RU2573937C1 - SET OF SYNTHETIC OLIGONUCLEOTIDES FOR SPECIES IDENTIFICATION OF BUPLEURUM MULTINERVE (Bupleurum multinerve DC) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a set of synthetic oligonucleotides for species identification of Bupleurum multinerve (Bupleurum multinerve DC.), comprising polymerase chain reaction with a fragment ITS2 of nuclear DNA. And for identification species-specific portions are used to create the forward, reverse primers and destroyed probe, where the forward primer is the DNA sequence with the following nucleotide structure: 5'-TATCCCGGCTCTCGCATCGA-3'; the reverse primer is the DNA sequence with the following nucleotide structure: 5'-GACGAGGCACGGGAGGTCAG-3', the destroyed probe DNA sequence with the following nucleotide composition (fluorescent label)-5'-CCGTGAACCATCGAGTTTTT-3'-(quencher).

EFFECT: invention enables the species identification of Bupleurum multinerve during screening of plant raw materials.

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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 affiliation of a medicinal plant of multifinished hairwort (Bupleurum multinerve DC.). The invention can be used to identify the species of this plant; during the screening of plant materials, to control the compliance 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 testing 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 have chosen a method based on a destructible probe, based on the fact that it relates to specific detection methods, the possibility of its free use without violating copyright and related rights (the system of destructible probe was originally proposed in 1991, however, all patents have now 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 large 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, Μ. (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 principal differences of the prototype from the claimed invention are as follows: medicinal plants with nucleotide sequences of specific primers that are different from those declared are identified. A set of synthetic oligonucleotides for identifying the species of a medicinal plant - multicircular volodushka (Bupleurum multinerve DC.), Designed for the polymerase chain reaction of a fragment of ITS2 nuclear DNA, including species-specific forward primers, reverse primers and a destructible probe to identify this plant:

as a direct primer, a DNA sequence with the following nucleotide composition is used: 5′-TATCCCGGCTCTCGCATCGA-3 ′,

as a reverse primer, a DNA sequence with the following nucleotide composition is used: 5′-GACGAGGCACGGGAGAGTCTC-3 ′,

a DNA sequence with the following nucleotide composition is used as a destructible probe: (fluorescent label) -5′-CCGTGAACCATCGAGTTTTT-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 the special software or manually selects the sequence of oligonucleotides used to carry out the PNR 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 specific purposes is proved.

Analysis of the nucleotide polymorphism of the ITS2 sequence based on NCBI () data and de novo sequenced sequences of species not located in the genebank allows the use of 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 (Holland Ρ Μ, 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 USA. 1991 August 15; 88 (16): 7276-7280).

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

Similar kits, reaction mixtures, primers for amplification and identification of species of multifinished hairworms (Bupleurum multinerve DC.) Are unknown.

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 ° С - 10 sec, 58 ° С - 30 sec (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 MgCl2; 1 μl of 10 mm each primer; 0.5 μl a 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. one.

A set of synthetic oligonucleotides for identifying the species of a medicinal plant - multicontained hairwort (Bupleurum multinerve DC.), Designed for polymerase chain reaction with real-time detection.). The kit includes species-specific sites for creating direct, reverse primers and a destructible probe, namely, a direct primer 5′-TATCCCGGCTCTCGCATCGA-3 ′, a reverse primer 5′-GACGAGGCACGGGAGGTCAG-3 ′, as a destructible probe (fluorescent label) -GT-CTT-CTT-CT 3 ′ - (quencher). The kit has high sensitivity and specificity, allowing you to quickly and reliably identify the considered medicinal plant.

Claims (1)

A set of synthetic oligonucleotides for identifying the species of a multifinished hairworm (Bupleurum multinerve DC.), Including carrying out a polymerase chain reaction with a fragment of ITS2 nuclear DNA, characterized in that species-specific sites are used for identification to create direct, reverse primers and destructible probe, where as direct primer — DNA sequence with the following nucleotide composition: 5′-TATCCCGGCTCTCGCATCGA-3 ′; the reverse primer is a DNA sequence with the following nucleotide composition: 5′-GACGAGGCACGGGAGGTCAG-3 ′, destructible probe DNA sequence with the following nucleotide composition: (fluorescent label) -5′-CCGTGAACCATCGAGTTTTT-3 ′ - (quencher).

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