Synthetic oligonucleotide kit for identifying human monocytic ehrlichiosis ehrlichia spp agent dna by real-time polymerase chain reaction

FIELD: medicine.

SUBSTANCE: invention refers to a synthetic oligonucleotide kit for identifying DNA of human monocytic ehrlichiosis (HME) agent - pathogenic representatives of Ehrlichia genus by a polymerase chain reaction. The offered invention can be used for the diagnostic purposes for detecting monocytic ehrlichiosis Ehrlichia spp. by the real-time polymerase chain reaction. Said kit includes primers as follows: 5'- GGG GAA AGA TTT ATC GCT ATT AG -3', 5'- CGG CAT AGC TGG ATC AGG CT -3' and a sample: (BHQl)-5'- CCC ACT GCT GCC (FdT)CC CGT AGG AGT CTG G - 3'P, where BHQ1 means a dark fluorescence killer attached to 5'-terminal nucleotide, while FdT is a fluorescent dye FAM attached to nucleotide T.

EFFECT: invention allows reliable identification of Ehrlichia representatives in the biological material.

1 ex

 

1. The technical field

The invention relates to biotechnology, in particular genetic engineering, and can be used for diagnostic purposes to detect macrophage of ehrlichiosis Ehrlichia spp. the polymerase chain reaction "in real time", and to address the research objectives for the study of macrophage pathogen of erlichiosis. Received highly specific primers, allowing to detect the DNA of Ehrlichia spp. when conducting a polymerase chain reaction in real time from various clinical samples. Selected unique oligonucleotides do not give cross-reactions with related species permit, limited to single-stage procedure PCR, quickly and reliably determine the presence or absence of the DNA of Ehrlichia spp.

2. The level of technology

The prior art, the following analogs of this invention are:

You know the invention under the application №2003121605 (published 27.01.2005), which is set to identify enterohemorrhagic Escherihia coli. The invention relates to biotechnology and can be used to identify E. coli serotypes O:H7, o:H11, O:H7, O:N8, O:n, O:H14. The set consists of immunomagnetic separation test systems and test systems for the production of a multiplex polymerase chain reaction (PCR). Immunomagnetic separating the t-system contains media - magnetic latex particles and sensitin - protein a covalently associated with a specific rabbit immunoglobulins to polysaccharide fractions FSC O, o, O, O, O, O and protein flagellar antigens H7, H11, H8, n, H14. The set consists of six groups of 0.5% suspension of magnetic particles of latex protein-polysaccharide complex. Test-system for PCR buffer contains "master mix" with specific primers for the identification of genes stx1, stx2, rfb, fliC Enterohaemorrhagic E.coli. The set can be used for laboratory diagnosis of haemorrhagic colitis, haemolytic uraemic syndrome, as well as to identify enterohemorrhagic E. coli in foods. This invention involves carrying out PCR reactions involving reagents included in this kit. In particular, using the following primers specific for the target bacteria, which can detect the DNA of this bacterium in PCR:

Rfb for 5'-CAGGTGAAGGTGGAATGGTTG-3'

Rfb rev 5'-GAGTACATTGGCATGGTGTGG-3'

stx2 for 5'-ATCAGCAATGTGCTTCCGGAG-3'

stx2 rev 5'-CTGAGCACTTTGCAGTAACGG-3'.

The result found Enterohaemorrhagic Escherihia coli.

Also known similar invention "Method and kit for detecting a DNA of bacteria diseases Rimskogo borreliosis - borrelia burqdorferi" (application No. 2004105688 published 10.08.2005). The method of detecting DNA of bacteria diseases Rimskogo the Borra is Lisa-rrli burgdorferi, including the isolation of DNA from biological material, the synthesis of primers and setting polymerase chain reaction using synthetic primers, characterized in that when setting polymerase chain reaction is used to detect the DNA of bacteria in cell cultures, samples of peripheral blood and the grip of two primers:

BBs14.F/BBs14.R

-5'-AAGAATACATTAAGTGCGATATT-3',

5'-CAATCCACTTAATTTTTGTGTTAT-3',

encoding 16S rRNA, which is a component of ribosomes. The method, characterized in that the annealing of the primers is carried out at a temperature of 51-52°C for 10-30 C.

The method, characterized in that the number of cycles of amplification is 35-42 P.

Set for detecting a DNA of bacteria diseases Rimskogo borreliosis - Borrelia burgdorferi, including thermostable enzyme Tag polymerase, the buffer for the given reaction, a mixture of four deoxynucleotides, the positive control recombinant plasmid containing a fragment of the gene flagellin bacteria, and oil for polymerase chain reaction, characterized in that it contains primers BBs14.F/BBs14.R

-5'-AAGAATACATTAAGTGCGATATT-3',

5'-CAATCCACTTAATTTTTGTGTTAT-3', encoding 16S rRNA, which is a component of ribosomes, and 5-fold buffer for the given reaction.

Also the prior art "a Set of reagents for determination of DNA periodontopathogenic microbes Prevotella intermedia sensu stricto, Bacteroide forsythus, Treponema denticola, and Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis using multiplex polymerase chain reaction" (application No. 2005136997 published 10.06.2007). The invention relates to molecular biology and can be used in medicine. The proposed set of definitions for DNA periodontopathogenic microbes Prevotella intermedia sensu stricto, Bacteroides forsythus, Treponema denticola, and Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis using multiplex polymerase chain reaction kit includes reagents for sample preparation kit for amplification containing primers specific to the DNA of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Treponema denticola, Bacteroides forsythus, Prevotella intermedia sensu stricto, positive and negative control samples, and a set of analysis products. The use of the invention provides simultaneous, rapid, simple and accurate detection of 5 types of periodontopathogenic in one sample.

Also the prior art the following analogues of the present invention: patent # US 2002192672 "Defining a new variety ehlrichia spp. from a patient infected monocytic ehrlichiosis".

've got a new kind of Ehrlichia spp. from a patient infected monocytic ehrlichiosis. New found flavor was supposed to be like .canis, but clearly differed from E. canis. This invention describes methods and diagnostic kit for detection of monozy the ary erlichiosis in biological material of human, and describes the drug, which can be used to treat a new kind of Ehrlichia spp.

Human monocytic arlais is a new disease characterized by symptoms such as fever, headache, malaise, etc.

This invention "a Set of synthetic oligonucleotides for detection of the DNA of the causative agent of human monocytic srihitha Ehrlichia spp. the polymerase chain reaction "in real time"is a set of specific oligonucleotide primers, complementary to the conservative region of the genome of the bacteria Ehrlichia spp. - gene 16S RNA and used for DNA detection of bacteria Ehrlichia spp. PCR "real-time". The use of synthetic oligonucleotides to amplify a region of the genome of the bacteria Ehrlichia spp. polymerase chain reaction (PCR) in real time allows you to achieve maximum sensitivity and specificity of the analysis, i.e. the ability to detect a single copy of the DNA of pathogens erlichiosis person in the presence of DNA of other organisms and the DNA of the host body. The set is intended for use in clinical diagnosis and medical practice, its application provides fast, easy and accurate detection of the pathogen in a biological material (ticks, plasma samples of cervicalgia).

Task

The creation of synthetic oligonucleotides to identify the DNA of the causative agent of human monocytic ehrlichiosis EHRICHIA SPP. the polymerase chain reaction "in real time".

3. Disclosure of inventions

Bacteria of the genus Ehrlichia - small gram-negative polymorphic bacteria, characterized obligate parasitism.

Arlais human - natural focal, vector-borne infection that is caused by the intracellular microorganisms of the genus Ehrlichia and proceeds in the form of an acute febrile illness.

This kit is intended for use in medical diagnosis to identify the DNA of the causative agent of human monocytic ehrlichiosis (MACH) pathogenic members of the genus Ehrlichia.

The subject of the invention are specific oligonucleotide primers, complementary to the conservative region of the genome of the bacteria Ehrlichia spp. - gene 16S RNA and used for PCR. Their use as part of a set of reagents for PCR provides a rapid, simple and accurate detection of the pathogen in a biological material (ticks, samples of human blood plasma).

The set represents a set of synthetic oligonucleotides to amplify a region of the genome of the bacteria Ehrlichia spp. polymerase chain reaction (PCR) in real time.

Work on the creation of the primers used in these sets, is constructed generally as follows.

1) using open source and commercial databases of nucleotide sequences of the genomes of microorganisms either by self-determination of the nucleotide sequence of the studied microorganism selected region of the genome that is unique to this species (or species groups).

2) based On the selected region of the genome with the help of special software selected sequence of oligonucleotides used for PCR reactions (often 2 primer and probe).

At this stage the work is to create alignment of many genomic sequences and the selection of the sequence, where the number of mutations corresponds to the desired location of the primers.

Alignment of the genomic sequences means the comparison of the sequences of many organisms with each other, the unique search for the desired microorganism areas that are unmatched by any other microorganisms.

The match between the number of mutations and the location of primers means that the mutation is possible in this microogranisms, should not affect the selected primers for a region of the genome replacement in the genomic sequence (mu the purpose) may adversely affect the work of the primers.

3) the Manufacture of primers are ordered in a special service center.

4) using practical experiments proved the suitability of the selected sequences for specific purposes (for example, to determine the presence/absence of this microorganism in the biomaterial).

The present invention can detect the DNA of the causative agent of human monocytic ehrlichiosis (MACH) pathogenic members of the genus Ehrlichia.

Similar sets, as well as the reaction mixture, the primers and methods of detecting DNA of this bacterium is unknown.

The technical result, which is aimed by the invention, is the reliable detection of specified bacteria in biological material (Ixodes ticks, blood plasma of a person).

This result is achieved by the use in the formulation of PCR of a set of synthetic oligonucleotides for detection of the DNA of the causative agent of human monocytic ehrlichiosis (MACH) pathogenic members of the genus Ehrlichia, including primers:

5'- GGG GAA AGA TTT ATC GCT ATT AG -3'

5'- CGG CAT AGC TGG ATC AGG CT -3'

and try: (BHQ1)-5'- CCC ACT GCT GCC (FdT)CC CGT AGG AGT CTG G -3'P, where BHQ1 means attached to the 5'-terminal nucleotide dark quencher of fluorescence, a FdT - fluorescent dye FAM attached to the nucleotide T.

The specified set of synthetically the oligonucleotides is an integral part of the set of the following substances:

1) the Tubes with the reaction mixture, sealed with paraffin. This blend contains the specified primers and probe specific to the DNA of bacteria of the genus Ehrlichia - gene 16S RNA, a mixture of deoxyribonucleotides four types of reaction buffer (100 mm Tris-HCl (pH 8.8 at 25°C), 500 mm KCl, 0.8% R40, 20 mm MgCl2), the internal control sample (VK).

And the primers and probe are synthetic oligonucleotides. Primers specific to the marker region of the genome of the pathogen, are introduced into the reaction directly for amplification (operating time) of the product. Sample (too specific to a given region of DNA) is composed of a fluorescent label, a fluorescence intensity which indicates the amount of product formed, which in turn depends on the efficiency of the primers, the number of the starting material and other parameters of the reaction. In other words, the primers provide for the reaction, the sample is developing its results.

Internal control sample (VK) is a DNA sequence introduced into the reaction for evaluating the effectiveness of it. Time VK is independent from developments specific product using specific primers and samples.

2) Solution of the enzyme Taq polymerase.

3) a Positive control sample DNA recombinant plasmids with klonirovaniya size 198 BP the genome of the bacteria of the genus Ehrlichia. It is placed in a test tube (similar to the analyzed samples) in order to be able to compare the results obtained in the experimental test tubes, how it should be when "positive reaction". Accordingly added to the remaining tubes DNA samples are experienced.

4) a Negative control sample is a sample that is injected in the experiment to control for possible contamination of reagents products previously conducted reactions. A positive result for this sample demonstrates the need to replace the reagents and to rearrange the experiment.

The proposed set is different in that the synthetic primers and fluorescently-labeled probe specific to a marker for the exciter section of DNA, have the following nucleotide composition:

5'- GGG GAA AGA TTT ATC GCT ATT AG -3'

5'- CGG CAT AGC TGG ATC AGG CT -3'

(BHQ1)-5'- CCC ACT GCT GCC (FdT)CC CGT AGG AGT CTG G -3'P

BHQ1 means attached to the 5'-terminal nucleotide dark quencher of fluorescence, FdT means of the fluorescent dye FAM attached to the nucleotide T.

Oligonucleotide primers and fluorescently-labeled sample contained in the reaction mixture, which also includes a mixture of deoxyribonucleotides four types of reaction buffer (100 mm Tris-HCl (pH 8.8 at 25°C), 500 mm KCl, 0.8% R40, 20 mm MGCl2 ), the internal control sample (VK) and the enzyme Taq polymerase (2.5 u per sample).

This reaction mixture was added to the DNA sample and carry out PCR. In the case of formation of a specific product DNA sample is destroyed, which leads to an increased level of fluorescence, which is fixed by special devices - detection amplifiers.

Internal control sample (VK) is introduced into the reaction mixture to evaluate the effectiveness of the course of PCR. Samples used for detection of amplification products, the desired DNA and the internal control sample, in the state of fluorescent-labeled FAM and HEX, respectively, which enables you to separately record the results of amplification of specific NDP-product and VK.

Registration of the fluorescence signal is held by the device automatically during amplification.

4. The implementation of the invention

1) Biological material (ticks, samples of human blood plasma) before carrying out PCR using the proposed set of reagents is carried out through the procedure of sample preparation using a different set of reagents for sample preparation, is not the subject of this patent); in this procedure, from the biological material excreted DNA, which in turn is used for PCR;

2) the Required number of tubes prepared with actionnow mixture, containing specific synthetic oligonucleotide primers and a probe, labeled according to the number of samples;

3) all labeled test tubes without damaging the layer of wax is added to a solution of Taq polymerase;

4) all labeled tubes (except tubes To the-(negative control sample)To+ (positive control sample)) shall be allocated according to claim 1 DNA;

5) In a test tube, labeled K-recorded negative control sample;

6) To the tube labeled C+, making a positive control sample;

7) All tubes are installed in the unit amplifier, the amplification is carried out according to the modes prescribed in the instructions to the set. Detection results is carried out by detecting amplifier automatically during amplification (unit: amplifiers detecting DT-322, DT-96 (JSC "APF of DNA-Technology").

Analysis of results is carried out in accordance with the instructions to the device.

In the case of formation of a specific product DNA sample is destroyed, which leads to an increased level of fluorescence, which is fixed by special devices - detection amplifiers.

A set of synthetic oligonucleotides for detection of the DNA of the causative agent of human monocytic ehrlichiosis (MACH) pathogenic predstavitelyami Ehrlichia by polymerase chain reaction, characterized in that it includes primers:
5'- GGG GAA AGA TTT ATC GCT ATT AG -3'
5'- CGG CAT AGC TGG ATC AGG CT -3'
and try: (BHQ1)-5'- CCC ACT GCT GCC (FdT)CC CGT AGG AGT CTG G - 3'P, where BHQ1 means attached to the 5'-terminal nucleotide dark quencher of fluorescence, a FdT - fluorescent dye FAM attached to the nucleotide T.



 

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