RU2465332C1 - Synthetic oligonucleotides kit for dna detection of polyporus squamosus fungus - hardwood disease agent by polymerase chain reaction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents an synthetic oligonucleotides kit for DNA detection of the Polyporus squamosus fungus - a hardwood disease agent by polymerase chain reaction. Conducting a polymerase chain reaction with the use of said kit enables the reliable detection of DNA of the Polyporus squamosus fungus agent causing white wound heart rot in a biological material, particularly in protecte ground, seeds, plants, bark of elm, maple, linden, oak, poplar, birch and other hardwood.

EFFECT: invention may be used by phytoquarantine services for the purpose of instant diagnosing of the phytopathogens in PCR laboratories, as well as for laboratory purposes.

1 cl, 1 dwg

Description

Technical field

The invention relates to biotechnology, molecular genetic diagnosis of phytopathogens of the forest and is a set of synthetic oligonucleotides for detecting DNA of a causative agent of hardwood diseases - the fungus Polyporus squamosus by polymerase chain reaction. The invention can be used by phyto-quarantine services for express diagnostics of a phytopathogen in PCR laboratories and for research purposes.

State of the art

Polyporus squamosus (scaly tinder fungus) is a fungus that causes white wound sound rot of many hardwoods (elm, maple, linden, oak, poplar, birch and others). The fruit bodies of the fungus are annual, in the form of hats with slightly curved edges and most often a side leg. The surface of the cap is slightly depressed, yellowish, with large brown scales. The skin is fleshy, brown, with a black base. The tissue of the fruiting body in the fresh state is white, soft, yellowing and crumbles upon drying. The hymenophore is tubular, with very large, in the form of cells, angular, often split pores.

Infection of the trunk occurs through wounds in the cortex, freezing and other injuries. Rot, caused by a scaly tinder fungus, is sound, but extends to sapwood. It develops most often in the lower part of the trunk, sometimes it goes to the roots. In the final stage, the rot becomes white, often with dark lines, and numerous cracks appear in it, going in different directions, clusters of white mycelium form in the cracks.

A scaly tinder fungus is a typical wound parasite and is especially common in damaged old stands, forest parks and other stands with a high recreational load.

For effective control of fungi by forest phytopathogens, accuracy of determining a specific pathogen to a species is of the greatest importance. Recently, the practice of research includes the method of polymerase chain reaction (PCR). To this end, on the basis of PCR, we have developed methods for the molecular genetic identification of phytopathogenic fungi. The application of this approach in combination with the classical methods of taxonomy can significantly increase the speed of determination and achieve almost 100% accuracy of determination.

The ITS region is the most sequenced region in fungi. It is widely used in the molecular taxonomy of fungi at the species level and can serve as a marker even for intraspecific determination (for example, in geographically separated intraspecific types or strains).

Since these regions are not directly related to RNA coding, they are actively changing during evolution, which creates the prerequisites for using them as markers of evolutionary events.

At the same time, this region has been poorly studied in the fungus Polyporus squamosus; in the literature and patent databases we have not found primers selected for this region.

The implementation of the invention

Work on the creation of primers that are used in such sets is usually constructed as follows.

1. Using open and commercial databases of nucleotide sequences of the genomes of microorganisms, or as a result of independent determination of the nucleotide sequence of the studied microorganism, a portion of the genome is selected that is unique to this type of microorganism (or group of species). In this case, based on the analysis of the bases of the relevant sequences, we did not find, therefore, the ITS region of this fungus was first sequenced on an automatic sequencer. The sequence of the sequence is presented in Fig. 1.

2. Based on the sequenced portion of the genome using special software selects the sequence of oligonucleotides used to carry out the PCR reaction (2 primers). For this, the sequenced sequence is aligned with homologous sequences (closely related fungi) and primers are selected that are unique to the desired sequence.

3. The manufacture of primers is ordered in a special service center.

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

The present invention allows to detect the DNA of the phytopathogenic fungus Polyporus squamosus, which causes white wound core rot of many hardwoods (elm, maple, linden, oak, poplar, birch and others). However, similar kits, as well as reaction mixtures, primers and methods for detecting the DNA of this fungus, are not known.

The technical result, the achievement of which the present invention is directed, is the reliable detection of the indicated fungus in biological material (protected soil, seeds, plants, bark).

The specified result is achieved by using a set of synthetic oligonucleotides for PCR to identify DNA of the pathogen of vegetable diseases - the fungus Polyporus squamosus, which includes primers:

5'-TGT ATT GTG ATG TAA CGC ATS G-3 '

5'-TCC TCC GCT TAT TGA TAT GC-3 '

The specified set of synthetic oligonucleotides is an integral part of the set of the following substances.

1) Test tubes with a reaction mixture sealed with paraffin. This mixture contains the indicated primers and a sample specific to the site of the fungus Polyporus squamosus — ITS region, a mixture of four types of deoxyribonucleotide triphosphates, reaction buffer (100 mM Tris-HCl (pH 8.8 at 25 ° C), 500 mM KCl, 0.8% P40, 20 mM MGCl ₂ ), internal control sample (VK).

Primers are synthetic oligonucleotides. Primers specific for the marker region of the pathogen genome are introduced into the reaction directly for amplification (production) of the product. An internal control sample (VK) is a DNA sequence introduced into the reaction to evaluate the effectiveness of its course. The accumulation of VC is independent of the production of a specific product using separate primers and samples.

2) Taq polymerase enzyme solution.

3) A positive control sample — DNA of a recombinant plasmid with a cloned fragment of 453 bp the genome of the fungus Polyporus squamosus. It is introduced into the test tube (by analogy with the test samples) in order to be able to compare the results obtained in experimental tubes with how it should be obtained with a “positive reaction”. Accordingly, the DNA samples added to the remaining tubes are experimental.

4) Negative control sample - a sample that is introduced into the experiment to control possible contamination of reagents with products of previously conducted reactions. A positive result in this sample indicates the need to replace the reagents and rearrange the experiment.

This set is applied as follows.

1) Biological material (protected soil, seeds, plants, bark) before PCR using the proposed set of reagents is carried out through the sample preparation procedure using another set (a set of reagents for sample preparation is not the subject of this patent); during this procedure, DNA is extracted from the biological material, which in turn is used for PCR.

2) The required number of tubes with the prepared reaction mixture containing specific synthetic oligonucleotide primers and a sample is labeled according to the number of analyzed samples.

3) Taq polymerase solution is added to all labeled tubes without damaging the paraffin layer.

4) All labeled tubes (except K- (negative control sample), K + (positive control sample)) contain DNA isolated according to item 1).

5) A negative control sample is introduced into the tube labeled K-.

6) A positive control sample is added to the tube labeled K +.

7) All tubes are installed in the amplifier unit, amplification is carried out according to the following program: 1 cycle 95 ° C 5 minutes, 35 cycles 94 ° C 45 seconds, 55 ° C 45 seconds, 72 ° C 45 seconds, 1 cycle 72 ° C 10 minutes . Storage 4 ° C. Amplifiers of any manufacturers can be used.

8) Analysis of the reaction products is carried out using gel electrophoresis of the reaction products in a 1% agarose gel with the addition of ethidium bromide. A strip of 453 bp in length is visualized. using a transilluminator.

A positive PCR result is largely dependent on the quality of the DNA isolated from the test material. To solve the problem of isolating DNA from forest phytopathogenic fungi for the purpose of subsequent molecular genetic identification, it is best to use freshly collected samples or dried herbarium material.

It should be understood that a person skilled in the art can, without further experimentation, taking into account the present description, apply the present invention in practice in its entirety. Therefore, the description of the invention given in this context is presented for illustration, not reducing the scope of claims.

Claims (1)

A set of synthetic oligonucleotides for detecting DNA of a pathogen of hardwood diseases (elm, maple, linden, oak, poplar, birch and others) of the fungus Polyporus squamosus, which causes white wound core rot, by the polymerase chain reaction, characterized in that it includes primers:
5'-TGT ATT GTG ATG TAA CGC ATS G-3 '
5'-TCC TCC GCT TAT TGA TAT GC-3 '

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