RU2535064C1 - SET OF SYNTHETIC OLIGONUCLEOTIDES FOR SPECIES IDENTIFICATION OF SPINY ELEUTEROCOCUS, ELEUTHEROCOCCUS (Eleutherococcus senticocus (Rupr. et Maxim.) Maxim.) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of molecular genetics, genetic systematics and pharmacognosy, and is intended for species identification of spiny eleuterococus, eleutherococcus (Eleutherococcus senticocus (Rupr. Et Maxim.) Maxim.). The set of synthetic oligonucleotides for PCR with fragment ITS2 of nuclear DNA is proposed, comprising forward and reverse primers and a destructible probe.

EFFECT: set has a high sensitivity and specificity and enables to identify quickly and reliably the medicinal plant.

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Description

The invention relates to the field of molecular genetics, genosystematics and pharmacognosy. The use of a set of synthetic oligonucleotides allows one to reliably identify the species of a medicinal plant - a prickly free berry, Eleutherococcus senticocus (Rupr. Et Maxim.) Maxim.). The invention can be used to identify the species of this plant; during the screening of plant materials, to monitor the conformity of the composition declared by the manufacturer.

Among a large number of genetic diagnostic methods in order to identify the presence of DNA of a plant species of interest in a sample, the PCR format with real-time detection based on a destructible probe was adopted as the basis of our invention. Real-time PCR has an advantage over conventional PCR identification systems - there is no need for further analysis, which minimizes the risk of contamination in the laboratory. Hypersensitivity and the absence of a false positive result with non-specific annealing of primers are also characteristic. In addition, it should be noted that virtually all molecular genetic diagnostic laboratories are equipped with the equipment necessary for real-time PCR and the widespread use of the method in clinical diagnostics and state control bodies.

Based on the analysis of possible methods for detecting the product of the polymerase chain reaction, we chose a method based on a destructible probe based on the fact that it belongs to specific detection methods and the possibility of its free use without violating copyright and related rights (the system of destructible probe was originally proposed in 1991, with this all patents at the moment have expired).

The technical result of the claimed invention is the development of primers and destructible probes based on data of species-specific nucleotide sequences of plant nuclear DNA. As species-specific sites, we use the ITS2 fragment of nuclear DNA (internal transcribed spacer 2), which has a high copy number in the genome [Alvarez, I. & Wendel, J. F. (2003) Ribosomal ITS sequences and plant phylogenetic inference // Mol. Phylogenet. Evol. 29, 417-434] and used in the design of a DNA barcoding system. This region shows high variability and potential applicability as a marker site [Stoeckle, M. (2003) Use of DNA barcodes to identify flowering plants // Bioscience 53, 2-3].

Prototype - consider the identification of medicinal plants using the ITS2 fragment amplified with specific primers [Chiou SJ, Yen JH, Fang CL, Chen HL, Lin TY Authentication of medicinal herbs using PCR-amplified ITS2 with specific primers // Planta Med. 2007, Oct; 73 (13): 1421-6. Epub 2007 Oct 1]. The prototype uses specific sets of primers BEL-1 / BEL-3 and BEL-2 / BEL-3 to amplify the ITS2 region of the 55 rDNA of 55 medicinal plants.

The fundamental differences of the prototype from the claimed invention are as follows: a medicinal plant with nucleotide sequences of specific primers other than those declared is identified. A set of synthetic oligonucleotides for identifying the species of a medicinal plant - prickly free berries, eleutherococcus (Eleutherococcus senticocus (Rupr. Et Maxim.) Maxim.), Including polymerase chain reaction with a fragment of ITS2 nuclear DNA, where specific direct, reverse primers are used to identify this plant and a destructible probe, a DNA sequence with the following nucleotide composition is used as a direct primer: 5'-CGGCAACGGATATCTCGGCT-3 '; as a reverse primer, a DNA sequence with the following nucleotide composition: 5'-CGCACGACAGGAGAAGAGGGCTT-3 '; a DNA sequence with the following nucleotide composition is used as a destructible probe: (fluorescent label) -5'- CATCGAGTCTTTGAACGCAA-3 '- (quencher).

Work on the creation of primers is constructed as follows.

1. Using open and commercial databases of nucleotide sequences of various plant species, or as a result of independent determination of the nucleotide sequence of plants, a portion of the genome that is found in all species is selected.

2. Based on the selected region of the genome, using special software or manually selects the sequence of oligonucleotides used for the PCR reaction (2 primers and probe). At this stage, the work consists in creating the alignment of many sequences and selecting a portion of the sequence where differences are present to create forward, reverse primers and probe. Alignment of genomic sequences means comparing the sequences of many species with each other, searching for sites homologous to plants.

3. The manufacture of primers and probe is made on automatic synthesizers.

4. Using practical experiments, the suitability of the selected sequences for concrete purposes is proved.

Analysis of the nucleotide polymorphism of the ITS2 sequence based on the NCBI data (http://www.ncbi.nlm.nih.gov/) and de novo sequenced sequences of species that are not located in the genebank allows us to use these sequences as the basis for creating primers. For the detection of the accumulation of the PCR product during the reaction, a destructible probe technology is used (Holland PM, Abramson RD, Watson R, Gelfand D H. Detection of specific polymerase chain reaction product by utilizing the 5'-3 'exonuclease activity of Thermus aquaticus DNA polymerase .//Proc Natl Acad Sci US A. 1991 August 15; 88 (16): 7276-7280).

For example, FAM is used as a fluorescent label, BHQ1 as a quencher (other combinations of fluorophores and quenchers are possible and this is not subject to copyright).

Similar kits, reaction mixtures, primers for amplification and identification of species of the free spiny thorn, Eleutherococcus senticocus (Rupr. Et Maxim.) Maxim.) Are not known.

The progress using a set of synthetic oligonucleotides for amplification consists of the following steps.

Example

1. Plant material before PCR using the inventive kit is carried out through a sample preparation procedure using the Diamont DNA kit, in accordance with the manufacturer's instructions; during this procedure, DNA is extracted from the plant material, which in turn is used for PCR.

2. The polymerase chain reaction is carried out on a CFX96 thermocycler (Bio-Rad, USA). Amplification is carried out according to the following program: 1 cycle: 95 ° C - 3 min; 40 cycles: 95 ° C - 10 s, 58 ° C - 30 s (at this stage, the fluorescence level is scanned). The incubation mixture with a final volume of 25 μl contains: 14.1 μl of H ₂ O; 2 μl of DNA; 2.5 μl of 10X buffer; 2.5 μl 25 mM MgCl ₂ ; 1 μl of 10 mm each primer; 0.5 μl of probe; 1.2 μl of 20 mM dNTPs; 0.2 μl of Taq polymerase. The amplification result is determined by the increase in the fluorescence level, Fig. 1.

To identify the free thorny berry, Eleutherococcus (Eleutherococcus senticocus (Rupr. Et Maxim.) Maxim.), A DNA sequence with the following nucleotide composition is used as a direct primer: 5'-CGGCAACGGATATCTCGGCT-3 '; as a reverse primer, a DNA sequence with the following nucleotide composition: 5'-CGCACGACAGGAGAAGAGGGCTT-3 '; as a destructible probe, a DNA sequence with the following nucleotide composition is used: (fluorescent label) -5'-ATCGAGTCTTTGAACGCAA-3 '- (quencher).

The quencher is located at the 5'-end, and the fluorescent label at the 3'-end of the destroyed probes.

A new set of synthetic oligonucleotides has been developed to identify the species of a medicinal plant - a prickly free berry, Eleutherococcus senticocus (Rupr. Et Maxim.) Maxim.) - by real-time polymerase chain reaction method. The kit has high sensitivity and specificity, allowing quick and reliable identification of the considered medicinal plant.

Claims (1)

A set of synthetic oligonucleotides for detecting the species of a free thorny berry eleutherococcus (Eleutherococcus senticocus (Rupr. Et Maxim.) Maxim.), Including a polymerase chain reaction with a fragment of nuclear DNA ITS2, characterized in that for identification of a thorny eleutherococcus ( Rupr. Et Maxim.) Maxim.) Use species-specific sites to create direct, reverse primers and destructible probes, where as a direct primer is a DNA sequence with the following nucleotide composition: 5'-CGGCAACGGATAT CTCGGCT-3 '; reverse primer is a DNA sequence with the following nucleotide composition: 5'-CGCACGACAGGAGAAGAGGGCTT-3 '; destructible probe is a DNA sequence with the following nucleotide composition: (fluorescent label) -5'-CATCGAGTCTTTGAACGCAA-3 '- (quencher).

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