes. After that one can detect the RNA of Lujo on the basis of the presence and absence of intersection of the fluorescence curve in each of the channels used with the threshold line set at the appropriate level.

1 cl, 5 ex, 1 tbl, 7 dwg

Description

The invention relates to medicine, in particular to laboratory diagnostics, specifically to a method for determining the RNA of the Luyo virus (family Arenaviridae) by the method of reverse transcription and polymerase chain reaction (RT-PCR) in real time in material from people, animals and from environmental objects environment, and can be used in research and medical institutions, services of Rospotrebnadzor.

In August-September 2008, cases of hemorrhagic fever were detected in South Africa, which was accompanied by fever, headache, vomiting, diarrhea, arthralgia and hemorrhages. Mortality was 80%, the main route of transmission of the pathogen (except for the primary one, where the source of infection was never identified) was contact. Subsequently, it was proved that the etiological agent of this disease is a previously unknown virus designated as Lujo (Lujo mammarenavirus (LUJV)) and belonging to the Arenaviridae family [1, 2]. It was assumed that the natural reservoir of the pathogen in nature is rodents of unknown species, which excrete the virus in the urine [3].

Due to the increase in the number of our compatriots visiting African countries for work or tourist purposes, a large number of foreign students studying in higher educational institutions of Russia, it is possible that the causative agent of Luyo hemorrhagic fever (GLF) will be brought into our country, which shows the need to develop diagnostic methods this infection.

The laboratory diagnosis of Luyo virus fever is based on molecular genetic research methods, which are characterized by high sensitivity and specificity. Thus, V. Atkinson et al. [4] developed primers (size 100 bp) and probes that allow the detection of Luyo virus RNA in various types of material by RT-PCR in real time. It was shown that the sensitivity was 10 ⁵ copies / ml, and the specificity was 100%. However, this method of detecting the RNA of the Luyo virus does not allow laboratory analysis in a laboratory, because there is no kit of reagents and a control panel for analysis and registration of the reaction quality.

A known method for detecting the RNA of the Luyo virus using polymerase chain reaction with hybridization-fluorescence detection [5]. However, in this method, there are no positive control samples (K +) and an internal control sample (ICR), which made it possible to assess the quality of the studies.

The applicant's analysis of data on the development of drugs for the detection of RNA of the Luyo virus, including a search for patents and scientific and technical sources of information, did not reveal information about other methods, methods and diagnostic drugs that would allow the indication and identification of the causative agent of HLL.

The objective of the invention is to develop a method for detecting RNA of the Luyo virus by the method of reverse transcription and polymerase chain reaction, taking into account the results in real time.

The technical result of the claimed invention is to provide fast and reliable detection of Luyo virus RNA in samples of clinical and biological material.

The technical result is achieved by the method of detecting the RNA of the Luyo virus by the method of reverse transcription and polymerase chain reaction, taking into account the results in real time, which makes it possible to obtain a result with high accuracy and in a short time (5-6 hours), and is also characterized by high sensitivity, specificity and the presence of controls at all stages of research (VKO, K +, PKO).

To accomplish the task of the claimed invention, the known genetic sequences of the Luyo virus presented in the GenBank international database (https://www.ncbi.nlm.nih.gov/genbarik/) were analyzed. At the first stage of the work, conservative regions of the genome were selected and determined. Two conserved fragments of the polymerase gene were selected as targets for detection. The design of oligonucleotide primers and fluorescent probes was carried out; the length of the detected fragments was 108 and 133 base pairs, respectively. Sequences of oligonucleotides and probes used for research:

SEQ ID NO: 1

SEQ ID NO: 2

The optimal composition of the reaction mixture for PCR was experimentally established, the combination of primers and probes, the necessary and sufficient ratio of the components of the reaction mixture, the mode of setting up RT-PCR were selected, and control samples K +, VKO, PKO were developed.

To control the quality of the passage of the stages of RNA isolation, reverse transcription and PCR, the following were developed: internal control sample (ICR), ICR and recombinant positive control samples (K +). Due to the lack of the genetic material of the Luyo virus, the template for the creation of K + was prepared by a synthetic method based on an amplicon, which includes the first diagnostic target region and flanking nucleotide sequences. Amplicon was obtained by step-out PCR. The final PCR product was ligated into the pGEM-T plasmid vector (Promega, USA) under the control of the T7 RNA polymerase promoter and transformed into Escherichia coli (strain XL 1-Blue). Recombinant plasmids from individual clones were checked for the correct orientation of the target sequence and the absence of mutations in the region of primer and probe landing. The verification was carried out by the Sanger method using an ABI PRISM 3500x1 automatic capillary sequencing device (Applied Biosystems, USA). Recombinant plasmids meeting the specified criteria were used to prepare a positive control sample of the PCR stage (K +). For this, the concentration of DNA in the solution of the recombinant plasmid was determined and diluted with DNA buffer to a working concentration of 1 × 10 ⁶ - 1 × 10 ⁷ copies / ml.

Recombinant plasmids were used to create a recombinant RNA-containing positive control with a protective envelope of the MS2 phage (PCO). For the obtained product, the concentration was also determined, then it was diluted with RNA buffer (Interlabservice LLC, Russia) to a working concentration of 1 × 10 ⁶ - 5 × 10 ⁷ copies / ml, which was used as a PKO preparation.

The analytical sensitivity of the developed technique was assessed on dilutions of an RNA-containing recombinant positive control sample (POC), from which RNA was extracted using a set for the isolation of nucleic acids "Ribo-Prep" (LLC "Interlabservice", Russia), and then using specific primers and probes determined the minimum dilution, detected as positive in 100% of cases. The analytical sensitivity of the method determined in this way was 5 × 10 ³ GE / ml.

The specificity of the method was assessed using an RNA panel of 23 types of viruses belonging to 10 different families, as well as 30 samples of clinical material (blood plasma) from clinically healthy people. As a result of the study, cross-reactions were not recorded.

A method for detecting Luyo virus RNA by reverse transcription and polymerase chain reaction, taking into account the results in real time, includes the following steps:

1) Sample preparation and RNA isolation;

2) Conducting RT-PCR;

3) Analysis of the results.

Preparation and disinfection of samples is carried out in accordance with MU 1.3.2569-09 "Organization of the work of laboratories using methods of amplification of nucleic acids when working with material containing microorganisms of I-IV pathogenicity groups". Research material can be clinical and biological samples, as well as samples from environmental objects. A negative control sample (OKO), PKO is added to the total number of investigated samples. The ICO is added to all tubes. Isolation of RNA is carried out using commercial kits "Ribo-prep" or "Ribo-Sorb" (LLC "Interlabservice", Russia) in accordance with the instructions for the preparations.

After the stage of RNA isolation, the assembly of the reaction mixtures is started. To do this, select the required number of microtubes corresponding to the number of samples under study, as well as one tube each for positive and negative control. To simplify and standardize the preparation of reagents, two reaction mixtures are prepared before starting PCR.

The first reaction mixture for each reaction includes:

- diagnostic primers and primers for VKO at a concentration of 0.5 pmol / μl;

- fluorescent probes at a concentration of 0.3 pmol / μl;

- a mixture of deoxynucleoside triphosphates (dNTP): dATP, dTTP, dGTP, dCTP at a concentration of 0.44 mM;

- sterile water to a volume of 10 μl.

The first reaction mixture can be prepared in advance and must be stored at a temperature not exceeding minus 18 ° C.

The second reaction mixture per reaction contains:

- 5x reaction buffer for enzymes with MgCl ₂ at a concentration of 15 mM - 5 μl;

- reverse transcriptase - 0.25 μl;

- dithiothreitol at a concentration of 1 mM - 0.25 μl;

- TaqF polymerase - 0.5 μl.

The second reaction mixture is prepared immediately before setting up the reaction and is not subject to storage.

For RT-PCR, take the required number of 0.2 ml microtubes corresponding to the number of samples under study, as well as two tubes for positive and negative controls. Next, according to the number of test samples, the reaction mixtures are mixed: 10 μl of the first and 5 μl of the second for each reaction. 15 μl of the resulting mixture is added to prepared test tubes, then 10 μl of RNA sample extracted from the test material is added.

Prepare 4 control reactions. To do this, 10 μl of K + is added to the tube for the positive control of PCR, 10 μl of the sample extracted from the PCR into the tube for the positive control of reverse transcription, 10 μl of sterile water to the tube for the negative control of PCR, and 10 μl of the sample extracted from the OKO. The final volume of the reaction mixture is 25 μl. The microtubes are transferred to a rotary-type RT-PCR device and amplified under the following temperature conditions:

1.reverse transcription at a temperature of 50 ° С - 15 min (1 cycle);

2. warming up at a temperature of 95 ° С - 5 min (1 cycle);

3.polymerase chain reaction (42 cycles): 95 ° С - 10 s, 60 ° С - 20 s.

The fluorescent signal is detected at 60 ° C using the Green (FAM) and Yellow (JOE) fluorophore channels.

3) Accounting for results

The accounting and analysis of the results is carried out using the software of the device based on the absence or presence of a fluorescence signal in the test samples through the detected channels. The interpretation of the reaction results is carried out in accordance with table 1.

If the obtained results do not correspond to the data in the table, the reaction must be rearranged starting from the stage of RNA extraction.

The claimed invention is illustrated by the following examples.

Example 1. Detection of Luyo virus RNA in human blood.

To identify the RNA of the Luyo virus, 265 blood serum samples from Guinean residents observed in medical organizations for fever of unknown etiology were examined. Samples were taken according to the generally accepted method in the amount of 5 ml. RNA was isolated from 100 μl of the sample, which was added to lysis buffer based on 6 M guanidine isothiocyanate in the volume specified in the instructions for the kit for RNA isolation, and incubated for 15 minutes at a temperature of (65 ± 1) ° C. Into all tubes, including the release control, 10 μL of the internal control sample (ICR) was added, and the mixture was heated for another 5 minutes at a temperature of (65 ± 1) ° С. Isolation of RNA was carried out using the kits "Ribo-prep" or "Ribo-Sorb" (LLC "Interlabservice", Russia) in accordance with the instructions for the preparations. As a result of the studies carried out, negative results were obtained in all cases. Figure 1 shows the results of a study of blood sera from people collected on the territory of the Republic of Guinea.

Example 2. Detection of Luyo virus RNA from tick suspensions

Ectoparasites were identified to species, combined into samples in accordance with the collection site, sex, stage of development, and degree of fatness. A total of 83 mite pools of 3 species were studied. Sample preparation was carried out in accordance with MU 1.3.2569-09. Then, RNA was isolated from 100 μl of the obtained samples, RT-PCR was carried out as described in example 1. The results were taken into account in real time by the intensity of accumulation of the fluorescent signal on a rotary device. No positive samples were found. An example of the research is shown in Fig. 2.

Example 3. Detection of Luyo virus RNA from organs of small mammals (lungs + kidneys, liver + spleen, brain) captured in the Republic of Guinea.

Sample preparation was carried out in accordance with MU 1.3.2569-09. Then, RNA was isolated from 100 μl of the sample and real-time PCR was performed with a reverse transcription step, as described in example 1. The results were recorded by the hybridization-fluorescence method. No positive samples were found. FIG. 3 shows the results of a study of suspensions of organs of small mammals (lungs + kidneys), Fig. 4 - the results of the study of suspensions of organs of small mammals (liver + spleen), Fig. 5 - results of investigation of suspensions of organs of small mammals (brain).

Example 4. Detection of RNA of Luyo virus in blood serum of cattle.

The blood serum of cattle collected on the territory of the Republic of Guinea was examined. In total, the software of the samples was investigated. Sample preparation was carried out in accordance with MU 1.3.2569-09. Isolation of RNA was carried out from 100 μl of the sample, followed by PCR in real time with a stage of reverse transcription and hybridization-fluorescent accounting of the results, in the same way as in example 1. No positive samples were found. The results of the study are shown in FIG. 6.

Example 5. Study of various biological samples, artificially contaminated with RNA-containing recombinant positive control sample (PCO)

5 samples of blood serum from healthy people, 5 blood serum of cattle, 5 samples of organ suspensions (liver + spleen) from small mammals (laboratory mice) were used as the test material, for which 10 μl of PCO was added to 100 μl of the sample at a concentration of 5 × 103 GE / ml. The stage of RNA isolation and the production of PCR in real time with the stage of reverse transcription and hybridization-fluorescent registration of the results was carried out in the same way as in example 1. In all cases, the result was positive. The results of the study are shown in FIG. 7.

BIBLIOGRAPHY

1. Paweska JT, Sewlall NH, Ksiazek TG, Blumberg LH, Hale MJ, Lipkin WI, Weyer J., Nichol ST, Rollin PE, McMullan LK, Paddock CD, Briese T., Mnyaluza J., Dinh T.N., Mukonka V., Ching P., Duse A., Richards G., de Jong G., Cohen C., Ikalafeng B., Mugero C., Asomugha C., Malotle MM, Nteo DM, Misiani E., Swanepoel R. , Zaki SR Nosocomial outbreak of novel arenavirus infection, southern Africa. Emerging Infectious Diseases, Vol. 15, No. 10, October 2009, doi: 10.3201 / eidl510.090211.

2. Briese T, Paweska JT, McMullan LK, Hutchison SK, Street C, Palacios G, Khristova ML, Weyer J, Swanepoel R, Egholm M, Nichol ST, Lipkin WI. Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa. PLoS Pathog. 2009 May; 5 (5): e1000455. doi: 10.1371 / journal.ppat.l000455. Epub 2009 May 29.

3. Ishii A, Thomas Y, Moonga L, Nakamura I, Ohnuma A, Hang'ombe BM, Takada A, Mweene AS, Sawa H. Molecular surveillance and phylogenetic analysis of Old World arenaviruses in Zambia. J Gen Virol. 2012 Oct; 93 (Pt 10): 2247-51. doi: 10.1099 / vir.0.044099-0. Epub 2012 Jul 18.

4. Atkinson B, Chamberlain J, Dowall SD, Cook N, Bruce C, Hewson R. Rapid molecular detection of Lujo virus RNA. J Virol Methods. 2014 Jan; 195: 170-3. doi: 10.1016 / j.jviromet.2013.09.006. Epub 2013 Oct 4.

5. Naydenova E.B., Dedkov V.G., Safonova M.B., Senichkina A.M., Agafonov D.A., Zakharov K.S., Shcherbakova S.A., Kutyrev V.V. Development of a method for detecting Luyo virus RNA using polymerase chain reaction with hybridization-fluorescence detection // Proceedings of the International Scientific and Practical Conference "Molecular Diagnostics-2018", Minsk, pp. 453-454.

Claims (9)

A method for detecting Luyo virus RNA by reverse transcription polymerase chain reaction with hybridization-fluorescence accounting of the results, providing for reverse transcription and PCR using primers and probes with a set of SEQ ID NO: 1

designed for the detection of an internal control sample (ICR), and a set of SEQ ID NO: 2

designed to detect specific fragments of the RNA of the Luyo virus with the subsequent assessment of the results by the presence and absence of the intersection of the fluorescence curve in each of the channels used with the threshold line set at the appropriate level: the accumulation of the fluorescent signal along the JOE channel in the samples under study indicates the presence of RNA of the Luyo virus, while the result is considered valid if, in parallel, the accumulation of the fluorescent signal of the ITP is recorded in the same samples via the FAM channel.

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