Methods and kits for identifying vaccinium uliginosum

Abstract

The present invention provides methods for identifying Vaccinium uliginosum in a plant composition and kits specifically designed for the practice of the methods. The methods of the present invention are based on the detection of a nucleic acid fragment in a genomic region of Vaccinium uliginosum using PCR amplification, wherein the genomic region is a genomic region in the Internal Transcribed Spacer 1, 5.8S ribosomal RNA genomic region, and Internal Transcribed Spacer 2.

Description

Technical Field

[0001] This invention provides a method for identifying Vaccinium myrtillus in plant compositions using PCR amplification to detect specific genomic fragments. The invention also provides a kit specifically designed to carry out the method of this invention. Background of the Invention

[0003] Plant extracts are widely used in the medical, health product, cosmetic, and food industries. One of the main challenges in processing plant extracts is determining not only their chemical composition but also their plant origin to eliminate the risk of counterfeiting.

[0004] Genetic-based methods for determining the plant origin of plant material are known in the art (Parker, T, et al., Field-based species identification of closely-related plants using real-time nanopore sequencing. Sci Rep, 2017.7(1):p.8345; Group, CPW, A DNA barcode for land plants. Proc Natl Acad Sci USA, 2009.106(31):p.12794-7; Fazekas, AJ, et al., DNA barcoding methods for land plants. Methods Mol Biol, 2012.858:p.223). Such methods are based on comparing DNA present in plant material with known DNA sequences available in publicly available databases. For example, WO 2006 / 020147 (The Regents of the University of California) discloses a method for identifying individual biological genetic components present in plant mixtures, based on a combination of genome locus-specific PCR, single-strand conformational polymorphism (SSCP), and sequence analysis. This method is said to provide information about the biological composition of a composition without prior knowledge of which plants may be present, and to detect and identify any unknown biological components that may be present in the mixture.

[0005] Methods for genetic identification of plants from plant samples are also disclosed in CN102146477, CN106119394, CN1372005, CN107142329, CN107653330, CN105624291, CN105603107, ES2176066, CN104673930, CN102222969, CN102732513, CN105063203, and JP2007282626. In some cases, methods for identifying plant species are based on PCR amplification to detect specific sequences of ITS-1 and / or ITS-2, which are internal transcription spacers (ITS) located at nuclear ribosomal RNA coding loci. In some cases (CN1052429, CN105603107, ES2176066), these methods are intended to identify adulteration in commercial products containing plant material.

[0006] Jaakola L et al., Food Chemistry vol.123, no.2 (2010) pp.494-500, disclosed a method combining DNA barcoding and HRM (high-resolution melting) analysis to identify commercially important berry species using designed primer pairs capable of species-specific identification of wild berries. For black blueberry, primers ITSVm2f and ITSVm2r were used to obtain an amplicon located in the ITS (internal transcription spacer) region, which was then identified by HRM analysis.

[0007] CN108642207 discloses the construction of an allele map of blueberry plants and a method for identifying blueberry varieties and related species using primer-specific PCR amplification.

[0008] Marieschi M. et al., Food Chemistry vol.202(2016)pp.438-444, disclose a sequence characteristic amplification region (SCAR)-based method for detecting the presence of black blueberries and adulterants that can be used for multi-batch analysis.

[0009] Koskima Ki JJ et al., published in European Journal of Plant Pathology, Kluwer Academic Publishers-vol.125no.4(2009)pp.629-640, used SYBR-green as a fluorescent reporter molecule to quantify the relative expression of blueberry genes by real-time PCR.

[0010] When processing plant materials, especially during extraction operations, DNA degrades, producing fragments of varying sizes and numbers depending on the extraction method. These fragments cannot be directly compared with known DNA sequences, making it difficult, if not practically impossible, to apply genomic identification methods that can be used for starting materials to extracts.

[0011] Black bilberry extract is widely used in pharmaceuticals, cosmetics, nutritional supplements, and dietary products due to its known health benefits. The clinical benefits of black bilberry as a dietary supplement and therapeutic agent have been attributed to the presence of abundant flavonoids and anthocyanins. For extract manufacturers, ensuring that black bilberry extract meets the required specifications in terms of chemical composition and claimed pure plant origin is crucial. Therefore, it is desirable to provide a method that allows for the identification of black bilberry in plant compositions, such as plant extracts, thereby ensuring a high level of accuracy and species specificity, particularly when black bilberry is mixed with closely related contaminant species. Invention Details

[0013] These objectives are achieved by the present invention, which provides a method for specifically and accurately identifying black blueberry in a plant composition by detecting nucleic acid fragments contained in residual DNA of black blueberry extract.

[0014] Specifically, the method of the present invention includes detecting a black blueberry-specific nucleic acid fragment in a plant composition sample, the nucleic acid fragment being located in internal transcription spacer 1, a 5.8S ribosomal RNA gene, and internal transcription spacer 2, wherein the nucleic acid fragment consists of SEQ ID NO:1 or consists of a sequence selected from SEQ ID NO:2, 3, and 4 containing SEQ ID NO:1.

[0015] In a preferred embodiment, the primers used for PCR amplification are selected from the following primer pairs:

[0016] (i) SEQ ID NO:5 and SEQ ID NO:6;

[0017] (ii) SEQ ID NO:7 and SEQ ID NO:8;

[0018] (iii) SEQ ID NO:9 and SEQ ID NO:10;

[0019] (iv) SEQ ID NO:11 and SEQ ID NO:12.

[0020] In a particularly preferred embodiment, the PCR is real-time PCR (rtPCR) and the method of the present invention includes the following steps:

[0021] (a) Isolation of nucleic acids from a sample of a plant composition;

[0022] (b) Perform rt-PCR on the isolated nucleic acids, using:

[0023] -Selected from the following primer pairs:

[0024] (i) SEQ ID NO:5 and SEQ ID NO:6;

[0025] (ii) SEQ ID NO:7 and SEQ ID NO:8;

[0026] (iii) SEQ ID NO:9 and SEQ ID NO:10;

[0027] (iv) SEQ ID NO:11 and SEQ ID NO:12;

[0028] and

[0029] - A probe annealed within the nucleic acid region amplified by primers, the probe having the sequence SEQ ID NO:13;

[0030] (c) Confirm the presence of amplification products.

[0031] The detection of amplification products indicates the presence of black bilberry in the plant composition.

[0032] According to the present invention, the plant composition is a mixture of plants or parts thereof (e.g., leaves, fruits, bark, roots) intended for edible or therapeutic purposes, including plant extracts, particularly fruit extracts. In a preferred embodiment, the plant composition is a product containing a fruit extract of black blueberry, alone or in combination with related species, such as Empetrum nigrum, Sambucus nigra, Vaccinium oxycoccos, Vaccinium corymbosum, and Vaccinium macrocarpon.

[0033] The isolation of nucleic acids involves separating and purifying them from other components of plant mixtures or extracts, which can be done using commercially available kits and conventional techniques. In particular, genomic DNA can be isolated using extraction-precipitation protocols, silica membrane-based procedures, or anion exchange processes.

[0034] Real-time PCR technology is known in the art, combining polymerase chain reaction chemistry with the use of fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. Amplification of the target DNA is achieved by repeating cycles of denaturation, followed by primer and probe annealing, and then primer extension catalyzed by DNA polymerase. DNA amplification is monitored in each PCR cycle by measuring a fluorescence signal, which can be generated, for example, by a nonspecific fluorescent dye intercalating into double-stranded DNA or by a sequence-specific DNA probe composed of oligonucleotides labeled with a fluorescent reporter molecule, allowing detection upon hybridization of the probe to its complementary DNA target. Suitable intercalation dyes include… (Green I, Green II, Gold), LC SYTO-(9, 13, 16, 60, 62, 64, 82), BOBO-3, LC POPO-3, BEBO, T0-PR03, Pico SYTOX orange and similar commercially available fluorescent dyes (fluorophores).

[0035] Oligonucleotide probes are labeled with a fluorescent reporter molecule (fluorophore) at one end and a fluorescent quencher at the other end. The 5' exonuclease activity of the polymerase cleaves the probe, releasing the reporter molecule, resulting in an increase in fluorescence intensity. Examples of fluorophores include 5- or 6-carboxyfluorescein (5- or 6-FAM), tetrachlorofluorescein (TET), hexachloro-6-carboxyfluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimide ester (JOE), tetramethylrhodamine (TAMRA), 5-carboxytetramethylrhodamine (TAMRASE), carboxy-X-rhodamine (ROX), and 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL). Examples of quenchers include BHQ (Black Hole fluorescein). ) series, NFQ-MGB (non-fluorescent quencher and small groove binder), QSY 7 or 21 carboxylic acid succinimide ester.

[0036] The parameters and conditions of rtPCR, such as the denaturation and annealing temperatures and lengths per cycle, as known to those skilled in the art, can be adjusted according to the nucleic acid fragment to be amplified, the primer set used in the amplification, and other variables. In a preferred embodiment of the invention, the nucleic acid fragments disclosed herein are amplified with primers (i) to (iv) under the following conditions:

[0037] -Initial denaturation step: 95℃ for 180 seconds;

[0038] The two-step cycle of -95℃ for 15 seconds (step one) and 62-68.5℃ for 15 seconds (step two) is repeated forty (40) to fifty (50) times.

[0039] The specific combination of primers and probes according to the invention allows for the specific identification of black blueberry in plant compositions containing closely related species such as *Empetrum nigrum*, *Sambucus nigra*, *Vaccinium oxycoccos*, *Vaccinium corymbosum*, and *Vaccinium macrocarpon*. As reported in the Experimental Section, using the same primers and rt-PCR conditions disclosed above, but with a probe different from the black blueberry-specific probe SEQ ID NO:13 and similarly annealed with fragments SEQ ID NO:1-4, disrupts the system's ability to recognize black blueberry mixed with *Empetrum nigrum*. This demonstrates the specificity of the primer and probe combination selected according to the invention and the effectiveness of the rt-PCR conditions according to the invention.

[0040] Another aspect of the invention relates to a kit for identifying black blueberry in a plant composition. The kit of the invention comprises at least one pair of primers selected from (i) to (iv) and a probe as defined above. Furthermore, the kit may contain reagents required for running (rt) PCR, particularly deoxynucleotides and DNA polymerase, as well as reagents for isolating, purifying, and optionally quantifying DNA, in a separate container. The kit may also contain black blueberry DNA as a positive control, nuclease-free water or buffer as a negative control, and a printed instruction manual with instructions for performing the PCR assay.

[0041] In a preferred embodiment of the present invention, the kit comprises:

[0042] - A tube or vial containing all the reagents required to perform the analysis (DNA polymerase, dNTPs, buffer, probe chemistry, primers, and probes);

[0043] - A tube or vial containing a positive control (blueberry DNA);

[0044] - A tube or vial containing negative control DNA (nuclease-free water).

[0045] The kit is compatible with all commercially available real-time quantitative PCR systems. Attached Figure Description

[0046] Figure 1 rt-PCR amplification protocol.

[0047] Figure 2rt-PCR amplification results (a) and melting curve analysis (b) of genomic DNA isolated from frozen blueberry fruit.

[0048] Figure 3 Standard curve analysis of black blueberry.

[0049] Figure 4 Probe-based rt-PCR amplification using the M-FAM probe specific to blueberry. NTC: Negative control.

[0050] Figure 5 (a) In the mixed samples with the ratios reported in the legend, probe-based rt-PCR amplification was performed using the M-FAM probe, which is specific to black blueberry. NTC: negative control; (b) In the mixed samples with the ratios reported in the legend, probe-based rt-PCR amplification was performed using the E-HEX probe, which is specific to rock blueberry. NTC: negative control.

[0051] Figure 6 Correlation between the average Cq value and the percentage of the target species for black blueberry.

[0052] Figure 7 : Experimental protocol for rt-PCR analysis of dried extract samples.

[0053] Figure 8 rt-PCR amplification of residual DNA isolated from dried blueberry extract samples. The positive control was gDNA extracted from frozen blueberry fruit. a) Primer set L; b) Primer set S.

[0054] Figure 9 Agarose gel analysis of rt-PCR amplicon.

[0055] Figure 10 Alignment analysis of sequenced amplicon (top) and relative sequence identity matrix (bottom).

[0056] Figure 11 rt-PCR of 36% of E. ET from black blueberry. Amplification and melting curves.

[0057] Figure 12 36% rt-PCR of black blueberry (E. ET) using a probe-based method. PTC: positive control (gDNA extracted from frozen black blueberry fruit); NTC: negative control.

[0058] Experimental Section - General Procedure

[0059] Extraction of genomic DNA (gDNA)

[0060] Using supplier descriptions Plant II protocol (Macherey nagel.Cat.740770.250-July 2014 / Rev.09) was used for DNA extraction.

[0061] Purification of residual DNA from dry extract

[0062] The first purification uses the supplier's description. The plant II Maxi protocol (Machereynagel.Cat.740770.250-July 2014 / Rev.09) includes the following modifications.

[0063] Weigh 3-5 grams of the dry extract into a 50 ml conical tube.

[0064] Add 3 ml of distilled water

[0065] Add 9ml of lysis buffer.

[0066] - Vortex for 30 seconds

[0067] - Transfer the sample to Filter Maxi

[0068] Centrifuge at 4500x g for 5 minutes, collect the clarified cross-flow, and discard the NucleoSpin Filter Maxi.

[0069] Add 20ml of binding buffer.

[0070] - Vortex for 30 seconds

[0071] - Load the sample into Plant II Maxi column

[0072] Centrifuge at 4500 x g for 3 minutes and discard the flow-through.

[0073] Repeat the loading process for all remaining samples.

[0074] Add 4 ml of washing buffer (PW1) to Plant II Maxi column

[0075] Centrifuge at 4500 x g for 3 minutes and discard the flow-through.

[0076] Add 10 ml of wash buffer (PW2) to Plant II Maxi column

[0077] Centrifuge at 4500 x g for 3 minutes and discard the flow-through.

[0078] Add 2 ml of wash buffer (PW2) to Plant II Maxi column

[0079] Centrifuge at 4500 x g for 12 minutes and discard the flow-through.

[0080] -Will Place the Plant II Maxi column into a new collection tube (50 ml).

[0081] Transfer 1000 ml of elution buffer (PE) (65°C) onto the membrane.

[0082] -Incubate at 65℃ Plant II Maxi column 5 minutes

[0083] Centrifuge at 4500x g for 3 minutes to elute DNA.

[0084] The second purification used the ReliaPrep kit. TM DNA Clean-UP and the concentration system described by the supplier (Promega.Cat.A2893).

[0085] DNA quantification

[0086] via NanoQuant Plate TM The instrument quantifies DNA. This quantification is performed using a UV method. Absorbance at 260 nm is used to quantify DNA, where 1 OD at 260 nm corresponds to 50 μg / ml DNA. The 260 nm / 280 nm absorbance ratio is determined to assess DNA purity.

[0087] rt-PCR and melting curve analysis

[0088] rt-PCR amplification was performed using SYBR Green or probe-based chemiluminescence as described by the supplier (SsoAdvanced). TM Universal Green Supermix, BioRad Cat.N.1725272; SsoAdvanced TM Universal Probes Supermix (BioRad Cat.N.1725281) was implemented using a 3-step amplification protocol, such as... Figure 1 As shown.

[0089] Real-time PCR

[0090] Prepare the mixture as follows, with a final volume of 20 μl:

[0091] Probe Mastermix (BioRad or equivalent) 2X 10μl Primer F 10 μM 0.5μl Primer R 10 μM 0.5μl Probe M-FAM 10μM 0.5μl DNA 0.5–30 ng / μl 2μl Nuclease-free water 6.5μl

[0092] Load the sample into a real-time instrument (BioRad or equivalent) and set the following method:

[0093]

[0094] Data is collected after the second step of the loop.

[0095] DNA sequencing

[0096] The amplified DNA was purified on an agarose gel, and the purified fragments were sequenced, generating two sequences for each sample: one using a forward primer and the other using a reverse primer. Each sequencing tube was prepared by mixing the purified DNA with 5 mM TRIS-HCl at pH 8.0 to obtain the concentration required for sequencing (2–5 ng / μL depending on the sequence length).

[0097] Sequences were analyzed using BioEdit or BLAST software for comparison and identification. Example

[0098] Example 1 - Method Validation

[0099] The gDNA of frozen black blueberry fruit and its contaminated / related species was purified and quantified (Table 1), as described below:

[0100] Table 1. Quantification of gDNA extracted from all species tested in this report

[0101]

[0102] The settings of rt-PCR reaction parameters, in terms of Cq (quantitative cycling) and Tm (melting temperature) peaks, were initially evaluated using gDNA extracted from frozen blueberry fruit. Figure 2 rt-PCR results showed that, for all primer sets, the designed primers allowed for the amplification of single DNA regions (Tables 2 and 3).

[0103] Table 2

[0104]

[0105] Table 3

[0106]

[0107] rt-PCR was also performed on DNA isolated from black blueberry contamination / related species, and the results showed that different DNAs could be distinguished by using the primer set, especially the small 2 primer (Table 4).

[0108] Table 4 - Peak values ​​of melting curves

[0109] sample 2nd year Small 2 big Small Black Bilberry 90.50 90.50 90.50 89.50 Iwako Ran 88.50 88.00 90.50 89.00 / 89.50 Western elderberry 88.50 87.50 89.50 87.50 Cranberry moss 91.00 91.00 91.00 89.50 / 90.00 Highbush Blueberry 89.00 89.00 89.00 88.00 Large-fruited blueberries 91.50 91.50 91.00 90.50

[0110] The linearity of the amplification curve was also evaluated by generating a standard curve for black blueberry using a small 2-primer set. Figure 3 As can be seen, linearity was ensured within the tested concentration range (approximately 0.0625-8.00 ng / μl).

[0111] To improve the ability of methods to distinguish black blueberry from contaminated / related species, rtPCR was performed using a minor groove binding probe (M-FAM-SEQ ID NO:13), which was specifically designed to allow amplification of the black blueberry sequence.

[0112] In the comparative experiment, rtPCR was performed simultaneously using the minor groove binding probes SEQ ID NO:13 (M-FAM) and SEQ ID NO:14 (E-HEX).

[0113] To test this probe-based method, experiments were conducted in different subgroups, summarized in the table below.

[0114] Table 5

[0115]

[0116]

[0117] The amplification results are directly proportional to the content of the target species. Figure 6 ).

[0118] Example 2 - Identification of residual DNA in dried black blueberry extract

[0119] For each sample, two independent residual DNA isolations (biological replicates) were performed; and for each extracted DNA, three technical replicate tests were conducted. Figure 7 .

[0120] The entire process initially involved four samples: 32549 / H76, 32549 / H80, 32549 / H83, and 32549 / H84. After residual DNA isolation and quantification (Table 6), the rt-PCR amplification characteristics (Cq and Tm) of these samples were analyzed compared to the positive control. Figure 8 (and Table 7).

[0121] Table 6 - Quantification of Residual DNA

[0122]

[0123] Table 7 - Summary of rt-PCR Results

[0124] sample DNA (ng / μL) Cq. Average value Cq. Standard deviation melting temperature 32549 / H76_2 1.0 32.05 0.180 89.50 32549 / H83_1 0.7 35.26 0.181 89.00 32549 / H84_1 10.3 28.96 0.119 89.50 32549 / H84_2 10.6 28.44 0.092 89.50 negative control 0.0 none none none Positive control 24.8 23.60 0.040 89.50

[0125] The rt-PCR amplification results for all samples showed:

[0126] - DNA was amplified in both the positive control and all test samples;

[0127] - The negative control (no DNA) showed no amplification signal;

[0128] The positive control and the sample showed comparable Tm peaks.

[0129] This result indicates that these amplicones share similar characteristics in terms of length and / or nucleotide base composition.

[0130] Furthermore, the Cq results are correlated with the amount of DNA tested, which means that the amplification is specific to the selected target.

[0131] To verify that the generated amplicon had the same sequence as the positive control, all amplified sequences were purified on agarose gels. Figure 9 The purified fragments were then sequenced. Figure 10 ).

[0132] Agarose gel analysis confirmed the difference in amplicon length: primer set S produced a fragment of approximately 130 bp, while primer set L produced a fragment of approximately 270 bp. Furthermore, the presence of nonspecific rt-PCR products was also observed in the agarose gel analysis, such as... Figure 9 As shown, lane 4 of sample 32549 / H83_1 shows two bands, which is in good agreement with the Tm peak results. Figure 8 b).

[0133] All generated sequences were aligned by considering only the portions with high-quality sequencing parameters. Sequencing results ( Figure 10 The results showed that all amplicon sequences (small and large) were identical to the sequences in the black blueberry standard reference.

[0134] Example 3 - Identification of residual DNA in 36% dry ethanol extract (E.ET.) of black blueberry

[0135] Residual DNA analysis was also performed on samples 32788 / M1, 32786 / M2, and 32788 / M2 that had Indena code 9042202, MIRTILLO (black blueberry) E.ET. 36% after the dry powder mixing stage. Previous samples 32549 / H76, 32549 / H80, and 32549 / H83 were tested again as control samples.

[0136] To optimize the purification process, ReliaPrep was used after the first step of DNA purification. TM The kit (Promega) was used to treat the isolated residual DNA. The results of DNA quantity (ng / μL) and quality (260 / 280 ratio) at the two purification steps (Table 8) indicate that introducing the second step resulted in better concentration and purification.

[0137] Table 8 – Quantification of Residual DNA – E.ET. 36%

[0138]

[0139] According to the above scheme, by using SYBR Green ( Figure 11 ) and probe-based methods ( Figure 12 rt-PCR analysis was performed.

[0140] The results show that:

[0141] (a) When analyzed using the SYBR Green method, all test samples showed the same Tm peak as the positive control. Figure 11 (and Table 9):

[0142] Table 9 - Summary of rt-PCR results, SYBR Green Method - E.ET. 36%

[0143] sample DNA (ng / μL) Cq. Average value Cq. Standard deviation melting temperature 32549 / H76 2.3 31.59 0.099 89.50 32549 / H80 0.2 31.87 0.107 89.50 32549 / H83 2.1 33.61 0.276 89.50 32788 / M1 3.4 31.85 0.286 89.50 32786 / M2 2.5 31.88 0.011 89.50 32788 / M2 3.6 31.36 0.144 89.50 negative control 0 0 0 none Positive control 24.8 23.75 0.016 89.50

[0144] (b) When using probe-based methods for analysis, using probes specific to the black blueberry sequence, all test samples were detected. Figure 12 (and Table 10).

[0145] Table 10 - Summary of rt-PCR results, probe-based methods - E.ET. 36%

[0146] sample DNA (ng / μL) Cq. Average value Cq. Standard deviation 32549 / H76 2.3 31.52 0.306 32549 / H80 0.2 31.38 0.073 32549 / H83 2.1 33.44 0.326 32788 / M1 3.4 31.10 0.098 32786 / M2 2.5 31.52 0.156 32788 / M2 3.6 30.53 0.197 negative control 0 0 0 Positive control 7.45 24.84 0.177

[0147] Example 4 - Kit for Analysis

[0148] The kit consists of the following parts:

[0149] - A 1.5ml test tube containing all the reagents needed for the analysis (DNA polymerase, dNTPs, buffer, probe chemistry, primers, and probes).

[0150] - A 1.5ml test tube containing a positive control (blueberry DNA).

[0151] - A 1.5ml test tube containing negative control DNA (nuclease-free water).

[0152] The kit is compatible with all commercially available real-time PCR systems (especially: BioRad CFX96). TM BioRadCFX96 TM , Roche 480, etc.

[0153]

[0154]

Claims

1. A method for identifying Vaccinium uliginosum in a plant composition, comprising detecting a Vaccinium uliginosum nucleic acid fragment from a sample thereof by PCR amplification, said nucleic acid fragment being located in Internal Transcribed Spacer 1, 5.8S ribosomal RNA genomic region and Internal Transcribed Spacer 2, said method comprising the steps of: (a) isolating nucleic acids from said sample; (b) performing real-time PCR on the isolated nucleic acids, wherein: - a primer set selected from the group consisting of: (i) SEQ ID NO: 5 and SEQ ID NO: 6; (ii) SEQ ID NO: 7 and SEQ ID NO: 8; (iii) SEQ ID NO: 9 and SEQ ID NO: 10; (iv) SEQ ID NO: 11 and SEQ ID NO: 12; and - a probe annealing within the nucleic acid region amplified by the primers, said probe consisting of the sequence SEQ ID NO: 13 are used; (c) determining the presence of an amplification product, wherein the detection of an amplification product indicates the presence of Vaccinium uliginosum in the plant composition.

2. The method of claim 1, wherein primer set (i) is used in step (b).

3. The method of claim 1, wherein said real-time PCR is performed under the following conditions: - an initial denaturation step at 95°C for 180 seconds; - two-step cycles repeated 40 to 50 times, wherein the first step is at 95°C for 15 seconds and the second step is at 62-68.5°C for 15 seconds.

4. The method of any one of claims 1 to 3, wherein the plant composition is a plant extract.

5. A kit for identifying Vaccinium uliginosum in a plant composition, comprising a primer set and a probe, wherein: - said primer set is selected from the group consisting of: (i) SEQ ID NO: 5 and SEQ ID NO: 6; (ii) SEQ ID NO: 7 and SEQ ID NO: 8; (iii) SEQ ID NO: 9 and SEQ ID NO: 10; or (iv) SEQ ID NO: 11 and SEQ ID NO: 12; and - said probe consists of the sequence SEQ ID NO:

13.

6. The kit of claim 5, further comprising a DNA polymerase, a deoxynucleotide (dNTP) mixture, a buffer solution.

7. The kit of any one of claims 5 to 6, further comprising in separate containers a Vaccinium uliginosum nucleic acid sample as a positive control and nuclease-free water or a buffer solution as a negative control. ​

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