Oligonucleotide primers for b.mallei genetic typing by polymerase chain reaction

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns oligonucleotide primers for B.mallei genetic typing. The presented primers are complementary to a differentiation fragment BMA0416 of an equinia agent genome, possess activity of upstream and downstream primers in an amplification reaction and have the following structure: 5'-TGGCGGAGTATGGATGCTG-3' - BmVAT6-Ch2s 5'-GAACGAGAACACCTACGACCTGAT-3' - BmVAT6-Ch2as.

EFFECT: presented invention may be used in practical healthcare for detection of the differentiation DNA fragment BMA0416 of the equinia agent.

3 dwg, 3 ex

 

The invention relates to biotechnology, molecular biology, molecular epidemiology, and can be used in medicine to detect differential DNA fragment WMA in the scheme genotyping of the causative agent of glanders Burkholderia mallei as for practical health, service, Rospotrebnadzor, and for scientific research.

The causative agent of glanders (Burkholderia mallei) is an aerobic gram-negative non-fermentative bacterium belonging to the genus Burkholderia and related to potential agents of bioterrorism group C. the SAP of especially dangerous zoonotic infection. The need for research aimed at genotyping of strains .mallei, the persistence of the threat of a disaster or biological situations, due to the occurrence of man-made disasters, natural disasters and potential terrorist attacks with the use of this agent.

Genotyping - a comprehensive analysis unique to each living organism genotype based on the study of its DNA. Method for genotyping on the basis of the analysis variable amplicons (VAT - variable amplicon typing) is a series of polymerase chain reactions with primers flanking fragments unique DNA targets, existing only in certain strains, which allows for intraspecific differentiation.

Method floor is Mersey chain reaction is a direct method of detecting target DNA segments. The method is based on PCR lies the natural process of DNA replication is complementary completing DNA matrix, carried out by the enzyme DNA polymerase.

The process of doubling the nucleic acids can be used for copies of a short DNA segments that specific strains of specific microorganisms, i.e. to carry out a targeted search for such specific areas, which is the purpose of genotyping causative agent of glanders.

For efficient PCR required primers synthetic oligonucleotides of a certain size that are specific to certain strains of the causative agent of glanders. Primers complementary to DNA sequences on the left and right boundaries of the differentiating portion and are oriented so that the finished construction of a new DNA chain flows only between them. In the PCR is a manifold increase in the number of copies (amplification) of a specific gene, catalyzed by the enzyme DNA polymerase. The choice of a specific fragment and selection of primers plays a crucial role in the specificity of carrying out amplification, which affects the quality of the analysis of the studied microorganisms.

The closest analogue is the scheme genotyping using the differential amplification of fragments for who is odites melioidosis, proposed Kwanjit Duangsonk al. in 2006 [Use of a Variable Amplicon Typing Scheme Reveals Considerable Variation in the Accessory Genomes of Isolates of Burkholderia pseudomallei, Kwanjit Duangsonk, Daniel Gal, Mark Mayo, C.Anthony Hart, Bart J.Currie, and Craig Winstanley]. For the agent of glanders these earlier studies were not conducted.

The aim of the present invention to provide oligonucleotide primers to identify differentiating genome fragment WMA .mallei by polymerase chain reaction.

The objective is achieved by designing specific oligonucleotides for the identification of variable DNA fragment strains of the causative agent of glanders, which has active forward and reverse primers in the amplification reaction having the following structure:

5'-TGG CGG AGT ATG GAT GCT G-3'-BmVAT6-Ch2s

5'-GAA CGA GAA CAC CTA CGA GAT CCT-3'-BmVAT6-Ch2as

Characterization of oligonucleotide primers and plot amplificare DNA.

Based on the data presented in the GenBank NCBI (National Center for Biotechnology Information, USA), primers were selected, marked BMVAT6-CH2S/BMVAT6-CH2AS, complementary differentiating fragment VMAA for intraspecific typing .mallei. The estimated length of the specific fragment is 606 BP

Testing of primers and probe was carried out on a set of strains of the causative agent of glanders collection centre (yrc Volgograd scientific research anti-plague Institute.

Examples of Kratovo execution.

Example 1. The method of designing oligonucleotide primers for detection of differentiating fragment WMAA the DNA of the causative agent of glanders by the method of Poland.

On the basis of theoretical study sequenced the nucleotide sequences of the four strains of the causative agent of glanders, present in the databases (EMBL, Genbank, DDBJ), for design of primers was selected variable sequence WMA detected in silico in the first chromosome of strains .mallei NCTC 10229, .mallei NCTC 10247 and .mallei ATCC 23344. In the genome of strain .mallei SAVP1 this fragment was not detected. This leads us to consider the sequence WMA as differentiating fragment suitable for genotyping of the causative agent of glanders. The estimated length of the DNA fragment, flankiruemogo offer primers - 606 BP

When using computer programs, we have analyzed the structure of the selected primer pairs (formation of dimers, hairpins and other secondary structures) and shows their theoretical suitability for the successful initiation of the amplification reaction.

Example 2. Amplification of specific DNA fragments using designed primers for intraspecific differentiation of causative agent of glanders.

In the composition of the reaction mixtures consisted of complementary specific fragment primers, deoxyribo nucleosidase, buffer solution and the enzyme Taq polymerase. To prevent evaporation in the process of amplification on multicyclone "Terzic" on the surface of the mixture was layered 20 ál of mineral oil. For the negative control in a test tube instead of a sample made of the same volume of distilled water. Amplification for a period of 40 cycles were carried out in microcentrifuge tubes (0.5 ml) at multicyclone "Terzic" (JSC "APF of DNA technology", Moscow) in a volume of 25 µl using a "hot start".

The conditions of the reaction: initial denaturation of DNA at 95°C for 5 min, then for 40 cycles denaturation of DNA at 95°C, 10 sec; annealing of primers at 60°C, 10 sec; chain elongation at 72°C for 10 sec, with a final polymerization for 1 minute

Analysis of PCR products was performed by electrophoresis in 3% agarose gel by comparing their mobility with the mobility of the bands of the molecular weight markers (figure 1). The specificity of the band of amplified DNA was confirmed by its position in relation to the control marker fragments (ladder 100 BP DNA Interlabservice LLC", Moscow) and DNA (DNA isolated from .mallei C-5 at a concentration of 1×105M.K./ml).

Example 3. The use of oligonucleotide primers BMVAT6-CH2S/BMVAT6-CH2AS in the scheme of differentiation of strains of the causative agent of glanders on the Museum strains collectible is the center of live cultures Volgograd scientific research anti-plague Institute.

The specificity of the amplification reaction with designed specific primers were evaluated in the study samples of DNA isolated from bacterial suspensions of cells grown on solid nutrient media in 4 ml of 0.15 M NaCl at a concentration corresponding to 1×109M.K./ml standard sample turbidity gisk named after. Laurasia (CCA 42-28-85 P). Disinfection of the material was performed in accordance with MU 1.3.1794-03 and MU 3.5.5.1034-01.

Disinfection of the investigated samples produced by adding a solution of thimerosal sodium to a final concentration of 0.1% and heating for 40 minutes at a temperature of 56°C. the isolation of DNA from pure cultures Burkholderia was performed using the method of nucleosil on silica gel in the presence of guanidinoacetate (R.Boom et al. - 1990). The formulation of the PCR reaction was carried out as described in example 2.

When testing culture collections .mallei Volgograd scientific research anti-plague Institute developed using oligonucleotide primers for the amplification product was synthesized with DNA of all studied strains of the causative agent of glanders (Fig.2).

Despite the fact that the primers VAT6-Ch2 not differentiated studied Museum strains .mallei, they were allowed to differentiate strains of the causative agent of glanders, is represented in the genetic databases that testified to their principal who is agnosti use for genotyping .mallei. On the basis of the analysis variable amplicons, we have developed a genotyping scheme of the causative agent of glanders, consisting of 9 reactions of amplification. The combination of positive and negative PCR results forms VAT-patterns that are unique to each strain of the causative agent of glanders (fig.3a).

Thus, using the designed primers BMVAT6-CH2S/BMVAT6-CH2AS in the scheme of intraspecific differentiation of causative agent of glanders method VAT, managed to split the 18 investigated strains .mallei 15 types (Fig.3).

Oligonucleotide primers for genotyping .mallei by polymerase chain reaction with active forward and reverse primers in the amplification reaction having the following structure:
5'-TGGCGGAGTATGGATGCTG-3'-BmVAT6-Ch2s
5'-GAACGAGAACACCTACGACCTG-3'-BmVAT6-Ch2as,
complementary differentiating fragment of the genome of the causative agent of glanders WMA.



 

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