RU2814814C2 - Method and kit for identification of vaccinium myrtillus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of identifying Vaccinium myrtillus in a botanical mixture by PCR amplification is described. A V. myrtillus nucleic acid fragment located within internal transcribed spacer 1, the 5.8S ribosomal RNA gene region and internal transcribed spacer 2 is determined in the mixture sample. Nucleic acids are isolated from the specified sample. Real-time PCR is performed on the isolated nucleic acid using a set of primers selected from the following group: SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:11 and SEQ ID NO:12. The probe is annealed to the region of the nucleic acid amplified by the primers, wherein the said probe consists of the sequence SEQ ID NO:13. The presence of an amplification product is determined. Detection of an amplification product indicates the presence of Vaccinium myrtillus in the botanical composition.

EFFECT: invention expands the range of agents for identifying Vaccinium myrtillus.

7 cl, 12 dwg, 10 tbl, 4 ex

Description

The present invention relates to a method for identifying Vaccinium myrtillus in a botanical composition, which is based on the detection of specific genomic fragments using PCR amplification. Furthermore, the invention relates to a kit specifically designed for carrying out the method of the invention.

Prior Art

Plant extracts are widely used in the medical, nutraceutical, cosmetic and food industries. One of the main problems that arise when working with plant extracts is determining not only their chemical composition, but also determining their botanical origin, in order to eliminate the risk of counterfeiting.

Genetic methods for determining the botanical origin of plant materials are known in the field (Parker, J., et al., Field-based species identification of closely-related plants using real-time nanopore sequencing. Sci Rep, 2017. 7(1): p. 8345; Group, C. V. W., ADNA barcoding methods for land plants. Proc Natl Acad Sci USA, 2009. 106(31): p. 12794-7; Fazekas, A. J., et al., DNA barcoding methods for land plants. Methods Mol Biol. 2012. 858: p. 223). Such methods rely on comparing DNA present in plant material with known DNA sequences present 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 a botanical mixture, the method being based on single-strand conformational polymorphism (SSCP) analysis and sequence analysis. It is believed that the method can provide information about the biological components of the composition without prior knowledge of what botanicals are present, and can also detect and identify unknown biological components that may be present in the mixture.

Methods for genetic identification of plants from botanical specimens are also disclosed in CN102146477, CN106119394, CN1372005, CN107142329, CN107653330, CN105624291, CN105603107, ES2176066, CN104673930, CN10 2222969, CN102732513, CN105063203, JP2007282626. In some cases, methods for identifying botanical species are based on the detection of specific sequences by PCR amplification located within a nuclear ribosomal RNA coding locus containing the ITS-1 and/or ITS-2 internal transcribed spacer (ITS) regions. In some cases (CN1052429, CN105603107, ES2176066), the methods are aimed at identifying impurities in commercial products containing plant materials.

Jaakola L et al., Food Chemistry vol. 123, no. 2 (2010) pp.494-500, discloses the identification of commercially important berry species through an integrated approach involving DNA barcoding and high-resolution melting curve (HRM) analysis using designed primer pairs that allow the identification of specific wild berry species. Vaccinium myrtillus is identified by HRM analysis of the amplicon located in the ITS (internal transcribed spacer) region obtained using primers ITSVm2f and ITSVm2r.

CN108642207 discloses the construction of a blueberry plant allelic map and a method for identifying blueberry cultivars and related species using primer-specific PCR amplification.

Marieschi M. et al, Food Chemistry vol. 202 (2016) pp. 438-444 disclose a method based on sequence characterization of amplified regions (SCARs) to detect the presence of V. myrtillus and contaminant species, useful for the analysis of multiple groups.

Koskima Ki JJ et al., European Journal of Plant Pathology, Kluwer Academic Publishers - vol. 125 no. 4 (2009) pp. 629-640 disclose relative blueberry gene expression quantified by real-time PCR with SYBR-green dye as a fluorescent reporter.

When processing plant materials, and in particular when extraction procedures are used, DNA is degraded, producing fragments of varying sizes and quantities depending on the extraction method, and these fragments cannot be directly compared with known DNA sequences, thereby making it difficult to even making it virtually impossible to apply genome identification methods to extracts that can be applied to starting materials.

Vaccinium myrtillus extracts are largely used in pharmaceutical, cosmetic, nutraceutical and dietary products due to their known health-promoting properties. The clinical benefit of V. myrtillus as both a dietary supplement and a therapeutic agent may be due to the presence of excess flavonoids and anthocyanins. For the extraction method, it is important that the V. myrtillus extracts have the required characteristics in terms of chemical components and declared pure botanical origin. Therefore, it is desirable to provide a method that allows the identification of V. myrtillus in a botanical composition, such as a plant extract, providing high levels of accuracy and species specificity, especially when V. myrtillus is mixed with closely related contaminant species.

Description of the invention

These objects are achieved by the present invention, which provides a method for specifically and accurately identifying Vaccinium myrtillus in a botanical composition by identifying a nucleic acid fragment that is contained in the residual DNA of V. myrtillus extracts.

In particular, the method of the invention includes detecting in a sample of a V. myrtillus botanical composition a specific nucleic acid fragment of a 5.8S ribosomal RNA gene within internal transcribed spacer 1 and internal transcribed spacer 2, wherein said nucleic acid fragment consists of SEQ ID NO: 1 or sequence containing SEQ ID NO: 1, which is selected from the group of SEQ ID NOs: 2, 3 and 4.

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

(1) SEQ ID NO: 5 and SEQ ID NO: 6;

(2) SEQ ID NO: 7 and SEQ ID NO: 8;

(3) SEQ ID NO: 9 and SEQ ID NO: 10;

(4) SEQ ID NO: 11 and SEQ ID NO: 12.

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

(a) isolating nucleic acids from a sample of the botanical composition;

(b) performing rt-PCR (rt-PCR) on the isolated nucleic acid using:

- a pair of primers selected from the group consisting of:

(1) SEQ ID NO: 5 and SEQ ID NO: 6;

(2) SEQ ID NO: 7 and SEQ ID NO: 8;

(3) SEQ ID NO: 9 and SEQ ID NO: 10;

(4) SEQ ID NO: 11 and SEQ ID NO: 12; And

- annealing of the probe in the region of the nucleic acid amplified using primers, said probe having the sequence SEQ ID NO: 13;

(c) determining the presence of the amplified product,

in this case, the detection of an amplification product indicates the presence of Vaccinium myrtillus in the botanical composition.

According to the invention, the botanical composition is a mixture of plants or parts thereof, for example leaves, fruits, barks, roots, including plant extracts and especially fruit extracts, which are intended for food consumption or therapeutic use. In a preferred embodiment, the botanical composition is a product containing an extract of the fruit of Vaccinium myrtillus, alone or in combination with related species such as Empetrum nigrum, Sambucus nigra, Vaccinium oxycoccos, Vaccinium corymbosum and Vaccinium macrocarpon.

Isolation of nucleic acids involves their separation and purification from other components of the plant mixture or extract, and can be carried out using conventional methods using commercially available kits. In particular, genomic DNA can be isolated using extraction-precipitation protocols, silica membrane-based methods, or anion exchange approaches.

Real-time PCR technology is known in the art, and it combines 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 through multiple cycles of denaturation followed by primer and probe annealing and DNA polymerase-catalyzed primer extension. DNA amplification is monitored at each PCR cycle by measuring the fluorescent signal, which is produced, for example, by non-specific fluorescent dyes that interact with double-stranded DNA, or by DNA-specific probes consisting of oligonucleotides labeled with a fluorescent reporter, which provides detection of the result of hybridization of the probe with its complementary target DNA. Suitable intercalating dyes include SYBR® (Green I, Green II, Gold), LCGreen®, SYTO-(9, 13, 16, 60, 62, 64, 82), BOBO-3, LCGreen®, POPO-3, BEBO, TO-PR03, PicoGreen®, SYTOX Orange and similar commercially available fluorescent dyes (fluorophores).

The oligonucleotide probe is labeled with a fluorescent reporter (fluorophore) at one end and a fluorescence quencher at the opposite end of the probe. 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 succinimidyl ether (JOE), tetramethylrhodamine (TAMRA), 5-carboxytetramethylrhodamine (TAMRASE), carboxy-X-rhodamine (ROX), 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL). Examples of quenchers include the BHQ family (Black Hole Quencher®), NFQ-MGB (non-fluorescent quencher and minor groove binding), QSY 7 or 21 carboxylic acid N-succinimidyl ester.

rtPCR parameters and conditions, such as temperature and duration of each denaturation and annealing cycle, can be adjusted depending on the nucleic acid fragment being amplified, the set of primers used for amplification, and other variables as known to those skilled in the art. In a preferred embodiment of the invention, the nucleic acid fragments described herein are amplified using primers (1) by (4) applying the following conditions:

- initial stage of denaturation at 95°C for 180 sec

- 2-stage cycles: 15 sec at 95°C (1st stage) and 15 sec at 62-68.5°C

(2nd stage), repeated from forty (40) to fifty (50) times.

The specific primer and probe combinations of the invention allow specific identification of Vaccinium myrtillus in botanical compositions containing closely related species such as Empetrum nigrum, Sambucus nigra, Vaccinium oxycoccos, Vaccinium corymbosum and Vaccinium macrocarpon. As reported in the experimental section, the use of a probe other than the probe specific to Vaccinium myrtillus SEQ ID NO: 13, and similar annealing with fragments SEQ ID NOs: 1-4, eliminates the system's ability to identify Vaccinium myrtillus mixed with Empetrum using the same primers and the same rt-PCR conditions disclosed above. This indicates the specificity of the selected combination of primers, probe and the effectiveness of the gSCR conditions in accordance with the invention.

Another aspect of the invention relates to a kit for identifying Vaccinium myrtillus in a botanical composition. The kit according to the invention contains at least a pair of primers selected from (1) to (4) and a probe as described above. In addition, the kit may contain, in separate containers, the reagents necessary to perform hCR, in particular deoxynucleotides and DNA polymerase, as well as reagents for DNA isolation, purification and possibly quantification. The kit may also contain DNA from Vaccinium myrtillus as a positive control and nuclease-free water or buffer as a negative control, as well as instructions for performing the PCR assay.

In a preferred embodiment of the invention, the kit contains:

- one tube or vial containing all reagents needed to perform the assay (DNA polymerase, deoxynucleotides (dNTPs), buffer, probe reagents, primers and probe)

- one tube or vial containing a positive control (Vaccinium myrtillus DNA)

- one tube or vial containing negative DNA control (nuclease-free water)

The kit can be used with all commercially available real-time PCR systems.

Brief description of graphic materials

Fig. 1: RT-PCR amplification protocol.

Fig. 2: rt-PCR amplification results (a) and melting curve analysis (Tm)

(b) genomic DNA isolated from frozen fruits of Vaccinium myrtillus.

Fig. 3: Calibration curve analysis for Vaccinium myrtillus.

Fig. 4: rt-PCR amplification based on the V. myrtillus-specific M-FAM probe. NTC: negative control.

Fig. 5: (a) rt-PCR amplification based on the V. myrtillus-specific M-FAM probe in mixed samples with the ratio indicated in the legend. NTC: negative control;

(b) rt-PCR amplification based on the E-HEX probe specific for E. nigrum in mixed samples with the ratio indicated in the legend. NTC: negative control.

Fig. 6: Correlation between Cq Mean (average) and percentage of target species for V. myrtillus.

Fig. 7: Experimental design used for rt-PCR analysis of dry extract samples. rt-PCR rt-PCR.

Fig. 8: rt-PCR amplification of residual DNA isolated from dry extract samples of Vaccinium myrtillus. The positive control was genomic DNA extracted from frozen Vaccinium myrtillus fruits. a) primer set L; b) primer set S.

Fig. 9: RT-PCR analysis of amplicons in agarose gel.

Fig. 10: Alignment analysis of sequenced amplicons (top) and relative identity identification matrix (bottom)

Fig. 11: RT-PCR of 36% dry ethanol extract of Vaccinium myrtillus. Amplification and melting curves.

Fig. 12: RT-PCR of 36% dry ethanol extract of Vaccinium myrtillus with probe-based method. PTC: positive control (genomic DNA extracted from frozen V. myrtillus fruits); NTC: negative control.

EXPERIMENTAL SECTION - general procedures Genomic DNA extraction (genomic DNA)

DNA extraction was performed using the NucleoSpin® plant II protocol as described by the supplier (Macherey nagel. Cat. 740770.250 - July 2014/Rev.09).

Purification of residual DNA from dry extract.

The first purification was performed using the NucleoSpin® plant II Maxi kit protocol as described by the supplier (Macherey nagel. Cat. 740770.250 - July 2014/Rev.09), with some modifications presented below.

- Weigh 3-5 g of dry extract in a 50 ml conical flask

- Adding 3 ml distilled water

- Add 9 ml lysis buffer

- Mix on a Vortex mixer for 30 seconds

- Transfer the sample to the NucleoSpin® Filter Maxi

- Centrifuge for 5 min at 4500 g, collect the clean eluate and remove the NucleoSpin Filter Maxi

- Add 20 ml binding buffer

- Mix on a Vortex mixer for 30 seconds

- Application of the sample to the NucleoSpin® Plant II Maxi column

- Centrifugation for 3 minutes at 4500 g and removal of the eluate

- Repeat application for all remaining samples

- Add 4 ml wash buffer (PW1) to the NucleoSpin® Plant II Maxi column

- Centrifugation for 3 minutes at 4500 g and removal of the eluate

- Add 10 ml wash buffer (PW2) to the NucleoSpin® Plant II Maxi column

- Centrifugation for 3 minutes at 4500 g and removal of the eluate.

- Add 2 ml wash buffer (PW2) to the NucleoSpin® Plant II Maxi column

- Centrifugation for 12 minutes at 4500 g and removal of the eluate

- Place the NucleoSpin® Plant II Maxi column in a new sample collection tube (50 ml)

- Add 1000 µl of elution buffer (PE) (65°C) to the membrane from a pipette

- Incubate the NucleoSpin® Plant II Maxi column for 5 minutes at 65°C

- Centrifugation for 3 minutes at 4500 g to elute DNA

Secondary purification was performed using the ReliaPrep™ DNA Clean-UP kit and concentration system protocol as described by the supplier (Promega. Cat. A2893).

DNA quantification

The amount of DNA was determined using a NanoQuant Plate™ instrument. Quantitative assessment is performed using the UV method. Absorbance at 260 nm was used for DNA quantification, assuming that 1 OD at 260 nm corresponds to 50 μg/μl DNA. The absorbance ratio of 260 nm/280 nm was determined to evaluate DNA purity.

rt-PCR and melting curve analysis

rt-PCR amplification was performed using SYBR Green or probe-based as described by the supplier (SsoAdvanced™ Universal SYBR® Green Supermix, BioRad Cat. N. 1725272; SsoAdvanced™ Universal Probes Supermix, BioRad Cat. N. 1725281), with 3 -amplification-based step protocol as indicated in FIG. 1.

Real-time PCR

Prepare the mixture as follows, final volume 20 µl:

Load the sample into a real-time PCR instrument (BioRad or equivalent) and install the following program:

Receiving data after the second stage of cycles.

DNA sequencing

The amplified DNA was purified using an agarose gel and the purified fragment was sequenced by generating two sequences for each sample: one obtained by using a forward primer and the other by using a reverse primer. For sequencing, purified DNA and TRIS-HC1 5 mM pH 8.0 were mixed to obtain the concentration required for sequencing (depending on sequence length, 2-5 ng/μl).

Sequences were analyzed using BioEdit or BLAST software for sequence comparison and identification.

Examples

Example 1 - Method Validation

Genomic DNA from frozen fruits of Vaccinium myrtillus was purified and quantified (Table 1), and its contaminating/related species are listed below:

Establishment of rt-PCR reaction parameters relative to Cq peak (cycle quantification) and Tm (melting temperature) were initially assessed using genomic DNA isolated from frozen fruits of Vaccinium myrtillus (Figure 2). The rt-PCR results showed that the designed primers allowed amplification of one DNA region for all primer sets (Tables 2 and 3).

rt-PCR was also performed using DNA isolated from V. myrtillus contaminant/related species, and the results demonstrated that it was possible to distinguish different DNAs through a set of primers and, in particular, small 2 primers (Table 4).

The linearity of the amplification curve was also assessed by generating a standard curve for Vaccinium myrtillus using the small 2 primer set (Figure 3). It is clear that linearity was achieved over the tested concentration range (almost 0.0625 8.00 ng/µL).

To improve the ability of the method to distinguish between Vaccinium myrtillus and contaminating/related species, rtPCR was performed using a minor groove binding probe (M-FAM - SEQ ID NO: 13) specifically designed for amplification of the V. myrtillus sequence.

In a comparative experiment, rtPCR was performed using simultaneous probes binding to the minor groove of SEQ ID NO: 13 (M-FAM) and SEQ ID NO: 14 (E-HEX).

To validate the probe-based method, various experimental methods were carried out, summarized in the table below.

Amplification results were proportional to the content of target species (Fig. 6)

Example 2 - Identification of residual DNA in dry extract of Vaccinium myrtillus

For each sample, two independent residual DNA extractions (biological replicates) were performed, and three technical replicates were tested for each DNA extraction, FIG. 7.

The entire procedure was initially performed on four samples: 32549/H76, 32549/H80, 32549/H83, 32549/H84. After isolation and quantification of residual DNA (Table 6), these samples were analyzed for their rt-PCR amplification characteristics (Cq and Tm) compared to those of the positive control (Figure 8 and Table 7).

The results of rt-PCR amplification with all samples showed that:

DNA was amplified for both the positive control and all samples tested;

negative control (no DNA) showed no amplification signal;

the positive control and samples showed equal values for melting temperature (Tm) peaks.

This result indicates that the amplicons have the same characteristics in terms of length and/or nucleotide base composition.

In addition, Cq results correlate with the amount of DNA tested, meaning that amplification is specific to the selected target.

To verify that the generated amplicons had the same positive control sequence, all amplified sequences were purified on an agarose gel (Figure 9) and the purified fragments were sequenced (Figure 10).

Agarose gel analysis confirmed the difference in amplicon length: the fragment generated with primer set S showed a length of approximately 130 bp, while the fragment generated with primer set L showed a length of approximately 270 bp. o.. In addition, during the analysis of the agarose gel, the presence of non-specific rt-PCR products can also be detected, as in Fig. 9, in track 4 for sample 32549/H83_1, where two bands are visible in good agreement with the results of the Tm peak (Fig. 8, b).

All obtained sequences were aligned by considering only a subset using high-quality sequencing parameters. The sequencing results (Fig. 10) showed that all amplicon sequences (small and large) were identical to the Vaccinium myrtillus reference sequence pattern.

Example 3 - Identification of Vaccinium myrtillus from 36% dry ethanol extract (D.E.T.) residual DNA

Residual DNA analysis was also performed on samples with company code mdena 9042202, MIRTILLO (V. MYRTILLUS) E. ET. 36% after dry powder mixing phase, 32788/M1, 32786/M2, 32788/M2. Previous samples 32549/H76, 32549/H80 and 32549/H83 were tested again as control samples.

To optimize the purification procedure, after the first DNA purification step, isolated residual DNA was processed using the ReliaPrep™ kit (Promega). The results in terms of DNA quantity (ng/µl) and quality (ratio 260/280) for the two-step purification (Table 8) revealed that the concentration as well as purification was better when a second step was introduced.

rt-PCR analysis was performed using SYBR green (Figure 11) and probe-based methods (Figure 12) according to the protocols described above. The results show that:

(a) all test samples have the same Tm peak of the positive control (Fig. 11 and Table 9) when analyzed by the SYBR green method:

Table 9 - Summary of rt-PCR results, SYBR green method - E. ET. 36%

(b) All test samples were analyzed with a V. myrtillus sequence-specific probe (Figure 12 and Table 10) using a probe-based method.

Example 4 - Assay Kit

The set consists of:

One 1.5 ml tube containing all reagents needed to perform the assay (DNA polymerase, dNTPs, buffer, probe reagents, primers and probe)

One 1.5 ml tube containing positive control (Vaccinium myrtillus DNA)

One 1.5 ml tube containing DNA negative control (nuclease-free water)

The kit can be used with all commercially available real-time PCR systems (eg BioRad CFX96™, BioRad CFX96™, bCube®, Roche LightCycler® 480, etc.)

Claims (24)

1. A method for identifying Vaccinium myrtillus in a botanical mixture by means of PCR amplification, including determining in a sample of this mixture a fragment of the V. myrtillus nucleic acid located within the internal transcribed spacer 1, the region of the 5.8S ribosomal RNA gene and the internal transcribed spacer 2, where the specified the method includes the following stages: (a) isolating nucleic acids from said sample; (b) performing real-time PCR on the isolated nucleic acid using: - a set of primers selected from the group consisting of: (1) SEQ ID NO:5 and SEQ ID NO:6; (2) SEQ ID NO:7 and SEQ ID NO:8; (3) SEQ ID NO:9 and SEQ ID NO:10; (4) SEQ ID NO:11 and SEQ ID NO:12; And - annealing the probe in the region of the nucleic acid amplified using primers, where the specified probe consists of the sequence SEQ ID NO:13; (c) determining the presence of an amplification product, wherein detection of the amplification product indicates the presence of Vaccinium myrtillus in the botanical composition. 2. The method according to claim 1, where the set of primers (1) is used in step (b). 3. The method according to claim 1 or 2, where the specified real-time PCR is performed under the following conditions: - initial stage of denaturation at 95°C for 180 s; - 2-stage cycles, where the 1st stage is 15 s at 95°C, and the 2nd stage is 15 s at 62-68.5°C, are repeated 40 to 50 times. 4. Method according to any one of paragraphs. 1-3, where the botanical mixture is a plant extract. 5. A kit for the identification of Vaccinium myrtillus in a botanical mixture, containing a set of primers selected from the group consisting of: (1) SEQ ID NO:5 and SEQ ID NO:6; (2) SEQ ID NO:7 and SEQ ID NO:8; (3) SEQ ID NO:9 and SEQ ID NO:10; (4) SEQ ID NO:11 and SEQ ID NO:12; and a probe consisting of the sequence SEQ ID NO:13. 6. The kit according to claim 5, additionally containing DNA polymerase, a mixture of deoxynucleotides, and buffer solutions. 7. The kit of claim 5 or 6, further containing in separate containers a sample of Vaccinium myrtillus nucleic acid for use as a positive control and water or a nuclease-free buffer solution as a negative control.