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WO1998014607A1 - Methode pour detecter une espece vegetale particuliere dans un produit - Google Patents

Methode pour detecter une espece vegetale particuliere dans un produit Download PDF

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Publication number
WO1998014607A1
WO1998014607A1 PCT/GB1997/002688 GB9702688W WO9814607A1 WO 1998014607 A1 WO1998014607 A1 WO 1998014607A1 GB 9702688 W GB9702688 W GB 9702688W WO 9814607 A1 WO9814607 A1 WO 9814607A1
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WO
WIPO (PCT)
Prior art keywords
seq
dna
sequence
plant species
product
Prior art date
Application number
PCT/GB1997/002688
Other languages
English (en)
Inventor
Angus Ian Knight
Original Assignee
The Minister Of Agriculture Fisheries And Food
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9620498.7A external-priority patent/GB9620498D0/en
Priority claimed from GBGB9624083.3A external-priority patent/GB9624083D0/en
Application filed by The Minister Of Agriculture Fisheries And Food filed Critical The Minister Of Agriculture Fisheries And Food
Priority to AU45627/97A priority Critical patent/AU4562797A/en
Priority to GB9907356A priority patent/GB2333361B/en
Publication of WO1998014607A1 publication Critical patent/WO1998014607A1/fr

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Classifications

    • 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
    • C12Q1/6876—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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a method for detecting or identifying plants or plant derived material within food products , to reagents and in particular oligonucleotide primers for use in said methods .
  • the methods may be used to detect certain fruit species in particular citrus fruits such as mandarin, for example within fruit containing food products or juice products , or particular oil components such as olive oil , to determine the authenticity of said products .
  • RAPD Polymorphic DNA analysis
  • oils which may be derived from a wide variety of plant sources.
  • the quality and the cost of oils produced from different plants varies a great deal and thus adulteration of oils with cheaper varieties is also a problem.
  • the present invention provides a method for detecting a material derived from a particular plant species in a product, such as a food product, said method comprising extracting chloroplast DNA from said product and analysing said DNA to detect a sequence which is characteristic of said particular plant species.
  • the characteristic sequence selected does not exceed 250 base pairs in length. This is due to the degradation of DNA during processing of products for example, in food processing.
  • Chloroplast DNA can be extracted from products such as fruit juices, concentrates, jams, yoghurts and oils using techniques known in the art.
  • kits are available commercially which will yield DNA from food products such as fruit juices and oils as well as from leaves and fruits of known species which may be required in the methods of the invention for comparison purposes .
  • the sequences which are characteristic of the particular plant species are identified using Cleavable Amplifiable Sites Analysis (CAPS) .
  • Cleavable Amplifiable Sites Analysis Cleavable Amplifiable Sites Analysis
  • amplification primers are prepared based upon known conserved sites which flank variable regions within the DNA. The primers are designed so that in use, the variable region between the conserved sites is amplified. The amplification product may then be analysed.
  • heteroduplex analysis is used in order to detect and even to quantify the amount of particular species or adulterant present.
  • amplification primers are selected from conserved chloroplast DNA. Specifically they are selected so that they produce PCR products from different plant species that differ by sequences for example of from 3-10 base pairs which are the result of small insertion/deletion events. Amplification using these primers results in amplification of both the authentic sample and adulterant with equal efficiency. The amplification of authentic samples in the presence of adulterant results in the formation of heteroduplexes The heteroduplexes can be detected and resolved on non-denaturing gels such as polyacrylamide gels (PAGE) and quantified by known fluori etric methods. The ratio of heteroduplex:homoduplex product allows a simple quantitative measure of adulteration. Chloroplast DNA is ideally suited to this type of analysis as naturally occurring sequences capable of forming heteroduplexes of this type occur frequently as illustrated hereinafter.
  • RNA samples can be carried out using amplification techniques such as the Polymerase Chain reaction (PCR) or the ligase chain reaction, followed by analysis of the amplified product, for example using restriction fragment length polymorphism (RFLP) analysis, in which the product is digested using restriction enzymes and the restriction fragments are detected on a gel such as an agarose gel using conventional methods.
  • PCR Polymerase Chain reaction
  • RFLP restriction fragment length polymorphism
  • oligonucleotide probes such as radiolabelled probes or fluorescently labelled probes can be used to detect particular sequences as is understood in the art. These methods are particularly useful if, once the variable regions have been identified, they are sequenced and polymorphisms between species identified. These polymorphisms may then be detected in the products under test using methods conventional in the art.
  • sequence of the variable region can be used to design primers which will amplify a region which is specific to only one plant such as one fruit, for example mandarin.
  • Particular sequences which are useful in the analysis comprise all or part of the DNA sequence between the chloroplast genes rJc (large subunit of ribulose biphosphate carboxylase) and psal or between tRNA genes trnT and fcraL. In citrus species, these regions are generally of the order of 3. Okb and l.lkb in length respectively.
  • Suitable primers for amplification of these particular regions include the following:
  • SEQ ID NOs 3 and 4 are novel and these primers form a further aspect of the invention.
  • the region between tRNA genes trnT and trnL and the primers thereof have previously been identified (Taberlet et al., Plant Molecular Biology (1991) 17, 1105-1109).
  • the 3. Okb region between the rbc and psaJ genes are particularly useful as this contains sequences which are characteristic of most fruits, including berries such as raspberries, strawberries blackberries etc., citrus fruits, such as oranges, mandarins, grapefruit and lemons, and others such as apples, pears, plums, bananas etc. This region is long enough to provide characteristic restriction fragment length polymorphisms for all such species.
  • restriction endonucleases for the analysis of this region are ApoJ and Hinf I or isoschizomers of these enzymes, although others may be employed.
  • the DNA region between the chloroplast tRNA genes trnT and trnL has been sequenced in orange (SEQ ID NO 1) and mandarin (SEQ ID NO 2) as illustrated hereinafter ( Figure 1), and the polymorphisms identified can be used to distinguish between these very similar fruit species.
  • the invention provides a method for detecting the presence of C. reticulata in a sample containing C sinensis which method comprises extracting DNA from said sample, carrying out an amplification reaction using primers which are able to amplify a region of DNA comprising part of the C. reticulata sequence shown in Figure 1 but not the corresponding part of the C. sinesis sequence shown in Figure 1, and detecting the presence of amplified product.
  • Polymorphisms identified in Figure 1 which can be used in primer design are designated 1 and 2.
  • Specific primers for use in this embodiment include the following:
  • SEQ ID NOs 7, 8,9 and 10 are novel and form a further aspect of the invention.
  • the PCR reaction of the invention may be carried out as part of a multiplex PCR reaction, for instance using primers specific for a conserved region to provide an internal standard.
  • a suitable internal standard is derived from the large subunit of the ribulose biphosphate carboxylase (rbc L) gene.
  • Particular primers to achieve this include the following:
  • Heteroduplex analysis as outlined above and illustrated below is a further possibility for allowing quantitative determination of adulterant in products.
  • Amplification across an indel sequence such as indel 1 or indel 2 as illustrated in Figure 1 would produce a suitable detectable heteroduplex product.
  • Specific primers for example for amplication of the indel 2 region of Figure 1 are as follows:
  • the PCR conditions are chosen so as to provide adequate specificity of the reaction. These conditions will be known to the skilled person or could be readily determinable using routine methods.
  • the reaction is suitably carried out in the presence of magnesium ions, (although the concentration of these ions has not been found to be particularly critical) and annealing temperatures in the range of 50-65°, preferably from 55-60°C depending upon the particular reaction being effected are employed. Reaction times can be varied, for instance between 15 seconds and 1 minute in the cycling, with longer reaction times of from 3 to 10 minutes preferred at the intial denaturatio.n and final polymerisation steps. Examples of suitable conditions include the following steps:
  • the methods of the invention may be suitably adapted to quantify the detected plant species, using technology known to the skilled person.
  • quantification may be effected using a fluorescence based PCR product quantification system such as the TAQMAN TM from Perkin-Elmer. This system uses selective cleavage of a probe oligonucleotide during the PCR in order to provide a "real-time" picture of the progress of the PCR reaction. It offers the potential for accurate quantification of PCR products.
  • an amplification reaction such as a PCR reaction may be quantified using fluorescence
  • an oligonucleotide which hybridises with the target sequence is immobilised on a waveguide of an evanescent wave detector, and a fluorescent label is provided which becomes bound to the hybridised product but not to the unhybridised immobilised oligonucleotide.
  • the levels of fluorescence generated at each cycle of the amplification reaction can be measured and related to the amount of target sequence present in the sample.
  • the methods of the invention provide a simple and robust method for determining the authenticity of plant products at the genus and species level.
  • the sensitivity of chloroplast based PCR reactions in particular is approximately 100 -fold greater than that of reactions based on genomic DNA. This provides a much greater level of sensitivity when applied to food products, particularly processed food products.
  • the methods may also be of use for phylogenetic analysis by plant breeders for example in fruit production, particularly by citrus fruit growers, for reliably determining the parentage of any particular line. For instance, the reaction using primer set A above has been used to demonstrate that chloroplast DNA is maternally inherited in citrus species.
  • Kits for use in the above described method form a further aspect of the invention.
  • Such kits will comprise at least some of the reagents necessary for carrying out the method, in particular, oligonucleotides which are suitable for amplification of a sequence which is characteristic of the particular plant species being detected, such as PCR primers. They may additionally comprise other reagents used in the amplification process such as enzymes like thermostable polymerase enzymes and/or buffers. In some cases, they may also comprise reagents used in the extraction of DNA from food products.
  • Figure 1 shows the partial sequences of orange and mandarin DNA amplified using PCR primers chl A and chl B;
  • Figure 2 shows the results of the application of CAPS analysis to Mandarin/Orange DNA mixes; in this Figure, Lanes 1 and 11 are 0X174 Hae III size markers, Lane 2 is 100% mandarin, Lane 3 is 75% mandarin, Lane 4 is 50% mandarin, Lane 5 is 25% mandarin, Lane 6 is 10% mandarin, Lane 7 is 5% mandarin, Lane 8 is 1% mandarin, Lane 9 is 0.5% mandarin and Lane 10 is 0% mandarin (100% orange) ;
  • Figure 3 illustrates PCR assays for mandarin (C. reticulata) ;
  • Figure 4 shows the PCR detection of mandarin DNA in mandarin/orange DNA mixes
  • Figure 5 shows the results of multiplex PCR analysis of concentrate derived fruit juices including commercially available fruit juices
  • Lanes 1 and 9 are 0X174 Hae III size markers
  • Lanes 2, 3 and 4 are concentrate derived orange juices obtained from retail outlets
  • Lanes 5, 6 and 7 are concentrate derived orange juices prepared from orange juice concentrates
  • Lane 8 is concentrate derived mandarin juice prepared from mandarin concentrate
  • Figure 6 is the results of heteroduplex analysis using PCR, in this Figure, Lanes 1 and 10 are 0X174 Hae III size markers, Lane 2 is 100% mandarin juice, Lane 3 is 50% mandarin juice, Lane 4 is 25% mandarin juice, Lane 5 is 15% mandarin juice, Lane 6 is 10% mandarin, Lane 7 is 5% mandarin juice, Lane 8 is 1% mandarin juice and Lane 9 is 0% mandarin juice (100% orange juice) .
  • PCR amplification reactions were performed in a total volume of 50 ⁇ l in 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25 °C) , 1% Triton X-100, 2.0 mM MgCl2 , 100 ⁇ M each of dATP, dCTP, dGTP, and dTTP;
  • the PCR reaction may be carried out using polymerase enzymes which are active only at high temperatures, for example at 95°C, such as Amplitaq Gold
  • Amplifications were for 3 min at 94°C followed by 35 cycles of 1 min at 94°C, 1 min annealing at 60°C and 1 min extension at
  • extension was carried on for 10 min at 72°C prior to holding at 18°C.
  • Amplification products (10 ⁇ l) were separated by electrophoresis in 1.5 % agarose gels and visualized after staining with ethidium bromide solution.
  • PCR primer pairs used were SEQ ID NOs 3 and 4 and SEQ ID NOs 5 and 6 given above.
  • the products of these reactions were designated the Region A and Region B products respectively.
  • PCR products were phenol/chloroform extracted and ethanol precipitated. DNA was resuspended in 30 ⁇ l TE (lOmM Tris pH 8.0,
  • Restriction fragments were either separated by electrophoresis using 2% high- resolution agarose gels (region B) , ('Metaphor' agarose, FNC, Rockland, USA, or conventional 1.5% agarose gels (region A) . Restriction fragments were visualised after staining with ethidium bromide.
  • Region B ( 1100bp ) RegionA ( 3000bp) trnT - trnL rbcL -psal
  • Regions A and B and particularly Region A could be used to di s tinguish frui t species using CAPS .
  • Multiplex PCR to detect mandarin specific sequences A multiplex PCR reaction using primer pair A from example 3 as well as primers for the large sub-unit of the highly conserved chloroplast ribulose biphosphate carboxylase (rbc L) gene were included at 75 ⁇ M. These primers were as SEQ ID NOs 11 and 12 above. Otherwise, the PCR conditions were the same as those described in Example 1 above.
  • DNA may be extracted directly from small quantities of juice (up to 200 ⁇ l) .
  • juice for highly acidic juices such as orange juice, the juice was first neutralised by the addition of 1/10 volume of 2M Tris HC1 buffer of pH 8.0.
  • DNA extraction was carried out using a modified version of the protocol supplied with the kit as follows:
  • Cell lysis was effected using the reagents supplied with the kit and following the manufacturers instructions for this step.
  • the lysed sample was placed on ice for 20 minutes and then extracted with chloroform (500 ⁇ l) .
  • the resulting aqueous phase was then extracted with chloroform (500 ⁇ l) and Nucleon Phytopure DNA extraction silica suspension (lOO ⁇ l) .
  • the mixture was shaken for
  • the DNA was then resuspended in TE buffer (20-50 ⁇ l) and 2 ⁇ l used for PCR.
  • PCR conditions used were as outlined in Example 1.
  • the amplication product spans indel 2 as shown in Figure 1.
  • the gel was subjected to circulation cooling to ensure that the temperature remained of the order of 20 °C although the temperature may be allowed to vary provided it does not rise high enough to cause significant denaturation.
  • Alternative conventional electrophoretic conditions e.g. Temperature or Non-denaturing Gradient Gel Electrophoresis
  • Heteroduplex bands are clearly visible in most adulterated samples .

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Abstract

L'invention concerne une méthode pour détecter une espèce végétale dans un produit, tel qu'un produit alimentaire, consistant à extraire l'ADN des chloroplastes dudit produit et à analyser ledit ADN pour détecter une séquence qui est caractéristique de l'espèce végétale particulière considérée. On a identifié les régions de l'ADN des chloroplastes qui contiennent des séquences présentant des caractéristiques appropriées. La méthode peut être adaptée pour pouvoir déterminer la quantité de l'espèce détectée dans un échantillon particulier. La méthode peut être utilisée pour mettre en évidence la présence d'une espèce végétale étrangère dans les produits dérivés de fruits et dans les huiles et elle est particulièrement utile pour identifier l'ADN des mandarines. L'invention concerne également de nouveaux réactifs et, en particulier de nouveaux oligonucléotides destinés à la méthode, ainsi que des matériels de diagnostic permettant de mettre en oeuvre la méthode de l'invention.
PCT/GB1997/002688 1996-10-02 1997-09-30 Methode pour detecter une espece vegetale particuliere dans un produit WO1998014607A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU45627/97A AU4562797A (en) 1996-10-02 1997-09-30 Method for detecting a particular plant species in a product
GB9907356A GB2333361B (en) 1996-10-02 1997-09-30 Method for detecting a particular plant species in a product

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB9620498.7A GB9620498D0 (en) 1996-10-02 1996-10-02 Analytical method
GB9620498.7 1996-10-02
GBGB9624083.3A GB9624083D0 (en) 1996-11-20 1996-11-20 Analytical method
GB9624083.3 1996-11-20

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19745196A1 (de) * 1997-10-13 1999-04-15 Alcum Gmbh Analyseverfahren für den Nachweis pflanzlicher Zusätze in Lebensmitteln
US6500616B1 (en) 1998-04-16 2002-12-31 Case Western Reserve University Methods of monitoring genomic integrity and detecting genomic destabilization of plant cells in tissue culture
ES2246614A1 (es) * 2002-12-12 2006-02-16 S.A.T. N. 3189 VIVEROS "SOL DE RIU" Procedimiento de identificacion molecular de una variedad de clementinas.
US7037653B2 (en) 2000-03-08 2006-05-02 Consejo Superior De Investigaciones Cientificas Molecular methods for detecting guar gum additions to locust bean gum
WO2008081052A1 (fr) * 2006-12-29 2008-07-10 Fundacion Azti-Azti Fundazioa Procédé de détection de mandarine
CN107904288A (zh) * 2018-01-08 2018-04-13 集美大学 一种鉴定橄榄油掺杂的检测方法
CN108070587A (zh) * 2018-02-11 2018-05-25 云南省烟草农业科学研究院 一种鉴定食品中植物源性成分的特异性引物对及其应用
CN110273023A (zh) * 2019-08-08 2019-09-24 广州中医药大学(广州中医药研究院) 肉桂及其近缘植物的鉴定或鉴别方法，和该方法所用的引物及试剂盒

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0678770A (ja) * 1992-09-03 1994-03-22 Kirin Bibaretsuji Kk 遺伝子増幅用プライマー
JPH0759577A (ja) * 1993-08-23 1995-03-07 Kirin Bibaretsuji Kk プローブ及びプライマーの製造方法
GB2283568A (en) * 1993-10-13 1995-05-10 Mini Agriculture & Fisheries Identification of the origin of fruit or fruit juice

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0678770A (ja) * 1992-09-03 1994-03-22 Kirin Bibaretsuji Kk 遺伝子増幅用プライマー
JPH0759577A (ja) * 1993-08-23 1995-03-07 Kirin Bibaretsuji Kk プローブ及びプライマーの製造方法
GB2283568A (en) * 1993-10-13 1995-05-10 Mini Agriculture & Fisheries Identification of the origin of fruit or fruit juice

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LURO F. ET AL.,: "DNA amplified fingerprinting, a useful tool for determinantion of genetic origin and diversity analysis in Citrus", HORTSCIENCE, vol. 30, no. 5, - August 1995 (1995-08-01), pages 1063 - 1067, XP002051823 *
PATENT ABSTRACTS OF JAPAN vol. 018, no. 334 (C - 1216) 24 June 1994 (1994-06-24) *
PATENT ABSTRACTS OF JAPAN vol. 095, no. 006 31 July 1995 (1995-07-31) *
TABERLET P. ET AL.,: "Universal primers for amplification of three non-coding regions of chloroplast DNA", PLANT MOLECULAR BIOLOGY, vol. 17, - 1991, pages 1105 - 1109, XP002051822 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19745196A1 (de) * 1997-10-13 1999-04-15 Alcum Gmbh Analyseverfahren für den Nachweis pflanzlicher Zusätze in Lebensmitteln
DE19745196C2 (de) * 1997-10-13 1999-12-30 Alcum Gmbh Oligonukleotid für den Nachweis pflanzlicher Zusätze in Lebensmitteln
US6500616B1 (en) 1998-04-16 2002-12-31 Case Western Reserve University Methods of monitoring genomic integrity and detecting genomic destabilization of plant cells in tissue culture
US6773889B2 (en) 1998-04-16 2004-08-10 Case Western Reserve University Method for detecting genomic destabilization arising during tissue culture of plant cells
US7037653B2 (en) 2000-03-08 2006-05-02 Consejo Superior De Investigaciones Cientificas Molecular methods for detecting guar gum additions to locust bean gum
ES2246614A1 (es) * 2002-12-12 2006-02-16 S.A.T. N. 3189 VIVEROS "SOL DE RIU" Procedimiento de identificacion molecular de una variedad de clementinas.
ES2246614B1 (es) * 2002-12-12 2007-04-01 S.A.T. N. 3189 VIVEROS "SOL DE RIU" Procedimiento de identificacion molecular de una variedad de clementinas.
WO2008081052A1 (fr) * 2006-12-29 2008-07-10 Fundacion Azti-Azti Fundazioa Procédé de détection de mandarine
CN107904288A (zh) * 2018-01-08 2018-04-13 集美大学 一种鉴定橄榄油掺杂的检测方法
CN108070587A (zh) * 2018-02-11 2018-05-25 云南省烟草农业科学研究院 一种鉴定食品中植物源性成分的特异性引物对及其应用
CN110273023A (zh) * 2019-08-08 2019-09-24 广州中医药大学(广州中医药研究院) 肉桂及其近缘植物的鉴定或鉴别方法，和该方法所用的引物及试剂盒
CN110273023B (zh) * 2019-08-08 2023-04-11 广州中医药大学(广州中医药研究院) 肉桂及其近缘植物的鉴定或鉴别方法，和该方法所用的引物及试剂盒

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