RU2439159C1 - OLIGONUCLEOTIDE PROBE FOR IDENTIFICATION OF CAUSATIVE AGENTS OF GLANDERS AND MELIOIDOSIS B. pseudomallei AND B. mallei - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: described is oligonucleotide probe with complementary end sequences by type of "molecular beacon", ensuring fluorescent detection in identification of causative agents of glanders and melioidosis B. pseudomallei and B. Mallei by method of polymerase chain reaction with oligonucleotide primers BTTS-sl/BTTS-as3, complementary to fragment of gene 23S pRNA B. pseudomallei and B. mallei: 5'-(FAM)-CGCGCACCGGCAGTGATGAGCCACGCGCG-(RTQ1)-3', where FAM is carboxyfluorescein, fluorescent dye, whose absorption wavelength constitutes 492 nm, and fluorescence wavelength is 520 nm, RTQ1 is fluorescence quencher with range of quenching 470-570 nm. ^ EFFECT: application of hybridisation probe makes it possible to identify causative agents of glanders and melioidosis in short term with high sensitivity and specificity in biological material and environment objects. ^ 3 ex

Description

The invention relates to biotechnology, molecular biology and can be used in medicine to identify the genetic material of glanders and melioidosis pathogens B. pseudomallei and B. mallei in samples both for diagnosis in practical health care and the Rospotrebnadzor service, and for scientific research.

The causative agent of glanders (Burkholderia mallei) and the causative agent of melioidosis (Burkholderia pseudomallei) are aerobic gram-negative non-fermenting bacteria belonging to the genus Burkholderia. Melioidosis is an infectious disease in humans and animals endemic to Australia and a number of Southeast Asian countries. Sap is a particularly dangerous zoonotic infection.

The polymerase chain reaction method is a direct method for detecting the DNA of these pathogenic burkholdery. And it has high specificity and sensitivity. The PCR method is based on the natural process of DNA replication - the complementary completion of DNA, a matrix, carried out using the enzyme DNA polymerase.

The nucleic acid doubling process can be used to obtain copies of short sections of DNA specific for specific microorganisms, i.e. to carry out a targeted search for such specific sites, which is the purpose of gene diagnostics to identify pathogens of glanders and melioidosis.

For effective PCR, primers are needed - synthetic oligonucleotides of a certain size, specific for each type of pathogen. The primers are complementary to the DNA sequences on the left and right borders of a specific fragment and are oriented in such a way that the completion of a new DNA chain proceeds only between them. As a result of PCR, there is a multiple increase in the number of copies (amplification) of a specific gene region catalyzed by the enzyme DNA polymerase. The choice of a specific fragment and the selection of primers plays a crucial role in the specificity of the amplification, which affects the quality of the analysis of the studied microorganisms.

The use of special fluorescent labels eliminates the stage of electrophoresis, which reduces the risk of cross-contamination with PCR products and, accordingly, reduces the number of false positive results. Since the results are recorded directly during the amplification reaction, the entire analysis can be carried out in one or two rooms of the laboratory by one employee. This approach allows automatic interpretation of the results and removes the problem of subjective assessment of electrophoregrams. There are many fluorescence technologies that differ in how reporter fluorescence is generated. In this work, probes with complementary terminal sequences of the molecular beacons type were used.

The closest analogue is the specific primers for the TTS1 gene cluster, proposed in standard PCR format for detection of the causative agent of melioidosis in clinical samples (blood, sputum, joint fluid, skin, pericardium) (Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis; Wuthiekanun V., Anuntagool N., White NJ et al .; Am. J. Trop. Med. Hyg. - 2002. - Vol.66. - P.759-761). The test system was 100% specific, but low sensitivity, especially when examining blood samples in contrast to the culture method.

R. Novak et al. (Development and evaluation of a real-time PCR assay targeting the type III secretion system of Burkholderia pseudomallei; Novak R.T., Glass M.V., Gee JE et al .; J. Clin. Microbiol. - 2006. - Vol.44. - P.85-90) described a faster and more sensitive real-time PCR method based on the orf2 target DNA, from a secretion type III gene cluster (TTS1), which detected 100% specificity culture of strains of B. pseudomallei.

An object of the present invention is to provide highly specific oligonucleotide primers and fluorescently-labeled probes for the identification of B. pseudomallei and B. mallei by polymerase chain reaction.

The goal is achieved by the construction of specific oligonucleotides for the identification of DNA of glanders and melioidosis pathogens with direct and reverse primer activity in the amplification reaction, having the following structure:

5'-GACTGACCGCGCTCAAAGCCG-3'-BTTS-s1

5'-TCCGGCCTGACAAACGTTTGG-3'-BTTS-as3

Fluorescence detection is carried out using the designed oligonucleotide probe with complementary terminal sequences of the type of "molecular beacon":

5 '- (FAM) -CGCGCACCGGCAGTGATGAGCCACGCGCG- (RTQ1) -3'

Where FAM is carboxyfluorescein, a fluorescent dye whose absorption wavelength is 492 nm and the fluorescence wavelength is 520 nm. RTQ1 is a fluorescence quencher with a quenching range of 470-570 nm.

Characterization of oligonucleotide primers, probe and amplified DNA site.

Based on the data presented in the GenBank database of NCBI (National Center for Biotechnology Information, USA), primers designated BTTS-s1 / BTTS-as3 complementary to the orf13 gene fragment from the secretion type III gene cluster (TTS1) were identified for B.pseudomallei identification and B.mallei. The estimated length of the specific fragment was 162 bp

As a positive control, experiments were carried out on a typical strain of B. pseudomallei 100, using disinfected microorganism suspensions at concentrations from 1 × 10 ⁹ m.k. / ml to 1 × 10 ¹ m.k. / ml for DNA extraction. Testing of the primers and probe was carried out on a set of strains of glanders and melioidosis pathogens of the collection center of the MLC of the Volgograd Research Anti-Plague Institute.

The sensitivity of the amplification reaction with end-point fluorescence detection with BTTS-s1 / BTTS-as3 primers was evaluated by studying DNA samples isolated from ten-fold dilutions of pure cultures of glanders and melioidosis pathogens, and amounted to 1 × 10 ³ mk / ml. When using real-time PCR, the sensitivity of the reaction was increased to 1 × 10 ² m.k. / ml.

To detect glanders and melioidosis pathogens by PCR with fluorescence detection, the possibility of using designed primers and probes for the analysis of biological material (blood, liver, spleen, lungs and lymph nodes) and in experimental infections was evaluated. It was shown that in the amplification reaction the pathogen was detected in organ suspensions from all golden hamsters infected with the culture of these microorganisms at all observation times.

Examples of specific performance.

Example 1. The method of designing oligonucleotide primers and a hybridization probe for identification of DNA pathogens glanders and melioidosis by PCR.

Based on a theoretical study of the sequenced nucleotide sequences of glanders and melioidosis pathogens present in the databases (EMBL, Genbank, DDBJ) for primer design, the orf13 gene sequence was selected from the secretion type III gene cluster (TTS1), whose size is 29814 bp (GenBank NCBI, GeneID: 14719861). The estimated length of the DNA fragment flanked by the proposed primers is 162 bp

When selecting probes, they were guided by the general requirements for oligonucleotide primers used in PCR. Using computer programs, the structure of the selected pairs of primers and probes (the formation of dimers, hairpins, and other secondary structures) was analyzed and their theoretical suitability for the successful initiation of the amplification and hybridization reaction was shown.

The primers and probe were also analyzed using the BLAST computer program on the web server of the National Center for Biotechnological Information (NCBI) (bttp: //www.ncbi.nlm.nih.gov/BLAST/) to establish homology between them and the nucleotide sequences of closely related burkholderies and heterologous microorganisms present in the databases (EMBL, GenBank, DDBJ). At the time of the computer analysis, no homology was detected.

Example 2. Amplification of specific DNA fragments using the designed primers and probe for the identification of DNA pathogen glanders and melioidosis was carried out in two formats: with the detection of results of the NDP "at the end point" and in real time.

The reaction mixtures included oligonucleotide probes complementary to the specific fragment labeled with FAM fluorophore and fluorescence quencher (RTQ1), as well as primers, deoxyribonucleoside triphosphates, buffer solution and Taq polymerase enzyme. The probe for internal control was used to test the test systems for specificity and in the analysis of DNA isolation methods. To prevent evaporation during amplification on the Tertsik multicycle, 20 μl of mineral oil was layered on the surface of the mixture. In PCR with detection at the "end point" used 5 μl of the sample, with real-time detection - 10 μl. For negative control, the same volume of distilled water was added to the tube instead of the sample.

Amplification lasting 45 cycles was carried out in microcentrifuge tubes (0.5 ml) on a Tertsik multicycle (ZAO NPF DNA Technology, Moscow) and on Rotor-Gene 6000 devices (Corbett Research, Australia) and "SmartCycler" (Cepheid, USA) in real time in microcentrifuge tubes (0.2 ml) in a volume of 25 μl using a "hot start".

The analysis of PCR products was carried out using the Gene fluorescence detector (NPF DNA Technology CJSC, Moscow) at the end point and in real time, Rotor-Gene 6000 (Corbett Research, Australia) and SmartCycler (Cepheid, USA). The results were recorded in tabular and graphical form using computer programs. In real time, the results were analyzed by the presence or absence of intersection of the fluorescence curve with the threshold line, which is determined by the value of the threshold cycle "Ct" in the corresponding column in the table of results.

At the same time, PCR products were analyzed by gel electrophoresis in a 1.5% agarose gel with staining of DNA fragments with ethidium bromide and visualization in UV light. The results were evaluated by the presence or absence in the gel after electrophoresis of DNA fragments of the required size.

The specificity of the amplified DNA band was confirmed by its position in relation to the control marker fragments (100 bp DNA leader, Interlabservis LLC, Moscow) and the DNA standard (DNA isolated from B.mallei 10230 or B. pseudomallei 100 at a concentration of 1 × 10 ⁵ m.k. / ml).

Example 3. Determination of the sensitivity and specificity of the amplification reaction using the developed oligonucleotide primers and probe for identifying DNA of glanders and melioidosis pathogens.

The sensitivity of the amplification reaction with the developed specific primers and probe was evaluated by studying DNA samples isolated from bacterial suspensions of cells grown on solid nutrient media in 4 ml of 0.15 M NaCl at a concentration corresponding to 1 × 10 ⁹ MK / ml according to standard model turbidity GISK them. L.A. Tarasevich (OSO 42-28-85 P), and raised to the required dilutions (from 1 × 10 ⁹ to 1 × 10 m.k. / ml). From a dilution of 1 × 10 ³ MK / ml, a control culture of 0.1 ml of each sample was made on Petri dishes with BHI agar to determine the number of CFU. Disinfection of the material was carried out in accordance with MU 1.3.1794-03 and MU 3.5.5.1034-01.

The disinfection of the test samples is carried out by adding a solution of sodium merthiolate to a final concentration of 0.1% and heating for 30 minutes at a temperature of 56 ° C. Isolation of DNA from pure cultures of burkholderia is carried out using the method of nucleosorption on silica gel in the presence of guanidine thiocyanate (R.Boom et al. - 1990). The PCR reaction is carried out as described in Example 2. When testing the culture collection of B.mallei and B. pseudomallei of the Volgograd Research Anti-Plague Institute using the developed oligonucleotide primers and probe, the amplification product was synthesized from DNA of all strains of glanders and melioidosis with a sensitivity of 1 × 10 ² -1 × 10 ³ m.k. / ml. With other types of closely related burkholderies and heterologous microorganisms in the PCR reaction with the developed primers, a negative result was obtained in 100% of cases.

Thus, the developed BTTS-s1 / BTTS-as3 primers and the probe can be used to identify glanders and melioidosis pathogens and allow to quickly detect glanders and melioidosis pathogens in biological material and environmental objects with high sensitivity and specificity.

Claims (1)

An oligonucleotide probe with complementary terminal sequences of the “molecular beacon” type, which provides fluorescence detection for identification of glanders and melioidosis pathogens B. pseudomallei and B. mallei by polymerase chain reaction with oligonucleotide primers BTTS-s1 / BTTS-p3n, as3 B.pseudomallei and B.mallei:
5 '- (FAM) -CGCGCACCGGCAGTGATGAGCCACGCGCG- (RTQ1) -3',
where FAM is carboxyfluorescein, a fluorescent dye with an absorption wavelength of 492 nm and a fluorescence wavelength of 520 nm; RTQ1 is a fluorescence quencher with a quenching range of 470-570 nm.