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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
  • A01H5/08—Fruits
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/36—Ericaceae, e.g. azalea, cranberry or blueberry
  • A01H6/368—Vaccinium, e.g. cranberry, blueberry
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2561/00—Nucleic acid detection characterised by assay method
  • C12Q2561/113—Real time assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/13—Plant traits
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention provides a method for the identification of Vaccinium myrtillus in a botanical composition, which is based on the detection of specific genomic fragments using PCR amplification.
• the invention further provides a kit specifically designed for implementing the method of invention.
• Plant extracts are widespreadly used in the medical, nutraceutical, cosmetic and food industry.
• One of the main issues encountered when dealing with plant extracts is that of determining not only their chemical composition, but also their botanical origin, in order to exclude the risk of counterfeit.
• WO 2006/020147 discloses a method for identifying individual biological genetic components present in a botanical mixture, said method being based on a combination of genomic-locus specific PCR, single strand conformation polymorphyspm (SSCP), and sequence analysis.
• the method is said to be able to provide information on the biologic components of the composition without requiring prior knowledge as to which botanicals may be present and to detect and identify unknown biologic components that may be present in the mixture.
• Methods for the genetic identification of plants from botanical samples are also disclosed in CN102146477, CN106119394, CN1372005, CN107142329, CN107653330, CN105624291, CN105603107, ES2176066, CN104673930, CN102222969, CN102732513, CN105063203, JP2007282626.
• the methods for the identification of botanical species are based on the detection, by PCR amplification, of specific sequences located within the nuclear ribosomal RNA-encoding locus containing the internal transcribed spacer (ITS) regions ITS-1 and/or ITS-2.
• the methods are aimed at identifying adulterations in commercialised products containing plant materials.
• Vaccinium myrtillus is identified through HRM analysis of an amplicon located in the ITS (Internal Transcribed Spacer) region obtained with primers ITSVm2f and ITSVm2r.
• CN108642207 discloses the construction of an allelic map of the bilberry plant, and a method for the identification of blueberry varieties and related species using primer-specific PCR-amplification.
• SCARs Sequence Characterized Amplified Regions
• Vaccinium myrtillus extracts are largely used in pharmaceutical, cosmetic, nutraceutical and dietary products due to their known health-beneficial properties.
• the clinical benefits of V myrtillus as both a dietary supplement and a therapeutic have been attributed to the presence of abundant amounts of flavonoids and anthocyanins.
• the present invention provides a method for the specific and accurate identification of Vaccinium myrtillus in a botanical composition through detection of a nucleic acid fragment which is contained in the residual DNA of V. myrtillus extracts.
• the method of invention comprises detecting, in a sample of botanical composition, a V. myrtillus-spec ⁇ f ⁇ c nucleic acid fragment located within the internal transcribed spacer 1, 5.8S ribosomal RNA gene and the internal transcribed spacer 2, wherein said nucleic acid fragment consists of either SEQ ID NO: l or a sequence comprising SEQ ID NO: l which is selected from the group of SEQ ID NOs:2, 3 and 4.
• the primers used for PCR-amplification are selected from the following pairs:
• the PCR is a real-time PCR (rtPCR) and the method of invention comprises the following steps:
• the botanical composition is a mixture of plants or parts thereof, e.g. leaves, fruits, bark, roots, including plant extracts and particularly fruit extracts, which are intended for consumption or therapeutic use.
• the botanical composition is a product containing an extract of fruits of Vaccinium myrtillus , alone or in combination with related species such as Empetrum nigrum , Sambucus nigra , Vaccinium oxycoccos, Vaccinium corymbosum and Vaccinium macrocarpon.
• the isolation of nucleic acids involves their separation and purification from other components of the plant mixture or extract and it can be conducted with conventional techniques using commercially available kits.
• the genomic DNA may be isolated using extraction-precipitation protocols, silica-membrane- or anion-exchange-based procedures.
• Real-time PCR technology is known in the art and it combines the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction.
• the amplification of the target DNA is obtained by repeated cycles of denaturation followed by primer- and probe annealing and by DNA polymerase-catalyzed primer extension.
• DNA amplification is monitored at each cycle ofPCRby measuring a fluorescent signal which is produced for instance by non-specific fluorescent dyes that intercalate with double-stranded DNA or by sequence specific DNA probes consisting of oligonucleotides labelled with a fluorescent reporter which allows for detection after probe hybridization with its complementary DNA target.
• Suitable intercalating dyes include SYBR® (Green I, Green II, Gold), LC Green®, SYTO-(9, 13, 16, 60, 62, 64, 82), BOBO-3, LCGreen®, POPO-3, BEBO, T0-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 quencher of fluorescence 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 of the fluorescence intensity.
• fluorophores examples include 5- or 6-carboxyfluorescein (5- or 6-FAM), tetrachlorofluorescein (TET), hexachloro-6- carboxyfluorescein (HEX), 6-carboxy-4 , ,5 , -dichloro-2 , ,7 , -dimethoxyfluorescein succinimidyl ester (JOE), tetramethylrhodamine (TAMRA),
• quenchers include those of the BHQ (Black Hole Quencher®) family, NFQ-MGB (non-fluorescent quencher and minor groove binder), QSY 7 or 21 carboxylic acid succinimidyl ester.
• the parameters and conditions of the rtPCR can be adjusted depending on the nucleic acid fragment to be amplified, on the set of primers used in the amplification and on other variables, as known to anyone skilled in the art.
• the nucleic acid fragments herein disclosed are amplified with primers (i) through (iv) applying the following conditions:
• primers and probe according to the invention allows for the 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.
• Vaccinium myrtillus-specific probe As reported in the experimental section, the use of a probe different from the Vaccinium myrtillus- specific probe
• kits for the identification of Vaccinium myrtillus in a botanical composition comprises at least one pair of primers selected from (i) through (iv) and the probe as above defined.
• the kit may comprise, in separate containers, reagents needed for running the (rt)PCR, particularly the deoxynucleotides and the DNA polymerase, and reagents for isolating, purifying and optionally quantifying DNA.
• the kit may also contain DNA of Vaccinium myrtillus as positive control and nuclease-free water or buffer as negative control, as well as a leaflet with the instructions for performing the PCR assay.
• the kit can be used with all commercially available Real-time PCR System.
• Figure 1 rt-PCR amplification protocol.
• Figure 2 rt-PCR amplification results (a) and melt curve analysis (b) of genomic DNA isolated from Vaccinium myrtillus frozen fruit.
• Figure 3 Standard curve analysis for Vaccinium myrtillus.
• Figure 4 Probe-based rt-PCR amplification with M-FAM probe specific for V. myrtillus NTC: negative control.
• Figure 5 (a) Probe-based rt-PCR amplification with M-FAM probe specific for
• NTC negative control
• Figure 7 Experimental scheme used for rt-PCR analysis of dry extract samples.
• Figure 8 rt-PCR amplification of residual DNAs isolated from Vaccinium myrtillus dry- extract samples.
• the positive control is the gDNA extracted from Vaccinium myrtillus frozen fruit a) Primer set L; b) Primer set S.
• Figure 9 Agarose gel analysis of rt-PCR amplicons.
• Figure 10 Alignment analysis of sequenced amplicons (top) and relative sequence identity matrix (bottom).
• Figure 11 rt-PCR of Vaccinium myrtillus E. ET. 36%. Amplification and melt curves.
• Figure 12 rt-PCR of Vaccinium myrtillus E. ET. 36% with probe-based method.
• PTC positive control (gDNA extracted from frozen fruit of V. myrtillus );
• NTC negative control.
• the DNA extraction was performed by using the NucleoSpin® plant II protocol as described by the supplier (Macherey nagel. Cat. 740770.250 - July 2014/Rev.09).
• the first purification was done by using the kit NucleoSpin® plant II Maxi protocol as described by the supplier (Macherey nagel. Cat. 740770.250 - July 2014/Rev.09), with some modification reported below.
• the second purification was done by using the kit ReliaPrepTM DNA Clean-UP and
• the DNA was quantified through the NanoQuant PlateTM instrument. The quantification was performed by using the UV-method. The 260 nm absorbance was used to quantify the DNA as 1 OD at 260 nm correspond to 50 pg/m ⁇ of DNA. The 260 nm/280 nm absorbance ratio was determined for the assessment of DNA purity.
• the rt-PCR amplification was performed by using the SYBR Green or probe based chemistry as described by the supplier (SsoAdvancedTM Universal SYBR® Green Supermix, BioRad Cat. N. 1725272; SsoAdvancedTM Universal Probes Supermix, BioRad Cat. N. 1725281), with 3 -step based amplification protocol, as reported in Figure 1.
• the amplified DNA was purified on agarose gel and the purified fragment was sequenced through the generation of two sequences for each sample: one is generated by using forward primer and the other one by using reverse primer.
• Each sequencing tube was prepared by mixing the purified DNA and TRIS-HCl 5 mM pH 8.0 in order to obtain the concentration requested for the sequencing (depending on the length of the sequence, 2-5 ng/pL).
• sequences were analysed by using BioEdit or BLAST software in order to compare and identify the sequences.
• the gDNA was purified and quantified (Table 1) for the Vaccinium myrtillus frozen fruit and its contaminant/related species hereafter reported:
• the rt-PCR was also performed with DNA isolated from V. myrtillus contaminant/related species and the results showed that it is possible to distinguish the different
• the rtPCR was conducted with the Minor Groove Binding- Probe (M-FAM -SEQ ID NO: 13) specifically designed to enable the amplification of V. myrtillus sequences.
• M-FAM -SEQ ID NO: 13 Minor Groove Binding- Probe
• the DNA was amplified for the positive control as well as for all tested samples; the negative control (no DNA) showed no amplification signal;
• the agarose gel analysis confirmed the differences of the amplicons length: the fragment generated with primer set S shows a length of about 130 bp, while the fragment generated with primer set L shows a length of about 270 bp. Moreover, from gel agarose analysis it is possible to see also the presence of unspecific rt-PCR products, as in Figure 9, lane 4 for the sample 32549/H83 1 where two bands are visible, in good accord with Tm peak results ( Figure 8, b).
• Example 3 Vaccinium myrtillus 36% dry ethanolic extract (E. ET.) residual DNA identification
• the kit is composed by:
• the kit can be used with all commercially available Real-time PCR System

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