RU2699180C1 - Set of oligonucleotide primers and a fluorescent-labeled probe for identifying rnas of burkholderia mallei sap excretion and burkholderia pseudomallei melioidosis based on transcription amplification (nasba) in real time mode - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention refers to biotechnology and molecular biology. Disclosed is a set of oligonucleotide primers and a fluorescent-labeled probe for identifying RNA of Burkholderia mallei sap excretion agents and Burkholderia pseudomallei melioidosis based on NASBA transcription amplification in real time.EFFECT: invention can be used for detection of RNA exciter of Burkholderia mallei sap and Burkholderia pseudomallei melioidosis in samples of pure cultures, biological material and environmental objects for health needs, service of Rospotrebnadzor and in scientific research.1 cl, 1 dwg, 1 tbl, 3 ex

Description

The invention relates to the field of biotechnology, molecular biology, and can be used to detect RNA pathogen glanders Burkholderia mallei and melioidosis Burkholderia pseudomallei in samples of pure cultures, biological material and environmental objects by specialists of institutions of both practical health care, the Rospotrebnadzor service, and in scientific research.

In connection with the development of transport links between countries, the expansion of tourist destinations, as well as significant migration flows, there is wariness regarding the importation into the territory of the Russian Federation of new or “forgotten” pathogens for the country, which include glanders and melioidosis. An increase in the number of confirmed cases of melioidosis is noted not only in the regions of Southeast Asia and Northern Australia endemic for this infection, but also in several countries of Europe, North and Latin America. The incidence of glanders in humans is rare, mainly professional in nature, but in a number of countries, including those adjacent to the Russian Federation (Mongolia, Turkey, Iran, etc.), outbreaks of the disease in animals continue to be recorded. It is possible that these pathogens, which belong to microorganisms of pathogenicity groups I-II, can be used as potential agents of bioterrorism. The variety of clinical manifestations of glanders and melioidosis significantly complicate the diagnosis and timely appointment of etiotropic therapy of these dangerous diseases. The combination of these factors determines the need for research aimed at improving laboratory methods for identifying pathogenic burkholderii.

Currently, among molecular genetic methods for diagnosing various infections, isothermal amplification of nucleic acids is increasingly used, due to their ability to detect the genetic material of a microorganism at a concentration lower than the lower limit of detection of polymerase chain reaction (PCR). Among these methods is the transcription-based amplification of nucleic acids NASBA (Nucleic Acid Sequence-Based Amplification). High sensitivity of the method is achieved by using as a target target molecules of matrix RNA (mRNA) or ribosomal RNA (rRNA) of microorganisms, the number of which can reach several tens of thousands per bacterial cell. In PCR, even multi-copy DNA regions used as targets do not exceed two tens per bacterial cell. Therefore, using the NASBA method, pathogens can also be detected in cases where their number is very small and insufficient for detection by PCR. This is especially true with a latent course of infection, against the background of the absence of clinical signs of the disease. Another important property of RNA as an amplification target is its lower stability compared to a DNA molecule, which determines the possibility of using the NASBA method to control the effectiveness of the therapy.

The NASBA transcriptional amplification method is based on the detection of an RNA fragment under isothermal conditions (at a temperature of 41 ° C) using two specific primers and the joint enzymatic activity of three enzymes: reverse transcriptase of avian myeloblastosis virus {avian myeloblastosis virus reverse transcriptase - AMV RT), RNase H Escherichia coli and RNA polymerase of phage T7. In this case, an exponential accumulation of single-stranded RNA occurs, which is a convenient target for hybridization detection methods. The use of fluorescently-labeled probes allows you to register a specific amplification product directly in the reaction process in real-time mode, which significantly reduces the analysis time, reduces the risk of contamination and increases the specificity of the method.

Real-time NASBA transcriptional amplification technology has been used to identify highly dangerous pathogens, such as Vibrio cholerae [Fykse EM, Skogan G., Davies W., et al. (2007) Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification. Appl. Environ. Microbiol. 73 (5), 1457-1466] and a number of RNA viruses - a highly virulent strain of the AH5N1 influenza virus, human immunodeficiency virus (HIV-1), SARS coronavirus, St. Louis encephalitis virus, hepatitis C virus, rabies virus, and also viruses dengue, West Nile and Chikungunya fever [Sidoti F., Bergallo M., Costa C, Cavallo R. (2013) Alternative molecular tests for virological diagnosis. Mol. Biotechnol. 53 (3), 352-362]. However, diagnostic reagents based on the NASBA transcriptional amplification reaction to detect B. mallei and B. pseudomallei have not been developed.

The closest analogues are panbacterial primers and a probe designed on the basis of the nucleotide sequence of the 23 S ribosomal RNA gene and designed to detect the target fragment of the bacterial nucleic acid by the NASBA method in real time in order to monitor the microbiological quality of the recirculated water aboard the international space station [Bechy -Loizeau AL, Flandrois JP, Abaibou N. (2015) Assessment of polycarbonate filter in a molecular analytical system for the microbiological quality monitoring of recycled waters onboard ISS. Life Sci. Space Res. (Amst). 6, 29-35]. However, the lack of determination of the species specificity of bacteria using the oligonucleotides used in the above prototype excludes the possibility of their use for identification of glanders and melioidosis.

The aim of the present invention is to develop a set of highly specific oligonucleotide primers and a fluorescently labeled probe for identification of B. mallei and B, pseudomallei by NASBA transcription amplification in real time.

The goal is achieved by creating a set of oligonucleotides for identifying the RNA of glanders and melioidosis pathogens, containing a pair of highly specific oligonucleotide polymers to the target sequence of the 23 S rRNA gene, with forward and reverse primer activity in the NASBA transcriptional amplification reaction, and with a fluorescently-labeled probe with complementary sequences molecular beacon ”, providing detection of reaction products in the“ real time ”mode, having the following structure:

5'-AAT TST AAT ACG ACT CAC TAT AGG GAG ACG GCT AAC AAT ACA AAT AAA GAG TA-3 '- Burk23SrRNA-T7-P1

5'-CCT TTT GGG TCA TCC TAG A-3 '- Burk23SrRNA-P2

5 '(FAM) -CCA CAC ATA GGT STA GTG AGG CGT GTG G- (RTQ-1) 3' - Burk23SrRNA-MM

where FAM is carboxyfluorescein, a fluorescent dye with an absorption wavelength of 490 nm and a fluorescence wavelength of 520 nm. RTQ-1 is a fluorescence quencher with a quenching range of 470-570 nm.

Characterization of oligonucleotide primers, probe and RNA target for hybridization.

Based on the data presented in the international GenBank database of the US National Center for Biotechnological Information (https://www.ncbi.nlm.nih.gov/genome), primers designated Burk23SrRNA-T7-P l / Burk23SrRNA-P2, complementary to the gene 23S pRNA and providing the amplification of a specific fragment of ribonucleic acid of 159 bp in the NASBA reaction

To detect NASBA amplification products in real-time mode, a molecular beacon probe, designated Burk23SrRNA-MM, was constructed at different ends of which a fluorophore and fluorescence quencher are located. Such a probe structure provides the maximum quenching effect and low background fluorescence, since the fluorophore and quencher molecules are brought together in space.

Testing of the developed set of oligonucleotides was carried out on a set of strains of glanders and melioidosis pathogens from the collection of FKUZ Volgograd Research Anti-Plague Institute of Rospotrebnadzor. The reference strains of B. mallei 10230 and B. pseudomallei C-141 were used as a positive control in the experiments. RNA was isolated from disinfected bacterial suspensions of microorganisms in concentrations from 1 × 10 ⁹ m.k. / ml to 1 × 10 ⁰ m.k. / ml. The specificity of amplification was further confirmed by sequencing.

Real-time NASBA reaction conditions were optimized using commercially available reagents, enzymes and devices intended for mass use in laboratory practice, which allows for the rapid and reliable use of this invention in medical and research laboratories.

Examples of specific performance.

Example 1. The method of designing oligonucleotide primers and fluorescently-labeled hybridization probe to identify RNA pathogens glanders and melioidosis by NASBA in real time.

Based on the in silico study of the structure of the ribosomal gene cluster (16SpPHK, 23SpPHK, 5S rRNA) as part of the sequenced nucleotide sequences of glanders and melioidosis pathogens, presented in the GenBank NCBI genetic database, the 23S rR9 gene was selected for constructing primers and probes (GeneID: 31 BPSLr08)) (tab. 1).

A pair of primers Burk23SrRNA-T7-P1 and Burk23SrRNA-P2, the nucleotide sequence length of which was 53 and 19 nucleotides, respectively, were selected for the synthesis of the target 23 S rRNA fragment by NASBA transcriptional amplification. The GC content of the hybridizing parts of the target primers was 32% for the Burk23SrRNA-T7-P1 primer and 47.4% for the Burk23SrRNA-P2 primer. When designing the Burk23SrRNA-T7-P1 primer to form the functional promoter of the T7 RNA polymerase, a sequence of 25 nucleotides was added to the 5'-terminal region. Between the promoter region of the Burk23SrRNA-T7-P1 primer and its portion hybridizing to the target, three purine nucleotides were additionally added to improve amplification. The estimated size of the 23S rRNA gene fragment flanked by the proposed primers is 159 bp

To detect NASBA reaction products in real-time mode, a Burk23SrRNA-MM probe in the molecular beacon format has been constructed, which is potentially capable of hybridizing with amplified 23 S rRNA targets of glanders and melioidosis pathogens with the formation of stable hybrids at a temperature of 41 ° C. Fluorescent labeling of the probe was carried out by combining the FAM dye at the 5'-end and the RTQ-1 quencher at the 3'-end. The designed fluorescently-labeled probe was a stem-loop-structured oligonucleotide and had 6 mutually complementary nucleotides in the stem region, while the loop size was 16 nucleotides. The secondary structure and thermodynamic parameters of the developed probe were evaluated online using the Mfold Web Server (https://mfold.rna.albany.edu).

Preliminarily, the specificity of the developed primers and probe was evaluated in silico by multiple sequence comparison using the BLAST NCBI resource to establish homology between them and the nucleotide sequences of closely related species of the genus Burkholderia and heterologous microorganisms. At the time of the computer analysis, no homology was detected.

Example 2. Amplification and detection of specific fragments of 23S rRNA using the developed set of primers Burk23SrRNA-T7-P1 / Burk23SrRNA-P2 and fluorescently labeled probe Burk23SrRNA-MM to identify causative agents of glanders and melioidosis by NASBA in real time.

For real-time NASBA with the developed Burk23SrRNA-T7-P1 / Burk23SrRNA-P2 primers and the Burk23SrRNA-MM oligonucleotide probe, it is necessary to separately prepare the enzyme solution and the reaction mixture containing all other reaction components.

The reaction mixture with a volume of 12.3 μl (per sample) included the following components: 40 mM Tris-HCl (pH = 8.5), 1 mM each of deoxynucleoside triphosphates (Fermentas, USA), 2 mm each of ribonucleoside triphosphates (Fermentas ”, USA), 12 mM MgCb (Dialat Ltd, Russia), 87 mM KCl (Sigma-ALDRICH, Germany), 0.5 mM DTT (Fermentas, USA), 15% DMSO (ArrNset, Germany) ), 0.125 μM forward and reverse primers, and 0.0625 μM fluorescently-labeled probe. The resulting reaction mixture was kept at 65 ° C for 5 minutes.

An enzyme solution with a volume of 2.7 μl (per sample) included the following components: 2.1 μg BSA (Fermentas, USA), 0.08 active units of RNase H (Thermo Scientific), USA, 6.4 active units of AMV Reverse Transcriptase (" Promega ", USA), 32 active units of T7 RNA Polymerase (" Thermo Scientific)), USA).

12.3 μl of the prepared reaction mixture and 5 μl of RNA extract of the analyzed samples were added to 0.2 ml microcentrifuge tubes, after which they were thermostated in the following mode: 65 ° С for 5 min, 41 ° С for 5 min. At the end of the incubation, an enzyme solution was added and the tubes were installed in an amplifier. As a negative control, RNA buffer was added to the reaction mixture.

The real-time NASBA transcriptional amplification reaction was performed on a Rotor-Gene Q instrument (QIAGEN GmbH, Germany) under isothermal conditions: 41 ° C for 5 min, then 95 cycles (41 ° C for 60 s) s detection of the intensity of the fluorescent signal.

The results were recorded in graphical and tabular form. The amplification product of the 23SpPHK region of B. mallei and B. pseudomallei for the FAM fluorophore was detected via the Green channel. The result was considered positive if the fluorescence accumulation curve for the corresponding sample had a characteristic "sigmoid" shape and crossed the threshold line set at a certain level, which corresponds to the presence of the threshold cycle value "Ct" in the corresponding column in the results table.

Example 3. Determination of the sensitivity and specificity of the reaction of transcriptional amplification of NASBA in real time using the developed set of oligonucleotide primers Burk23SrRNA-T7-P l / Burk23SrRNA-P2 and fluorescence-labeled probe Burk23SrRNA-MM for RNA identification of B. mallei and B. pseudomallei.

The sensitivity of the NASBA transcriptional amplification reaction with the developed set of Burk23SrRNA-T7-Pl / Burk23SrRNA-P2 oligonucleotide primers and the Burk23SrRNA-MM fluorescently labeled probe was evaluated by studying ten-fold dilutions of RNA preparations obtained from pure cultures of sap pathogens and sap. Burkholder strains were grown on solid nutrient media for 1-2 days. Bacterial cell suspensions were prepared in 4 ml of a 0.15 M sodium chloride solution at a concentration of 1 × 10 ⁹ MK / ml according to the industry standard sample of turbidity of 10 units of FSBI NTsESMP (OSO 42-28-85-P (10ME).

The preliminary inactivation of the samples and the isolation of nucleic acids were carried out under the conditions regulated by SP 1.3.3118-13 “Safety of work with microorganisms of pathogenicity groups I-II” and guidelines of MU 1.3.2569-09 “Organization of the work of laboratories using nucleic acid amplification methods during operation with material containing microorganisms of I-IV pathogenicity groups. " The procedure for isolating nucleic acids from the test material was carried out using the RIBO-prep kit (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) according to the manufacturer's instructions. The formulation of the NASBA reaction in real time was carried out as described in example 2.

RNA concentration was measured using a commercial Qubit ™ RNA HS Assay Kit (Invitrogen, USA) and a QUBIT 2.0 fluorimeter (Invitrogen, USA). The amount of total RNA was determined in samples of pure cultures of B. mallei and B. pseudomallei, isolated from a concentration of 1 × 10 ⁸ MK / ml, and then sequential ten-fold dilutions of nucleic acid samples were performed.

As a result, the sensitivity of the “real-time” NASBA transcriptional amplification reaction with the developed set of oligonucleotides was determined, which amounted to 0.4 fg / μl of total RNA in the sample, which corresponded to 10 ¹ μm / ml (Fig. 1).

Figure 1 shows a graph of the increase in fluorescence curves obtained by amplification of RNA of B. mallei 10230 and B. pseudomallei C-141 strains using the designed primers Burk23SrRNA-T7-P1 / Burk23SrRNA-P2 and the Burk23SrRNA-MM probe (1 - B. mallei 102 concentration of 10 ² mk / ml, 2 - B. pseudomallei С-141 at a concentration of 10 ² mk / ml, 3-B. mallei 10230 at a concentration of 10 ¹ mk / ml, 4 - V. pseudomallei C-141 at a concentration of 10 ¹ mk./ml, 5 - negative control).

The specificity of the developed set of oligonucleotides was evaluated on a collection of 58 strains of microorganisms, of which 14 strains of B. mallei, 20 strains of B. pseudomallei and 24 strains of heterologous microorganisms (12 strains of Burkholderia cepacia, 5 strains of Burkholderia thailandensis, and 1 strain of Pseudomonasasensugensaensugensaensugensaensugensa aerugensa Pseudomonas putida, Francisella tularensis, Yersinia pestis, Bacillus anthracis, Echerichia coli). Assessment of specificity showed the absence of cross-reactions with heterologous strains and the presence of specific amplification with all strains of B. mallei and B. pseudomallei.

Thus, the developed set of oligonucleotide primers Burk23SrRNA-T7-P1 / Burk23SrRNA-P2 and a fluorescently labeled probe Burk23SrRNA-MM can be used to identify glanders and melioidosis by NASBA transcription amplification in real time with a short time and allows sensitivity and specificity to detect RNA of B. mallei and B. pseudomallei in samples of pure cultures, biological material and environmental objects.

Claims (5)

A set of oligonucleotide primers and a fluorescently-labeled probe for identification of RNA pathogens of glanders Burkholderia mallei and melioidosis Burkholderia pseudomallei based on transcriptional amplification (NASBA) in real time, containing a pair of highly specific oligonucleotide g polymers having the direct sequence and 23R polymers with the target p sequence primers in the NASBA transcriptional amplification reaction, and a fluorescently-labeled probe with complementary terminal sequences of the type of "molecular beacon", both sintering detection of reaction products in the "real time" mode, having the following structure: 5'-AAT TCT AAT ACG ACT CAC TAT AGG GAG ACG GCT AAC AAT ACA AAT AAA GAG TA-3 '- Burk23SrRNA-T7-P1 5'-CCT TTT GGG TCA TCC TAG A-3 '- Burk23SrRNA-P2 5 '(FAM) -CCA CAC ATA GGT CTA GTG AGG CGT GTG G- (RTQ-1) 3' - Burk23SrRNA-MM, where FAM is carboxyfluorescein, a fluorescent dye with an absorption wavelength of 490 nm and a fluorescence wavelength of 520 nm. RTQ-1 is a fluorescence quencher with a quenching range of 470-570 nm.

Images