WO1998004737A1 - Detection of d genome in wheat species - Google Patents

Detection of d genome in wheat species Download PDF

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Publication number
WO1998004737A1
WO1998004737A1 PCT/GB1997/001988 GB9701988W WO9804737A1 WO 1998004737 A1 WO1998004737 A1 WO 1998004737A1 GB 9701988 W GB9701988 W GB 9701988W WO 9804737 A1 WO9804737 A1 WO 9804737A1
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Prior art keywords
sequence
seq
genome
nucleotide sequence
pcr
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PCT/GB1997/001988
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French (fr)
Inventor
Gordon Wiseman
Glenn James Bryan
Michael Dennis Gale
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The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
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Application filed by The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland filed Critical The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
Priority to AU36299/97A priority Critical patent/AU3629997A/en
Publication of WO1998004737A1 publication Critical patent/WO1998004737A1/en

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    • 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 certain species of wheat such as common wheat ( T ⁇ ticum aestivum), in particular when these are present in food products such as pasta, as well as nucleic acids which are useful in said method.
  • wheat such as common wheat ( T ⁇ ticum aestivum)
  • T ⁇ ticum aestivum common wheat
  • nucleic acids which are useful in said method.
  • the cultivated emmer wheats appear to have been subjected, whether intentionally or not to selective pressures that discriminated in favour of the wheat that had more durable seed heads and stayed on the stem during harvesting and were easier to thresh.
  • T. durum a wheat that produced a rough and particular flour (semolina) that was essentially devoid of fine flour panicles.
  • This wheat was grown particularly in countries adjacent to the Mediterranean and also in areas of America and has found particular use in the production of pasta and related products.
  • Pasta manufactured from durum wheat which is intended for export outside the EU may contain a maximum of 3% common wheat to allow for adventitious cross contamination du ⁇ ng the ag ⁇ cultural process. Such cross contamination is not unusual since there is a tendency to grow both types of wheat in similar environments and they are often grown in close proximity to one another. It has been the case for many years that common wheat has traded at a substantially lower p ⁇ ce than durum wheat and this has acted as an incentive to those adulterating pasta for financial gain.
  • a number of different analytical methods are available to address the problem of detecting the presence of T aestivum in T durum pastas. Most rely upon detecting the products of the D-genome. a chromosome group that is specific to Hexaploid wheat and hence is absent from T durum. These methods usually involve the extraction of water or alcohol soluble proteins (albumins or gliadins) p ⁇ or to their separation by either isoelect ⁇ c focusing (EEF) or by polyacrylamide gel electrophoresis (PAGE). The detection of albumins which are fractionated by IEF (P. Resmini, Tec Molitona (1968) 19, 145-150, P. Resmini et al. Tec. Molitona 27. 97- 109) have for many years formed the basis of the official Italian test method.
  • IEF isoelect ⁇ c focusing
  • PAGE polyacrylamide gel electrophoresis
  • Contamination can also be detected by PAGE of specific water extractable enzymes (polyphenol oxidases (Feillet. P and Kobrehel, K. ( 1972) Ann. Technol. Ag ⁇ c. 21, 17- 24, Feillet, P and Kobrehel, K. (1974). Cereal Chem. 51. 203-209) and esterases (Cooke, R.J.. Smith, T.M. and Ainsworth, C.C. (1986). Seed Sci Technol. 14, 693- 704) Alcohol extractable low mobility oma-ghadms (Kobrehel, K., Agaga, P., Autran, J.C ( 1985). Ann Fals Exp Chim.
  • PAGE of specific water extractable enzymes polyphenol oxidases (Feillet. P and Kobrehel, K. ( 1972) Ann. Technol. Ag ⁇ c. 21, 17- 24, Feillet, P and Kobrehel, K. (1974). Cereal Chem. 51. 203-209) and esterases (Cooke,
  • Cereal Chem, 62 (1), 72-74 which are encoded by the D-genome of non-durum wheats, when fractionated by PAGE have also been shown to be effective at detecting contamination.
  • Dgas44 is a sequence derived from Ae squarrosa, which has been identified previously (McNeil. D, et al , (1994) Genome 37, 320-327) and is shown hereinafter in Figure 1 (SEQ ID No I) Plant DNA was isolated and this sequence was cloned and identified It was thought to be highly repeated in the D-genome of hexaploid wheat, and appears to be virtually absent from the A and B genomes
  • the present invention provides a method for detecting the presence of wheat having a D-genome in a processed food product, which method comprises extracting DNA from said product and detecting the presence of a nucleotide sequence which is characteristic of the D-genome
  • Suitable food products from which DNA may be extracted include milled wheat products such as semolina or flour, or more extensively processed products, in particular pasta including dried pasta products
  • the nucleotide sequence which is detected comprises or is a D-genome specific derivative of the Dgas44 sequence
  • the sequence detected is the PSR128 sequence of the D-genome shown hereinafter in Figure 9
  • Single copy sequences are particularly prefe ⁇ ed where quantification of the amount of T aestivum is required
  • PSR128 includes a single copy sequence derived from the D-genome
  • the said nucleotide sequence may be detected by a vanety of methods which are well known in the art They may for example be detected using Southern or dot blotting techniques, or more preferably, by a preliminary amplification of the target sequence. followed by detection of the amplified product
  • a particularly suitable amplification technique uses the polymerase chain reaction (PCR) after which the amplified product is suitably detected following eiectrophoresis on agarose gels
  • the method of the invention involves the detection of all or part of the sequence of Figure 2, and more particularly, the sequence of Figure 3 Novel probes and primers which are useful is such a method form a further aspect of the invention
  • the invention further provides a nucleotide sequence which comprises or hyb ⁇ dises with all or part of the nucleotide sequence shown in Figure 2 Said sequence is suitably detected m the method of the invention
  • Suitable parts of the sequences defined above are sufficiently long to allow specificity and identification. In general therefore, the parts will be at least 15 bases in length and preferably at least 20 bases in length
  • the nucleotide sequence comprises or hvb ⁇ dises with all or part of the nucleotide sequence shown in Figure 3 (SEQ ID No 3) This sequence conesponds to bases 1864 to 2150 of the Dgas44 sequence
  • sequences used in any particular analytical technique will depend upon the nature of the technique, the degree of complementa ⁇ ty of the sequence, the nature of the sequence and particularly the GC content of the p ⁇ mer and the particular hyb ⁇ disation conditions employed Under high stringency, only sequences which are completely complementary will bind but under low stringency conditions, sequences which are 60% homologous to the target sequence, more suitably 80% homologous, will bind.
  • the sequence is used as a labelled probe (for example a radiolabelled or fluorescently labelled probe for example using Southern or Dot blotting) for direct detection of the target D-genome sequence
  • the probe length will be quite long, for example of 100 bases or more.
  • the nucleotide sequence of the invention is preferably from 10 to 30 nucleotides in length, suitably from 17 to 30 nucleotides in length. At least two such primers will be necessary in order to effect the PCR amplification as is well understood in the art. These will be non-complementary sequences which hybridise with the opposite strands of the target region of the genome when denatured and which flank the region to be amplified. Suitable primers for use in a nested PCR technique may also be derived for example from
  • Particularly preferred primers are as follows:
  • CTGAATGCCCCTGCGGCTTAAG SEQ ID NO 5
  • GTCCTATATCTTGAGGCCGCAAG SEQ ID NO 6
  • AACCCACTGTACCTGAGTATATATC (SEQ ID NO 7)
  • SEQ ID Nos 4 and 6 are suitably forward primers for PCR amplification whilst SEQ ID Nos 5 and 7 are reverse primers.
  • a particularly prefe ⁇ ed pair of p ⁇ mers comp ⁇ se SEQ Id Nos 4 and 5
  • said method comprises amplifying the sequence of Figure 2. or preferably of Figure 3. using at least two primers and detecting amplified product.
  • the methods of the invention is earned out so that the amount of said species of wheat present in the sample is quantified This may be done, simply by companng signals produced for example using PCR amplification with those produced from control samples of known contamination levels It has been found by carrying out PCR experiments using control pastas, that an increase in the amount of PCR product using various p ⁇ mers has been seen as the level of bread wheat contamination increases
  • an internal standard or reference product not arising from the D-genome that can be used to calibrate the PCR-assays internally, is used.
  • a second nucleotide sequence is also amplified by multiplexing with additional appropriate primers. This second sequence is one which is found on the A and B and D genomes. In this way, detection of the second nucleotide sequence will provide an internal standard, against which levels of the D-genome specific sequence can be assessed to give relative proportions of the components.
  • quantification is effected by amplifying said target sequence by the using a pair of primers in the presence of a known concentration of a further sequence which competes with the said sequence for a primer, but which is distinguishable from said sequence during detection, and comparing signals produced by said further sequence and said target sequence.
  • This method is based upon a known protocol (F ⁇ rster, E.( 1994). Biotechniques. 16, 20-24).
  • a schematic diagram of the method used is shown in Figure 4 hereinafter. Basically the method entails the generation of a "synthetic" PCR product, of slightly shorter length than the PCR product generated from the template DNA, but having the same p ⁇ mer sites at its termini.
  • This short-product is engineered by a two-stage PCR process, using the original full-length PCR product as template.
  • the method employs "chimeric" primers at one end of the PCR reaction, in our case the 3' terminus.
  • This "short product” is then added to PCR reactions in differing concentrations, in which it can compete against the template DNA for the primers.
  • the signal strength arising from a known amount of the short product is then used as a control to estimate the amount or number of copies of the original template, for example, the amount of contaminating Dgas44 sequence arising from D-genome contamination of durum wheat samples or pasta products.
  • This assay would be independent of the DNA concentration or level of DNA quality or average size
  • the applicants have generated a PCR product using primers of SEQ ID Nos 4 and 5. and a shortened version thereof ("short PCR product”)as illustrated hereinafter, and have used this in a quantification assay
  • cloned short-PCR product in circular form is used
  • plasmid borne is used
  • Such a product will be easy to generate in large quantities and may minimise contamination' of PCR procedures (such a sequence may be extremely "invasive" sequence, and extreme care has to be taken to ensure that this sequence does not contaminate other PCR reactions using closely related primers)
  • quantification is suitably effected using a fluorescence based PCR product quantification system such as the TAQMAN TM from Perkin-Elmer
  • a fluorescence based PCR product quantification system such as the TAQMAN TM from Perkin-Elmer
  • TAQMAN TM fluorescence based PCR product quantification system
  • the amplification reaction is effected in the presence of a probe which includes a quencher moiety and a fluorescence reporter, said probe being arranged to be selec ely cleaved du ⁇ ng the amplification reaction so as to produce a flurorescent signal
  • the signal which is generated by laser induction, is monitored by and the rate of increase used to calculate the amount of target sequence present in the sample
  • the probes will be refe ⁇ ed to only by reference to the characteristic sequence which they contain It should be understood that the fluorescent
  • Such reactions can be used to quantify the amount of f aestivum present in a sample of known wheat content
  • a PCR reaction and a TAQMANTM probe designed to amplify and detect a sequence which is common to all the genomes is earned out in parallel
  • Such a reaction in effect measures the amount of wheat DNA in the sample in the reaction.
  • the signal generated by the D-genome specific amplification reaction could then be standardised against this measurement, and so an accurate percentage figure for any contaminant T. aestivum can be established.
  • the standardising reaction may be effected in a separate tube containing an equal aliquot of DNA being investigated. Alternatively, use of distinguishable laser dye mixtures would allow the reactions to be effected in the same reaction tube.
  • a useful TAQMAN probe which has been successfully been used in quantitative PCR reaction to amplify the sequence of Figure 3 is a probe based upon base numbers 1 88- 2014 of the Dgas44 sequence.
  • the TAQMANTM probe comprises SEQ ID No .10
  • PSR128 is an intron sequence derived from a cDNA clone. Techniques for mapping this probe are described by D.X. Xie et al., Theor. Appl. Genet (1993) 87: 70-74. The size of the intron differs in each of the genomes of hexaploid wheat.
  • Figure 9 shows the PSR128 sequence found in the D genome (SEQ ID NO. 19).
  • the D-genome copy of the intron includes a unique sequence (bases 62-114).
  • primers which will achieve this would be determinable by the skilled person.
  • at least one of the primers is derived wholly or partly from the D-genome unique sequence.
  • the probe may itself be derived from said sequence. Examples of suitable primers for the amplification of the D-genome PSR 128 sequence are
  • a TAQMANTM probe may further be employed to quanitate the PCR reaction, for example as follows:
  • DNA could be isolated from these and other commercially available pastas through the use of conventional extraction procedures, for example using an extraction method based upon a phenol/chloroform mixture
  • the isolated DNA was detectable using the methods of the invention, and in particular, after suitable purification, it is suitable for both amplification using PCR and for use in hybridisation protocols.
  • the target DNA is preferably subjected to analysis as soon as possible since stored material may suffer from some degradation, possibly as a resuit of theco-pu ⁇ fication of nucleases which may further degrade the template
  • nucleases are removed from the DNA, for example using a solid-phase cleanup procedure as is known in the art
  • this technique may be employed to identify the presence of D-genome charactensmg nucleotide sequences from DNA from any source, and therefore may be used for instance in the identification of particular species.
  • the invention further provides a method for identifying the presence of a D-genome in a crop sample, which method compnses extracting DNA from said sample, and detecting the presence of a nucleotide sequence which comprises or hybridises with all or part of the nucleotide sequence shown in Figure 2. for example, by amplifying said sequence using PCR
  • PCR reactions will be effected with at least 25-30 cycles in the presence of a magnesium ion concentration of from 0.5 to 4 OmM. preferably about 2.0mM.
  • the polymerase enzyme used is a thermostable polymerase such as Taq polymerase It is suitably present in concentrations of from 0 25U to 0.5U/50 ⁇ l.
  • Figure 1 shows the nucleotide sequence of the Dgas44 sequence
  • Figure 2 shows the region from base no. 1532-2150 of the Dgas44 sequence
  • Figure 3 shows the region from base no 1864-2150 of the Dgas44 sequence
  • Figure 4 illustrates the method used for synthesis of a PCR competitor template
  • Figure 5 shows the results of a 25 ⁇ l touchdown PCR 68-60 reaction using pnmers of SEQ 4 and 5. in which lO ⁇ l PCR product was run on a 1 % agarose gel;
  • Figure 6 shows the results of a 25 ⁇ l touchdown PCR 68-60 reaction using p ⁇ mers of SEQ ID Nos 4 and 5 and also SEQ ID Nos 8 and 9, and also p ⁇ mers in which lO ⁇ l PCR product was run on a 1% agarose gel;
  • Figure 7 shows the results of a dot blot with different amount of 56 pasta DNA per well, which was hyb ⁇ dised to a radiolabelled probe de ⁇ ved from Dgas44 sequence;
  • Figure 8 shows the results of a touchdown 68-60 PCR reaction using p ⁇ mers de ⁇ ved from Dgas44 in a 50 ⁇ l reaction using 0.5 ⁇ l Taq per tube, and with lO ⁇ l PCR product run on a 1 % agarose gel.
  • Figure 9 shows the sequence of an intron sequence designated PSR128 in the D genomes of T aestivum:
  • Figure iO shows a portion of the sequences of Figure 9 with PCR p ⁇ mer and TAQMANTM probe underlined;
  • Figure 1 1 shows a typical result of quantification assay of d ⁇ ed durum pasta using the TAQMANTM methodology for a pure durum sample (0% adulteration) and a sample containing 1 % adulteration:- the method determines the cycle threshold value (Ct) which is the PCR cycle or part of a cycle when the detected fluorescence differs from the 0% standard by 6 standard deviations.
  • Ct cycle threshold value
  • Figure 12 shows typical plots similar to those of Figure 1 1 but using dried pasta with 0%, 1% and 2% adulteration (each in triplicate);
  • Figure 13 shows typical plots similar to those of Figure 1 1 but using dried pasta with 0%, 1 %, 2% and 3% adulteration (each in triplicate);
  • Figure 14 is the calibration plots for the three sets of standard pastas (56, 80 and 104°C dried) assessed using a TAQMANTM probe to Dgas44;
  • Figure 15 presents the calibration plots derived from all of the data presented in Figure 14 which would be used to assay pastas whose thermal history was unknown;
  • Figure 16 shows the calibration plots for the three sets of standard pastas (56, 80 and 104°C dried) assessed using the TAQMANTM probe to the single copy sequence PSR 128;
  • Figure 17 shows an individual calibration curve for the 104°C dried pasta standards, assessed using the the TAQMANTM probe to the single copy sequence PSR 128.
  • Example 1 The preparation of semolinas of known levels of adulteration from accredited samples of T. durum (cultivar, Neodur) and T. aestivum (cultivar. Soissons).
  • Durum grain was taken from its natural moisture content of 14.6% to a final moisture of 18.5% in two stages (a) an initial step of taking it to 17% moisture; and (b) a final addition of water to bring the moisture content to 18.5%. These steps were ca ⁇ ied out over 8 hours.
  • the grains were then milled using a Buhler (Switzerland) test mill which had been dismantled and thoroughly cleaned prior to use in order to remove all traces of any contaminating T. aestivum flour. Only the first half of the mill was used, that producing semolina.
  • a non-accredited (normal T. durum mill grist) was used to set the mill to the required settings i.e. that designed to remove the bran layer from the hard T. durum grains while yielding a semolina that was as representative as possible of the product from the larger mills. Once the desired settings were achieved, the mill was stripped down and vacuum cleaned before the test millings proceeded.
  • Neodur Deliberately adulterated Neodur was milled in ascending order of contamination with a thorough mill stripdown and vacuum clean between each test sample.
  • the pasta press Prior to the extrusion of the pasta dough, the pasta press was thoroughly cleaned by totally dismantling the dye and screw mechanism. All parts were soaked and cleaned as necessary with the mesh and dye being subjected to cleaning by pressurised jets of water in a commercial die wash The screw and die, once clean, were kept at 50°C prior to drying and assembly ready for the next experiment. This enabled the desired running temperature to be achieved more quickly (important for quality reasons).
  • the pasta produced by the above method was dried by one of three regimes, representing the cu ⁇ ent commercial practices within Europe. All drying profiles (with the exception of the 104°C step within the very high temperature profile) were performed in a Hedinair (Kingsbridge Road, Barking By-pass, Barking, Essex) programmable hot air oven with relative humidity control and humidification. The 104°C step within the very high temperature procedure was carried out using a calibrated Gallenkamp model OV- 160 hot air oven. The three temperature profiles used were as follows:-
  • a lOmg/ml protemase K stock solution ( lOO ⁇ l) was added per tube (final concentration 0 05 mg/ml)
  • the tubes were then incubated at 65°C for 1-2 h with the occasional inversion to mix the contents
  • a phenol/chloroform mixture (20ml) which had been prepared by mixing 1 part TEN buffer saturated phenol, pH 7 8 1 part chloroform isoamyl alcohol(24 1 ) was added to each tube and mixed gently by inversion to form an emulsion Cent ⁇ fugation at 2000rpm for 20 min separated phases although the addition of phenol/chloroform mixture and subsequent cent ⁇ fugation was repeated if necessary
  • Propan-2-ol (0.6 vol) was added and mixed by inversion DNA was recovered by centrifugmg at 10,000 rpm at 4°C The DNA was washed with cold 70% ethanol and air dried A further wash with 100% ethanol was performed to speed drying The DNA was suspended in lx TE buffer (5ml) (composed of lOmM T ⁇ s (pH 8 0) and ImM EDTA (pH 8 0)) and kept at 4°C Total solution can take several days, however, this can be speeded by careful warming at 65°C for up to 2hrs with gentle inversion RNase stock solution (lO ⁇ l) (comprising 1 mg/ml RNase A and lOOU/ml RNase Tl which had been heated to 100°C, lOmin to destroy DNase activity) was then added and the solution incubated at 37°C for lh The phenol/chloroform extraction was then repeated using phenol/chloroform (5ml), followed by gentle mixing by inversion and centrifuge at 2000
  • the product was extracted using an equal volume of chloroform, a step which was repeated if mate ⁇ al at the phase interface is excessive.10% volume sodium acetate (3M) (final concentration 0.3M) was added with mixing, followed by 2 vols ethanol (100%). After centrifugmg at lO.OOOrpm @ 4°C, and washing in cold 70% ethanol, the DNA product was air dried and redissolved in 1.8-2 ml TE buffer (depending upon the size of pellet) This solution was stored frozen at -20°C
  • AU ohgonucleotides used as primers were synthesised on Milhpore Expedite oligonucleotide synthesiser using Expedite ' chemistry. They were purified by first unc ⁇ mping the column and pouring glass beads into screw-capped Eppendorf mbe. 300 ⁇ l ammonia was added and the reaction tube incubated at 55°C for 30-60 minutes. The tube was then placed tube on ice for 5 minutes and centrifuged at 12000 m for 2 minutes. The supernatant was added to a new screw-cap tube, and 30 ⁇ l 3M ammonium acetate, and 3 volumes cold ethanol added.
  • CAGCTCCGAGTCAGAGGTAACC (SEQ ID NO 9)
  • Example 2 Since the amplification product using pnmers of SEQ Id Nos 8 and 9 is significantly larger than the within-domain D-genome specific products produced in Example 2, it was thought that this would provide a useful internal control for successful DNA amplification Example 2 was therefore repeated using the crop DNA as well as the pasta DNA but using a combination of two groups of p ⁇ mers comprising SEQ Id No 4, SEQ Id No5. SEQ Id No 8 and SEQ Id No 9 The results are shown in Figure 6
  • CTGAATGCCCCTGCGGCTTAAG SEQ ID NO 5
  • CTGAATGCCCCTGCGGCTTAAG SEQ ID NO 5
  • PCR in this instance was performed using a touchdown 58-54°C protocol using the product de ⁇ ved in step 2) as DNA template.
  • the touchdown PCR protocol can be summarised as follows: Programme No No of cycles Linked to programme No Segments
  • the short-PCR product has been cloned into the PGEM-T cloning vector (Promega, Madison. WI, USA).
  • T. durum cultivars were collected (55 accredited samples and 3 unaccredited) representing the major cultivars in common commercial usage and the growing areas around the world.
  • a number of accessions from the John Innes germplasm collection were added to the screening procedure. The origins of these rare cultivars were diverse, yet they represent the genetic diversity sometimes utilised within breeding programmes. They were therefore included to strengthen the robustness of the protocol particularly with respect to new cultivars that may be bred as a result of crosses with these varieties.
  • Tntordeum lines H chilense (a wild barley) crossed with either T turgidum or T aestivum
  • T durum accessions from the John Innes Centre germplasm collection that had been included as more diverse examples of the species did however produce an amplified product. This indicated that the D-genome was present in these materials Further analysis of these accessions by Acid-PAGE showed that in every case that the putative pure T durum cultivar that had been included in the germplasm collection as a T durum cultivar was either a T aestivum or a mixture of the two species.
  • DNA extracted from pasta as descnbed above was digested to completion with the following combinations of endonuc leases were used .-Taql, EcoRl/Bam l, EcoRV/Sstl,
  • Example 2 was also prepared in this way to act as positive controls.
  • a probe was prepared by PCR from the Dgas44 sequence using the following as pnmer sequences
  • SEQ ID No 12 compnses a forward p ⁇ mer based upon base 5 to 29 of the Dgas44 sequence and SEQ ID no 20 comprises a reverse primer based upon bases 408 to 431 of the Dgas 44 sequence.
  • Hybridisation was performed with a probe made by PCR from plasmids containing the Dgas44 sequence using the method of Southern, E.M (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis J.Mol.Biol. 98. 503- 517.
  • DNA extracted from pure T durum was used as a positive control as well as DNA extracted from the 56° pasta prepared as descnbed above
  • a nitrocellulose membrane was cut to 9 x 12cm and washed in a buffer comprising 175.2g sodium chloride, and 88 2g sodium citrate per litre, adjusted to pH 7 0 with NaOH (SSC) for 10-15 min. and drained well.
  • SSC NaOH
  • the membrane was placed on a sealing gasket which was fixed into dot blotting apparatus which was attached to a vacuum lOO ⁇ l 20x SSC was loaded to each well and then drained with a full vacuum for approx 5 min Subsequently DNA at concentrations of 1, 2, 5 and lO ⁇ g/well (in 60 ⁇ l 04 M NaOH solution) was loaded to each well After gentle vacuuming for 20 min until wells look empty and dry
  • P ⁇ mers of SEQ ID Nos 8 and 20 were used in a touchdown PCR reaction at conditions 68-58°C (Don R.H et al , (1991 ) Nucleic Acids Research 19, 4008) to amplify DNA prepared as descnbed in Example 2 above
  • the reagents used in a standard 50 ⁇ l reaction per tube was as follows:
  • Cresol red 1 O ⁇ l 1 Omg/ml dissolved in 60% sucrose
  • 1 Ox PCR buffer 5 ⁇ l Mg + , 15mM
  • DNTP's 2 ⁇ l 2.5mM
  • Forward p ⁇ mer 0.5 ⁇ l (20 ⁇ M)
  • Reverse p ⁇ mer 0.5 ⁇ l (20 ⁇ M)
  • NP40 0.5 ⁇ l
  • Tween-20 0.5 ⁇ l (5%)
  • PCR reactions were earned out using p ⁇ mers of SEQ ID Nos 4 and 5 as the forward and reverse primers respectively and in addition a TAQMAN TM probe of SEQ ID No 10.
  • step 8-10 a total of 3 times
  • samples prepared as described in Example 1 were subjected to quantitative PCR using the following reagents.
  • TAQMANTM probe 5 GCT AGA TGG CTG GCT TCT ATT TCA TGA TCC A 3 (SEQ ID No 19) Calibration curves similar to those described in Example 8 were prepared ( Figures 16 and 17). Reliable and accurate quantification of the amount of T. aestivum was possible using this method.
  • MOLECULE TYPE DNA (genomic)
  • CTGCAGCAAC TCCCCCTTAG CGGAGCACTT
  • GGCCAACACG GTCGGTCTCC CCGGCTCAAT 60
  • GGTACACTTC GCAAAAGACT TGGAAATTGC AGATGTTGGA TACGGAGTTG GGTCCTATAT 1740
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • TCTTTTTTCA AATATCCAGC TAGATGGCTG GCTTCTATTT CATGATCCAT CGTCTCTTCT 120
  • MOLECULE TYPE DNA (genomic)
  • TCTTTTTTCA AATATCCAGC TAGATGGCTG GCTTCTATTT CATGATCCAT CGTCTCTTCT 120

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Abstract

A method for detecting the presence of wheat having a D-genome in for example a processed food product is described. The method comprises extracting DNA from said product and detecting the presence of a nucleotide sequence which is characteristic of the D-genome and in particular is derived from the Dgas44 sequence or from the PSR128 intron. The sequence is suitably detected using PCR. A particular sequence within each of the Dgas44 and the single copy PSR128 intron has been identified as being a suitable target for detection. The method is useful in detecting contamination of flour, semolina, or pasta products which are made from T. durum by bread wheat. Reagents useful in the method as well as kits for conducting the method are also described and claimed.

Description

DETECTION OF D GENOME IN WHEAT SPECIES
The present invention relates to a method for detecting certain species of wheat such as common wheat ( Tήticum aestivum), in particular when these are present in food products such as pasta, as well as nucleic acids which are useful in said method.
Historically wheat is thought to have arisen as the product of a fortuitous' cross that is believed to have occurred about 10,000 years ago between a wild goat grass (Aegilops sp) and the ancient wild species of einkom ( . urartu). The new species (emmer wheat, T. dicoccoides) having two sets of 7 chromosomes i.e. 28 in total. This spontaneous and rare doubling of the chromosomes gave the new species the distinct advantage that instead of being a 'sterile' hybrid it was able to produce the normal pollen and egg cells required for seed production. It was this emmer wheat that essentially formed the basis of early agriculture. The cultivated emmer wheats appear to have been subjected, whether intentionally or not to selective pressures that discriminated in favour of the wheat that had more durable seed heads and stayed on the stem during harvesting and were easier to thresh. During the subsequent years a range of emmer-related wheats emerged and one of the developments was that of T. durum, a wheat that produced a rough and particular flour (semolina) that was essentially devoid of fine flour panicles. This wheat was grown particularly in countries adjacent to the Mediterranean and also in areas of America and has found particular use in the production of pasta and related products.
in addition to the developments that happened to the T. durum wheat during this period a significant development also occurred to the emmer-related wheats that were spreading throughout Europe. This progression also involved a further chromosomal transfer and lead ultimately to the emergence of the bread wheats (7. aestivum). This process once more arose by the process of hybridisation, most probably between a cultivated emmer wheat and a wild goat grass. The net result of the hybridisation was to introduce a further set of 7 chromosomes, which once more doubled to produce a plant with a total of 42 chromosomes. It is reasonably certain that the goat grass that was the donor of the chromosomes was A squarrosa (called T tauschu in North Ameπca). These 7 chromosomes are collectively known as the D' genome and represent a major advance in terms of wheat development with particular regard to its bread making quality and the technological properties of the flour These 7 chromosomes carry the alleles that encode for a group of proteins known as the glutenins. It is these proteins that produce the viscoelastic properties typically associated with bread dough, they therefore can have a major effect on the resulting bread quality They are however regarded as reducing the quality of pasta product
The majority of pasta products that are manufactured and offered for sale within the European community are made solely from Durum wheat (Tnticum durum Desf) semolina and are considered to be supeπor products to those manufactured from common wheat (Tnticum aestivum L), or mixtures of the two species of wheat The area is controlled by legislation, particularly in Europe. For instance, Italian law currently prohibits the manufacture of pasta containing T aestivum for sale in Italy but not for subsequent export Several European countries including Italy, Spam and France take the firm view that the inclusion of common wheat in pasta is effectively adulteration However, pasta made from the semolina of both wheat species is frequently manufactured for sale in Germany and Holland. Currently the United Kingdom does not have specific regulations regarding the composition of pasta. However, both the Food Safety Act 1 90 and the Food Labelling Regulations 1984 make it clear that it is an offence to misdescπbe a product or to offer it for sale in a misleading way, hence, the presence of common wheat in a pasta product must be declared on the product label
Pasta manufactured from durum wheat which is intended for export outside the EU may contain a maximum of 3% common wheat to allow for adventitious cross contamination duπng the agπcultural process. Such cross contamination is not unusual since there is a tendency to grow both types of wheat in similar environments and they are often grown in close proximity to one another. It has been the case for many years that common wheat has traded at a substantially lower pπce than durum wheat and this has acted as an incentive to those adulterating pasta for financial gain.
As a direct result of these problems a number of analytical methods have been devised to check pasta products and to veπfy the label claims that are made, in order to enforce the vaπous requirements of food legislation.
A number of different analytical methods are available to address the problem of detecting the presence of T aestivum in T durum pastas. Most rely upon detecting the products of the D-genome. a chromosome group that is specific to Hexaploid wheat and hence is absent from T durum. These methods usually involve the extraction of water or alcohol soluble proteins (albumins or gliadins) pπor to their separation by either isoelectπc focusing (EEF) or by polyacrylamide gel electrophoresis (PAGE). The detection of albumins which are fractionated by IEF (P. Resmini, Tec Molitona (1968) 19, 145-150, P. Resmini et al. Tec. Molitona 27. 97- 109) have for many years formed the basis of the official Italian test method.
Contamination can also be detected by PAGE of specific water extractable enzymes (polyphenol oxidases (Feillet. P and Kobrehel, K. ( 1972) Ann. Technol. Agπc. 21, 17- 24, Feillet, P and Kobrehel, K. (1974). Cereal Chem. 51. 203-209) and esterases (Cooke, R.J.. Smith, T.M. and Ainsworth, C.C. (1986). Seed Sci Technol. 14, 693- 704) Alcohol extractable low mobility oma-ghadms (Kobrehel, K., Agaga, P., Autran, J.C ( 1985). Ann Fals Exp Chim. 78, 109-1 17, Burgoon, A.C., Ikeda, H.S. and Tanner. S.N. ( 1985). Cereal Chem, 62 (1), 72-74 ) which are encoded by the D-genome of non-durum wheats, when fractionated by PAGE have also been shown to be effective at detecting contamination.
Other methods include the analysis of a the secondary metabolite sitosteryl palmitate by either thin layer chromatography (TLC)( Gilles, K.A. and Youngs, V.L. ( 1964). Cereal
Chem. 41, 502-513, Berry, C.P., Youngs, V.L. and Gilles, K.A. (1968). Cereal Chem, 45 (6), 616-626) or more recently by reverse phase high performance chromatography (RP-HPLC) (Sarwar, M. and McDonald, C.E. (1993). Cereal Chem, 70, 405-411) The analysis of fatty acid sterol esters by TLC following petroleum ether extraction, being the current recommended method for the detection of T aestivum adulteration of pasta within the EC
Immmunochemical assays based upon either water soluble proteins (EP-A-540,432, Piazzi. S.E. et al., ( 1969). Cereal Chem, 46, 642-646, Piazzi, S.E. et al, ( 1972). Cereal Chem. 49 ( 1 ), 72-78) or alcohoKMcCarthy, et al., ( 1992). Food Safety and Quality Assurance. (Morgan, M.R.A. and Smith, C.J. Williams, P A., Eds), Elsevier, London, UK, 19 1 (pub.1 92), 41 1 -416) soluble proteins have also been investigated. Investigations have been made using RP-HPLC, to detect and quantify the presence of T aestivum contamination in T durum semolina based on the fractionation and quantification of a group of γ/β gltadins (McCarthy, P.K., et al., ( 1990). J. Sci Food Agπc. 50. 21 1 -226). This method has recently been extended to include the analysis of pasta products dned at low and elevated temperatures (Barnwell, P et al.,( 1994). J Cereal Sci. 20, 245-252). Specific albumins present in T aestivum have also been detected in semolina using RP-HPLC (de Bemardi, I, de Noni., and Pellegπno. L ( 1994) Food Chem 51 325-329)
All the of the methods listed above have their shortcomings The pnnctpal limitation being that of protein denaturation occumng as a result of the drying processes dunng pasta manufacture. This denatunng effect of heat on the proteins assumes an increased importance due to the fact that pasta producers are tending to move to higher temperatures to effect quicker drying of the product. Such processing renders the enzymes inactive and hence the enzymic detection methods ineffective. Denaturation of many of the other proteins used as markers of T. aestivum contamination also occurs Denaturation of the γ/β gliadins causes band broadening and a shift in elution time when subjected to HPLC, making accurate quantification of the contamination impossible. The alcohol soluble omega-gliadins however, due to their lack of cysteine residues are considerably more heat stable and survive moderate thermal processing. While these proteins survive as markers of contamination, little is known about their subsequent quantification other than that their apparent recovery from high temperature processed pasta is decreased, making accurate quantification difficult. A further factor that adds to the difficulties encountered is the little studied effect of the environment on the expression of the proteins used as markers of contamination. It has been noted (Wrigley, C.W. and Shepherd, K.W. ( 1973). Ann. NY. Acad. Sci. 209, 154-162) that the albumins and globulins used by Piazzi et al (supra.) can be affected by the environmental growth conditions of the wheat, raising questions concerning the plausibility of the results. While the effect of the environmental growth conditions on other groups of wheat proteins is little studied, there is evidence (Blumenthal, C.S.. Barlow, E. W.R. and Wrigley, C.W. ( 1993). J. Cereal Sci. 18, 3) that this factor may be more important than was previously thought. The consequential effect on quantitative studies is therefore unknown.
In addition, there are intrinsic difficulties in the detection limits achievable using these methods. The limit of detection of the 7". aestivum specific omega-gliadins for instance, is about 2-3% and also that the accuracy of determination is about 2%. The International standard for durum wheat trading permits a total of 3% non-durum grains in a shipment of T. durum hence when considering if a durum pasta product is misdescribed all of these potential errors must be summed.
The problem is further compounded due to differential processing conditions used in pasta manufacture, the nature of which may not be known to the analyst. The precise effect of different thermal processing upon the analyses is at the present time unquantifiable. and so for any meaningful analysis, a set of 'control' pastas processed at standard conditions must be used to quantify the adulteration levels. However, this control would represent only a median position and would result in the under measurement of adulteration in pastas dried at higher temperatures. Conversely, adulteration in pastas dried using somewhat lower temperatures would be over estimated. Due to the problems associated with the conventional methods of analysis, the applicants addressed the detection and quantification of contamination in for example T durum pastas through the detection of DNA of the D-genome
A charctenstic sequence which would give an indication of contamination has been designated "Dgas44" which is a sequence derived from Ae squarrosa, which has been identified previously (McNeil. D, et al , (1994) Genome 37, 320-327) and is shown hereinafter in Figure 1 (SEQ ID No I) Plant DNA was isolated and this sequence was cloned and identified It was thought to be highly repeated in the D-genome of hexaploid wheat, and appears to be virtually absent from the A and B genomes
It was not clear however what the effect of the extensive processing of the wheat dunng for example pasta production, would have on the DNA. and whether it would be possible to identify any charactenstic sequences in such a product
The applicants have found however, that such a method is possible and provides an advantageous means of detecting contamination
By detecting contaminant wheat at the DNA level, the problems associated with the denaturation of the proteins and also the perceived problem of the differential expression of particular proteins as a function of the environmental growth conditions are avoided
The present invention provides a method for detecting the presence of wheat having a D-genome in a processed food product, which method comprises extracting DNA from said product and detecting the presence of a nucleotide sequence which is characteristic of the D-genome
Suitable food products from which DNA may be extracted include milled wheat products such as semolina or flour, or more extensively processed products, in particular pasta including dried pasta products In a preferred embodiment, the nucleotide sequence which is detected comprises or is a D-genome specific derivative of the Dgas44 sequence
In another preferred embodiment, the sequence detected is the PSR128 sequence of the D-genome shown hereinafter in Figure 9 Single copy sequences are particularly prefeπed where quantification of the amount of T aestivum is required When multiple copy sequences such as Dgas44 are used (-500 copies present in the average D- genome), it is not certain that the number of copies remains constant across all the varieties of T aestivum Unknown variation may impede accurate quantification However, PSR128 includes a single copy sequence derived from the D-genome
The said nucleotide sequence may be detected by a vanety of methods which are well known in the art They may for example be detected using Southern or dot blotting techniques, or more preferably, by a preliminary amplification of the target sequence. followed by detection of the amplified product A particularly suitable amplification technique uses the polymerase chain reaction (PCR) after which the amplified product is suitably detected following eiectrophoresis on agarose gels
McNeil et al (supra.) proposed three primers for use in the polymerase chain reaction (PCR) amplification of Dgas44 These are shown underlined in Figure 1 However, when these were used in an attempt to identify contaminant wheats in pasta samples, it was found that the bands were very feint and diffuse bands which made identification difficult and which could not be entirely overcome by varying the PCR conditions The problems are thought to be due to the high copy number of this sequence in the genome, giving rise to a large amount of PCR products, not all of the same exact length It is also possible that there is some degree of length polymorphism among genomic copies of this sequence There have also been problems due to very faint PCR products in T uratu. the durums and the negative controls, indicating that the primers are not completely specific for the D-genome. The applicants have identified a particular region of the sequence which is highly specific to the D-genome and from where advantageous probes or diagnostic primers may be derived This region comprises nucleotide 1732 to 2150 of the Dgas44 sequence and is shown hereinafter in Figure 2 (SEQ ID no. 2)
Thus in a preferred embodiment, the method of the invention involves the detection of all or part of the sequence of Figure 2, and more particularly, the sequence of Figure 3 Novel probes and primers which are useful is such a method form a further aspect of the invention
Thus the invention further provides a nucleotide sequence which comprises or hybπdises with all or part of the nucleotide sequence shown in Figure 2 Said sequence is suitably detected m the method of the invention
Suitable parts of the sequences defined above are sufficiently long to allow specificity and identification. In general therefore, the parts will be at least 15 bases in length and preferably at least 20 bases in length
In a particularly prefeπed embodiment, the nucleotide sequence comprises or hvbπdises with all or part of the nucleotide sequence shown in Figure 3 (SEQ ID No 3) This sequence conesponds to bases 1864 to 2150 of the Dgas44 sequence
The expression "hybπdises with" means that the nucleotide sequence will anneal to all or part of the sequence of Figure 2 under stπngent hybndisation conditions, for example those illustrated in "Molecular Cloning, A Laboratory Manual" bv Sambrook. Fπtsch and Maniatis, Cold Spnng Harbor Laboratory Press, Cold Spπng Harbor, N Y.
The length of the sequence used in any particular analytical technique will depend upon the nature of the technique, the degree of complementaπty of the sequence, the nature of the sequence and particularly the GC content of the pπmer and the particular hybπdisation conditions employed Under high stringency, only sequences which are completely complementary will bind but under low stringency conditions, sequences which are 60% homologous to the target sequence, more suitably 80% homologous, will bind.
Where the sequence is used as a labelled probe (for example a radiolabelled or fluorescently labelled probe for example using Southern or Dot blotting) for direct detection of the target D-genome sequence, the probe length will be quite long, for example of 100 bases or more.
In view of the relatively low levels of DNA which may be expected however, for example in contaminated pastas, an amplification technique such as PCR is suitably employed, and the amplified product detected. For use as a PCR primer, the nucleotide sequence of the invention is preferably from 10 to 30 nucleotides in length, suitably from 17 to 30 nucleotides in length. At least two such primers will be necessary in order to effect the PCR amplification as is well understood in the art. These will be non-complementary sequences which hybridise with the opposite strands of the target region of the genome when denatured and which flank the region to be amplified. Suitable primers for use in a nested PCR technique may also be derived for example from
Particularly preferred primers are as follows:
CTTCTGACGGGTCAGGGGCAC (SEQ ID NO 4)
CTGAATGCCCCTGCGGCTTAAG (SEQ ID NO 5)
GTCCTATATCTTGAGGCCGCAAG (SEQ ID NO 6)
AACCCACTGTACCTGAGTATATATC (SEQ ID NO 7)
SEQ ID Nos 4 and 6 are suitably forward primers for PCR amplification whilst SEQ ID Nos 5 and 7 are reverse primers. A particularly prefeπed pair of pπmers compπse SEQ Id Nos 4 and 5
In particular, said method comprises amplifying the sequence of Figure 2. or preferably of Figure 3. using at least two primers and detecting amplified product.
Preferably, the methods of the invention is earned out so that the amount of said species of wheat present in the sample is quantified This may be done, simply by companng signals produced for example using PCR amplification with those produced from control samples of known contamination levels It has been found by carrying out PCR experiments using control pastas, that an increase in the amount of PCR product using various pπmers has been seen as the level of bread wheat contamination increases
However, in practice, it may be necessary to carry out a quantification assay in pastas of unknown quality. In such cases, factors such as DNA concentration, DNA quality, etc. are likely to play a very large role in the observed amounts of amplification products Although it may be possible to compare the PCR product from such a test sample to one from a control pasta, since DNAs from the test and control would, in all likelihood, have been made from pastas produced under differing conditions, in different locations etc. it is unlikely that the DNAs would be of the same quality, size etc. It has been found in fact that a direct link exists between the amount of DNA used as a target for amplification and the temperature at which the pasta was dried The more elevated the drying temperature the more DNA is required to obtain the same level of specific product after amplification. This effect is probably attributable to the thermal degradation of the target DNA and the subsequent decrease in the number of DNA molecules able to act as a target for the amplification reaction
Given these factors, and the sensitivity of the required quantification assay it is necessary to adopt a more rigorous method for the quantification assay
In a preferred embodiment, an internal standard or reference product, not arising from the D-genome that can be used to calibrate the PCR-assays internally, is used. In cne such method, a second nucleotide sequence is also amplified by multiplexing with additional appropriate primers. This second sequence is one which is found on the A and B and D genomes. In this way, detection of the second nucleotide sequence will provide an internal standard, against which levels of the D-genome specific sequence can be assessed to give relative proportions of the components.
The skilled person, would be able to check to see if any particular sequence from a plant genome fulfilled this requirement using routine methodology. In fact, the applicants have found that suitable non-specific sequences may also be derived from the Dgas44 sequence. A particular example of such a sequence is that defined by bases 5 to 876 of the Dgas44 sequence as shown in Figure 1.
Alternatively, quantification is effected by amplifying said target sequence by the using a pair of primers in the presence of a known concentration of a further sequence which competes with the said sequence for a primer, but which is distinguishable from said sequence during detection, and comparing signals produced by said further sequence and said target sequence. This method is based upon a known protocol (Fόrster, E.( 1994). Biotechniques. 16, 20-24). A schematic diagram of the method used is shown in Figure 4 hereinafter. Basically the method entails the generation of a "synthetic" PCR product, of slightly shorter length than the PCR product generated from the template DNA, but having the same pπmer sites at its termini. This short-product is engineered by a two-stage PCR process, using the original full-length PCR product as template. The method employs "chimeric" primers at one end of the PCR reaction, in our case the 3' terminus. This "short product" is then added to PCR reactions in differing concentrations, in which it can compete against the template DNA for the primers. The signal strength arising from a known amount of the short product is then used as a control to estimate the amount or number of copies of the original template, for example, the amount of contaminating Dgas44 sequence arising from D-genome contamination of durum wheat samples or pasta products. This assay would be independent of the DNA concentration or level of DNA quality or average size The applicants have generated a PCR product using primers of SEQ ID Nos 4 and 5. and a shortened version thereof ("short PCR product")as illustrated hereinafter, and have used this in a quantification assay
Preferably, cloned short-PCR product in circular form, most preferably plasmid borne, is used Such a product will be easy to generate in large quantities and may minimise contamination' of PCR procedures (such a sequence may be extremely "invasive" sequence, and extreme care has to be taken to ensure that this sequence does not contaminate other PCR reactions using closely related primers)
In addition, when using a PCR amplification technique, quantification is suitably effected using a fluorescence based PCR product quantification system such as the TAQMAN ™ 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 This svstem offers the potential for accurate quantification of PCR products In essence, the amplification reaction is effected in the presence of a probe which includes a quencher moiety and a fluorescence reporter, said probe being arranged to be selec ely cleaved duπng the amplification reaction so as to produce a flurorescent signal The signal, which is generated by laser induction, is monitored by and the rate of increase used to calculate the amount of target sequence present in the sample When refeπed to hereinafter, the probes will be refeπed to only by reference to the characteristic sequence which they contain It should be understood that the fluorescent and the quencher moiety are included.
Such reactions can be used to quantify the amount of f aestivum present in a sample of known wheat content However, since there are limitations on the accuracy of most methods of determination of the total wheat content, it may suitably form part of a comparative study, in which a PCR reaction and a TAQMAN™ probe designed to amplify and detect a sequence which is common to all the genomes is earned out in parallel Such a reaction, in effect measures the amount of wheat DNA in the sample in the reaction. The signal generated by the D-genome specific amplification reaction could then be standardised against this measurement, and so an accurate percentage figure for any contaminant T. aestivum can be established. The standardising reaction may be effected in a separate tube containing an equal aliquot of DNA being investigated. Alternatively, use of distinguishable laser dye mixtures would allow the reactions to be effected in the same reaction tube.
A useful TAQMAN probe which has been successfully been used in quantitative PCR reaction to amplify the sequence of Figure 3 is a probe based upon base numbers 1 88- 2014 of the Dgas44 sequence. In other words, the TAQMAN™ probe comprises SEQ ID No .10
5TTG GGA GGC ATG GTG AAA GTT GGT GAT3' (SEQ ID NO 10)
However, as mentioned above, quantification is more accurately carried out using a single copy sequence such as that found in the D-genome specific region of PSR128. PSR128 is an intron sequence derived from a cDNA clone. Techniques for mapping this probe are described by D.X. Xie et al., Theor. Appl. Genet (1993) 87: 70-74. The size of the intron differs in each of the genomes of hexaploid wheat. Figure 9 shows the PSR128 sequence found in the D genome (SEQ ID NO. 19). The D-genome copy of the intron includes a unique sequence (bases 62-114). By utilising this unique sequence, for example using PCR methods, the single copy PSR 128 sequence of the D- genome can be detected which can then be be most readily and accurately quantified.
Primers which will achieve this would be determinable by the skilled person. In general, at least one of the primers is derived wholly or partly from the D-genome unique sequence. Where the TAQMAN™ system is used, the probe may itself be derived from said sequence. Examples of suitable primers for the amplification of the D-genome PSR 128 sequence are
5'ATG GCT GGC TTC TAT TTC ATG3' (SEQ ID No. 12) as the forward primer and
5 CAC CTA CTC CTC CAC ACT TTG3' (SEQ ID No. 13) as reverse primer.
The corresponding sites on the PSR128 D-genome sequence are underlined in Figure 9 hereinafter.
Alternative primers focus more closely on amplification of a smaller amplicon within the D-genome specific region. For example, a suitable pair of primers would be
5 AAG GAG CTC GCC AAC GG3' (SEQ ID No 14) as forward primer and
5 AAC CGA GGG TCC AGA AGA GAC G3' (SEQ ID No 15) as reverse primer.
These primers produce a small 116 base pair amplicon. Furthermore, a TAQMAN™ probe may further be employed to quanitate the PCR reaction, for example as follows:
5'GCT AGA TGG CTG GCT TCT ATT TCA TGA TCC A3' (SEQ ID No 16)
The position of these primers and probes is indicated by underlining in Figure 10 hereinafter. The use of a smaller amplicon is preferable in the present case, both for kinetic reasons associated with the PCR reaction and as a result of the degradation which may have occurred during food processing, for example of pasta. As illustrated hereinafter, a number of "control" pastas were made using precise levels of common wheat T aestivum adulteration The range of adulteration levels covered were those causing most concern both to industry and the regulatory bodies These were manufactured using three different drying regimes. These were 5(-°C, 80°C and 104°C representing the range of drying protocols widely used by industry
It was surpnsingly found that DNA could be isolated from these and other commercially available pastas through the use of conventional extraction procedures, for example using an extraction method based upon a phenol/chloroform mixture The isolated DNA was detectable using the methods of the invention, and in particular, after suitable purification, it is suitable for both amplification using PCR and for use in hybridisation protocols.
The target DNA is preferably subjected to analysis as soon as possible since stored material may suffer from some degradation, possibly as a resuit of theco-puπfication of nucleases which may further degrade the template When storage is necessary, it is preferable is nucleases are removed from the DNA, for example using a solid-phase cleanup procedure as is known in the art
Using the methods of the invention. It has been found that very low levels of T aestivum can be detected in pasta (as low as 0 1%) and that appropriate controls are required if accurate quantification is to be earned out
In order to check the veracity of the results, a large number of commercial cultivars of both T durum and T aestivum have been assembled from around the world and have been investigated using the PCR assay and the D-genome specific Dgas44 pnmers. In addition to these cultivars, a large number of related species and amphiploid lines of wheat have also been assessed The results showed that all of the T durum cultivars gave negative results in respect of the amplification of the D-genome specific Dgas44 product All T aestivum cultivars produced the specific PCR product The results suggest that the detection of the D-genome by the Dgas44 pnmers is highly unlikely to be compromised by cross-reactions with other unrelated genomes. This finding was underlined by the use of other Tnticum species and amphiploid (Tritordeum) lines.
Therefore this technique may be employed to identify the presence of D-genome charactensmg nucleotide sequences from DNA from any source, and therefore may be used for instance in the identification of particular species. Specifically the invention further provides a method for identifying the presence of a D-genome in a crop sample, which method compnses extracting DNA from said sample, and detecting the presence of a nucleotide sequence which comprises or hybridises with all or part of the nucleotide sequence shown in Figure 2. for example, by amplifying said sequence using PCR
The skilled person would understand that changes can be made to. for example, the precise PCR conditions illustrated herein and still produce acceptable results. Particular aspects which may be varied include the use of different PCR reagents, number of PCR cycles, buffer conditions such as magnesium ion concentration, pπmer concentration, enzymes and enzyme concentrations, and thermal cycling apparatus. In general however, the PCR reactions will be effected with at least 25-30 cycles in the presence of a magnesium ion concentration of from 0.5 to 4 OmM. preferably about 2.0mM. Suitably, the polymerase enzyme used is a thermostable polymerase such as Taq polymerase It is suitably present in concentrations of from 0 25U to 0.5U/50μl.
The invention will now be particularly descnbed by way of example with reference to the accompanying drawings in which.
Figure 1 shows the nucleotide sequence of the Dgas44 sequence,
Figure 2 shows the region from base no. 1532-2150 of the Dgas44 sequence,
Figure 3 shows the region from base no 1864-2150 of the Dgas44 sequence, Figure 4 illustrates the method used for synthesis of a PCR competitor template;
Figure 5 shows the results of a 25μl touchdown PCR 68-60 reaction using pnmers of SEQ 4 and 5. in which lOμl PCR product was run on a 1 % agarose gel;
Figure 6 shows the results of a 25 μl touchdown PCR 68-60 reaction using pπmers of SEQ ID Nos 4 and 5 and also SEQ ID Nos 8 and 9, and also pπmers in which lOμl PCR product was run on a 1% agarose gel;
Figure 7 shows the results of a dot blot with different amount of 56 pasta DNA per well, which was hybπdised to a radiolabelled probe deπved from Dgas44 sequence;
Figure 8 shows the results of a touchdown 68-60 PCR reaction using pπmers deπved from Dgas44 in a 50μl reaction using 0.5μl Taq per tube, and with lOμl PCR product run on a 1 % agarose gel.
Figure 9 shows the sequence of an intron sequence designated PSR128 in the D genomes of T aestivum:
Figure iO shows a portion of the sequences of Figure 9 with PCR pπmer and TAQMAN™ probe underlined;
Figure 1 1 shows a typical result of quantification assay of dπed durum pasta using the TAQMAN™ methodology for a pure durum sample (0% adulteration) and a sample containing 1 % adulteration:- the method determines the cycle threshold value (Ct) which is the PCR cycle or part of a cycle when the detected fluorescence differs from the 0% standard by 6 standard deviations. By plotting the Ct value against the known adulteration level within the standards, a calibration curve may be plotted : Figure 12 shows typical plots similar to those of Figure 1 1 but using dried pasta with 0%, 1% and 2% adulteration (each in triplicate);
Figure 13 shows typical plots similar to those of Figure 1 1 but using dried pasta with 0%, 1 %, 2% and 3% adulteration (each in triplicate);
Figure 14 is the calibration plots for the three sets of standard pastas (56, 80 and 104°C dried) assessed using a TAQMAN™ probe to Dgas44;
Figure 15 presents the calibration plots derived from all of the data presented in Figure 14 which would be used to assay pastas whose thermal history was unknown;
Figure 16 shows the calibration plots for the three sets of standard pastas (56, 80 and 104°C dried) assessed using the TAQMAN™ probe to the single copy sequence PSR 128; and
Figure 17 shows an individual calibration curve for the 104°C dried pasta standards, assessed using the the TAQMAN™ probe to the single copy sequence PSR 128.
Example 1 The preparation of semolinas of known levels of adulteration from accredited samples of T. durum (cultivar, Neodur) and T. aestivum (cultivar. Soissons).
25kg of accredited grains of the T. durum cultivar Neodur and a small amount of accredited T. aestivum (cultivar, Soissons) was obtained. Soissons is currently a major breadmaking variety of wheat and as such could be expected to be found adulterating T. durum grists destined for pasta production. Analysis showed the sample of Neodur to contain 1 1.4% protein and 14.6% moisture. The sample of Soissons contained 10.4% protein and 1 1.0% moisture. Electrophoretic analysis (Acid-PAGE) of the samples of Neodur and Soissons showed that both varieties were 100% pure. The accredited Neodur was adulterated with Soissons on a weight basis (as below) to produce 0, 1 %, 2%, 3%, 5%, 10% levels of contamination are shown in Table 1.
TABLE 1
% Adulteration Neodur (g) Soissons (g) (T. durum) (T. aestivum)
0 5000 0
1 3960 40
2 4900 100
4850 150
5 4750 250
10 2250 250
Durum grain was taken from its natural moisture content of 14.6% to a final moisture of 18.5% in two stages (a) an initial step of taking it to 17% moisture; and (b) a final addition of water to bring the moisture content to 18.5%. These steps were caπied out over 8 hours.
The grains were then milled using a Buhler (Switzerland) test mill which had been dismantled and thoroughly cleaned prior to use in order to remove all traces of any contaminating T. aestivum flour. Only the first half of the mill was used, that producing semolina. In preparation for milling the test samples a non-accredited (normal T. durum mill grist) was used to set the mill to the required settings i.e. that designed to remove the bran layer from the hard T. durum grains while yielding a semolina that was as representative as possible of the product from the larger mills. Once the desired settings were achieved, the mill was stripped down and vacuum cleaned before the test millings proceeded.
Deliberately adulterated Neodur was milled in ascending order of contamination with a thorough mill stripdown and vacuum clean between each test sample.
The yields of semolina are shown below in Table 2:
TABLE 2
Figure imgf000022_0001
The moisture content of the adulterated semolina was then determined. This was assessed using two semolina samples (0 and 10% T. aestivum content). Both samples were placed in a Newport 400 NMR analyser and the result obtained. The results were as shown in Table 3:- TABLE 3
T aestivum content (%) moisture %
0 16.76 10 16.92
An average moisture content of 16.80% was used in all subsequent work
The semolinas were then turned into pasta using a small scale pasta press for the production that was capable of being completely cleaned out between samples and which had a very low dead space (and hence wastage). Doughs produced commercially from semolina generally contain 32% water and this recipe was adopted for the subsequent work with water addition being calculated as follows -
dry solids (g) = wt semolina (g) - (wt semolina (g) * 16 8/100) water addition (ml) = (dry solids (g) /0 68) - wt semolina (g)
The relatively small amounts of semolina (300-1300g) that were used in the series of experiments allowed mixing to be achieved using a Kenwood Chef mixer The semolina was placed in the bowl of the mixer and while the blade revolved on the slowest setting, the water (@ 50°C) was slowly dribbled into the bowl Initially, only two thirds of the required water was added over a period of 2 minutes The blade was then stopped and the bowl scraped down before the mixer was restarted and the remaining one third of the water added Mixing was continued for a further minute before the mix was transferred to the mixing compartment of the pasta press where it was allowed to mix for a ftirther 3 minutes before extrusion commenced
Prior to the extrusion of the pasta dough, the pasta press was thoroughly cleaned by totally dismantling the dye and screw mechanism. All parts were soaked and cleaned as necessary with the mesh and dye being subjected to cleaning by pressurised jets of water in a commercial die wash The screw and die, once clean, were kept at 50°C prior to drying and assembly ready for the next experiment. This enabled the desired running temperature to be achieved more quickly (important for quality reasons).
The pasta produced by the above method was dried by one of three regimes, representing the cuπent commercial practices within Europe. All drying profiles (with the exception of the 104°C step within the very high temperature profile) were performed in a Hedinair (Kingsbridge Road, Barking By-pass, Barking, Essex) programmable hot air oven with relative humidity control and humidification. The 104°C step within the very high temperature procedure was carried out using a calibrated Gallenkamp model OV- 160 hot air oven. The three temperature profiles used were as follows:-
l .low temperature method (56°C)
a) 12 h, @56°C, ΔT 5°C, 76%RH.
moisture content (by NMR) at the end of the procedure was 10.4%
2.'Normal' high temperature method (8θC)
a) 40mm, @60°C, ΔT 7°C, 69% RH. b) 2h, @80°C, ΔT 7°C, 74% RH. c) 2.5h. @75°C, ΔT 5υC, 80% RH. d) 8h, @56°C, ΔT 5°C, 81 % RH.
Moisture content (by NMR) at the end of the procedure was 10.0%
3.Very high temperature drying method ( 104°C) a) 40mιn. @60°C, ΔT 7°C, 69% RH b) 1H, @80°C, ΔT 7°C, 74% RH. c) lh, @88°C, ΔT 8.5°C, 71% RH d) 1.5h, @104°C, ~90% RH e) 1.5h, @75°C, ΔT 5°C, 80% RH f) 6h, @56υC, ΔT 4°C, 81 % RH.
Moisture content (by NMR) at the end of the procedure was 10.25%
The constraints of ensuπng complete mtegnty of the samples of pasta that were produced and organising the three different drying regimes dictated that each semolina was turned into pasta on three different days. The production at the end of each day being dned by a particular temperature profile. This eliminated cross-contamination and allowed each drying regime to take place overnight
To eliminate cross-contamination during manufacture pastas were produced each day in an ascending order of T aestivum contamination
To assess the cleanliness and to check the levels of adulteration in the pastas produced as 'controls' for the extraction of DNA, all pastas were examined by the traditional method of analysis i.e omega-g adin analysis. The results of this analysis confirmed the initial purity of the Neodur and the proportional increase in omega-gliadin content as the level of adulteration increased although the detection limits of the technique (2-3% when assessed visually) were also clear.
Samples of each 'control' pasta (~10g) were stored in stoppered glass pharmaceutical bottles (125ml) fitted with a tamper evident cap to ensure sample integrity Each bottle had a numbered label indicating both the level of adulteration of the pasta and the drying conditions of the pasta sample it contained. Individual bottles are tracked via the use of a log book giving details of both the user and the date used. All samples were stored in a locked cold room, in the dark at 4UC
Example 2 DNA extraction from samples and detection using PCR
DNA was isolated from the control pastas produced in Example 1 and using the phenol/chloroform method of Sharpe et al. (1988). Theor Appl Genet. 75. 289-290 Each pasta(5g) was ground into flour by milling for approximately 20 min after light crushing with a hammer The flour was transfeπed to a 50 ml polypropylene tube and buffer *'S"(20ml) compnsing lOOmM Tπs Cl (pH 8 5), lOOmM NaCl, 50 mM EDTA (pH 8.0) and 2% SDS, was added. The mixture was thoroughly dispersed by vortex g
A lOmg/ml protemase K stock solution ( lOOμl) was added per tube (final concentration 0 05 mg/ml) The tubes were then incubated at 65°C for 1-2 h with the occasional inversion to mix the contents A phenol/chloroform mixture (20ml), which had been prepared by mixing 1 part TEN buffer saturated phenol, pH 7 8 1 part chloroform isoamyl alcohol(24 1 ) was added to each tube and mixed gently by inversion to form an emulsion Centπfugation at 2000rpm for 20 min separated phases although the addition of phenol/chloroform mixture and subsequent centπfugation was repeated if necessary
Propan-2-ol (0.6 vol) was added and mixed by inversion DNA was recovered by centrifugmg at 10,000 rpm at 4°C The DNA was washed with cold 70% ethanol and air dried A further wash with 100% ethanol was performed to speed drying The DNA was suspended in lx TE buffer (5ml) (composed of lOmM Tπs (pH 8 0) and ImM EDTA (pH 8 0)) and kept at 4°C Total solution can take several days, however, this can be speeded by careful warming at 65°C for up to 2hrs with gentle inversion RNase stock solution (lOμl) (comprising 1 mg/ml RNase A and lOOU/ml RNase Tl which had been heated to 100°C, lOmin to destroy DNase activity) was then added and the solution incubated at 37°C for lh The phenol/chloroform extraction was then repeated using phenol/chloroform (5ml), followed by gentle mixing by inversion and centrifuge at 2000rpm for 15 mm.
The product was extracted using an equal volume of chloroform, a step which was repeated if mateπal at the phase interface is excessive.10% volume sodium acetate (3M) (final concentration 0.3M) was added with mixing, followed by 2 vols ethanol (100%). After centrifugmg at lO.OOOrpm @ 4°C, and washing in cold 70% ethanol, the DNA product was air dried and redissolved in 1.8-2 ml TE buffer (depending upon the size of pellet) This solution was stored frozen at -20°C
Aliquois of each stock DNA thus produced was taken and puπfied using a column Chromatography method (Wizard PCR Preps, Promega, Madison, WI, USA). The punfied DNAs were diluted to 20ng/μl as a working solution. This process was repeated twice, the first occasion giving cleaner DNA and a greater recovery
In addition, more DNA was made from durum and hexaploid wheat plants were extracted using the phenol/chloroform method descπbed above Plants sown in batches of about 30 individuals of each sample yielded enough leaf material after two and a half weeks to provide leaf for two DNA extractions (approx. 5g leaf tissue per DNA extraction) The varieties sown were the hexaploids Chinese Spring and Soissons, and the durums Neodur and Pπmadur Two sets of stock DNAs were obtained from each extraction. The first sample, obtained by "spooling" a DNA precipitate, and a less pure sample obtained by centrifugauon of the residual sample
Samples of DNA produced in this way were mixed in known concentrations to provide comparative results
The DNA obtained in this way was then analysed using touchdown PCR with SEQ ID Nos 4 and 5 shown above as forward and reverse primers respectively This technique involves the gradual reduction of annealing temperature every few cycles duπng the amplification procedure. This has the effect of reducing primer-binding to target sequences in the template DNA which do not have a perfect complimentary sequence, thereby increasing the reaction specificity. In this case, the following conditions were employed:
Programme No No of cycles Linked to programme No Segments 8 1 1 3min@94°C
1 2 2 30s @ 94°C
1 min @ 68°C 30s @ 72°C
2 2 3 30s@94°C lmin( ;660C
30s @ 72°C
3 2 4 30s@94°C
1 min (a) 64°C 30s @ 72°C 4 2 5 30s@94°C
1 min @ 62°C 30s (^72°C
5 30 6 30s(α>94°C
1 min @ 60°C 30s @ 72°C
6 1 7 5min@72°C
7 1 0 99h(a4°C The following PCR reagents were used: Water 15μl Cresol red 5μl (10 mg/ml dissolved in 60% sucrose) lOx PCR Buffer (see below) 2.5μl (Mg2+ cone. 15mM) DNTP's l μl (2.5mM) Forward pnmer 0.25μl (20μM) Reverse primer 0.25μl (20μM) NP40 0.25 μl (5%) Tween-20 0.25μl (5%) lμl DNA O. lμl Taq
lOx PCR buffer - 100mM Tns.HCl pH 8.3 500mM KC1
15 mM MgCl2 0.1% (m/v) gelatin
AU ohgonucleotides used as primers were synthesised on Milhpore Expedite oligonucleotide synthesiser using Expedite ' chemistry. They were purified by first uncπmping the column and pouring glass beads into screw-capped Eppendorf mbe. 300μl ammonia was added and the reaction tube incubated at 55°C for 30-60 minutes. The tube was then placed tube on ice for 5 minutes and centrifuged at 12000 m for 2 minutes. The supernatant was added to a new screw-cap tube, and 30μl 3M ammonium acetate, and 3 volumes cold ethanol added. The mixture was placed in -70°C freezer for 30-60 minutes and then spun at 12000 φm 15 minutes at 4°C. The supernatant was aspirated and the resultant pellet washed briefly with cold (-20°C) 80% ethanol. After allowing the pellet to dry, it was resuspended in 200μl ddH?O and the concentration measured. The results are shown in Figure 5 The PCR showed increasing band strength on the gel as the level of contamination with hexaploid wheat increased
In similar expenments. the pnmers SEQ Id Nos 4 and 6 and reverse primers 5 and 7 used in more or less any combination, appear to allow significantly enhanced D-genome specific amplification, than any of the other pπmer combinations, including the primers used in the McNeil et al paper
Example 3 PCR using Multiplex PCR
Duπng investigation of the Dgas44 sequence for suitably PCR pnmers, it was found that certain pnmers were none specific and would detect both the A & B as well as the D genome One such pair of pπmers comprises SEQ ID No 8 and 9 as follows
\GCAACTCCCCCTTAGCGGAGCAC (SEQ ID NO 8)
CAGCTCCGAGTCAGAGGTAACC (SEQ ID NO 9)
(SEQ Id no 8 is similar to that disclosed in McNeil et al , supra but without the 3'T) These primers are based upon the sequences found from base Nos 5-28 and 855-876 respectively in theDgas44 sequence (see Figure 1)
Since the amplification product using pnmers of SEQ Id Nos 8 and 9 is significantly larger than the within-domain D-genome specific products produced in Example 2, it was thought that this would provide a useful internal control for successful DNA amplification Example 2 was therefore repeated using the crop DNA as well as the pasta DNA but using a combination of two groups of pπmers comprising SEQ Id No 4, SEQ Id No5. SEQ Id No 8 and SEQ Id No 9 The results are shown in Figure 6
It is clear that the addition of the two non-specific primers provides a useful internal standard or control against which the level of adulteration may be assessed Example 4
Quantification using short PCR product
Using PCR conditions substantially as described above and following the scheme shown in Figure 4 and described in Forster et al. supra, short PCR products have been generated using the following steps:
1 ) As forward primer:
CTTCTGACGGGTCAGGGGCAC (SEQ ID NO 4)
and as the reverse primer
CTGAATGCCCCTGCGGCTTAAG (SEQ ID NO 5);
2. As forward primer
CTTCTGACGGGTCAGGGGCAC (SEQ ID NO 4)
and as the reverse primer in place of SEQ ID No 5
GCGGCTTAAGCTGTAGATTTCCGGCT (SEQ ID NO 17)
or GCGGCTTAAGTTCATAAGCCGCCACC (SEQ ID NO 18)
based upon regions 2050-2065, and 2077-2092 as well as 2129-2138 of the Dgas 44 sequence as shown in Figure 1.
PCR in this instance was performed using a touchdown 58-54°C protocol using the product derived in step 1 ) as DNA template. 3. As forward primer:
CTTCTGACGGGTCAGGGGCAC (SEQ ID NO 4)
and as the reverse primer:
CTGAATGCCCCTGCGGCTTAAG (SEQ ID NO 5).
PCR in this instance was performed using a touchdown 58-54°C protocol using the product deπved in step 2) as DNA template.
The touchdown PCR protocol can be summarised as follows: Programme No No of cycles Linked to programme No Segments
Figure imgf000032_0001
1 2 2 30s (w 94°C 1 min (w 58°C 30s (ω 72°C 30s (fl) 94°C 1 min (a) 56°C 30s @ 72°C 30s @ 94°C 1 min @ 54°C 30s @ 72°C
4 7 5 min @. 72°C
7 0 99 h (a), 4°C
The existence of the desired short-PCR product was confirmed on a 4% agarose gel When these products were used as a competitor in PCR reactions using pasta DNA obtained as described above in Example 2, it was found that increasing the amount of short-PCR product, reduces the amount of the full-length PCR product. Since the amount of short-PCR product added to the reactions was known, the original amount of contaminating Dgas44 present in the pasta samples can be calculated by a compaπson of the signals.
The short-PCR product has been cloned into the PGEM-T cloning vector (Promega, Madison. WI, USA).
Example 5
Cultivar screening
To assess the applicability of the protocol to determine the presence of the D-Genome in a uniform manner a wide range of T. durum and T. aestivum cultivars were gathered from around the world. T. durum cultivars were collected (55 accredited samples and 3 unaccredited) representing the major cultivars in common commercial usage and the growing areas around the world. In addition, a number of accessions from the John Innes germplasm collection were added to the screening procedure. The origins of these rare cultivars were diverse, yet they represent the genetic diversity sometimes utilised within breeding programmes. They were therefore included to strengthen the robustness of the protocol particularly with respect to new cultivars that may be bred as a result of crosses with these varieties. The large number of cultivars allowed an assessment of the uniformity of detection of the D-Genome. It is known that the Dgas44 sequence is repeated many times within the D-Genome, however, the exact number of repeats and the question of whether or not the number of repeats is constant between cultivars is unknown.
In an attempt to cover the major sources of T. aestivum contamination a large collection (66) of cultivars have been gathered. Most of the cultivars are in current commercial cultivation however where these were not available then slightly older cultivars have been substituted. The collection represented the most common wheat growing areas of the world and hence covers the most probable sources of contamination/adulteration
In addition, a selection of 38 samples of the related Tnticum species and suspected donors of the vanous genomes were included in the screen m order to evaluate the unlikely possibility that other genomes may interfere with the specific detection of the D-genome. These samples were examined 'blind'
In addition to the species and cultivars descnbed above a limited number of Tntordeum lines (H chilense (a wild barley) crossed with either T turgidum or T aestivum) were examined due to their potential use within the food industry and the possibility of cross contamination of the Spanish durum crop
DNA was extracted from all of the cultivars shown by the method described in Example 2 above for pasta
Following the analysis of the extracted DNA for the presence of the D- genome by the PCR method as descnbed above and using SEQ ID Nos 4 and 5 as primers, the results were as follows -
1 DNA samples extracted from T aestivum cultivars all produced the characteπstic amplification product indicative of the presence of the D-genome when subjected to PCR
2. All the DNA's extracted from accredited! durum cultivars failed to produce the amplified product indicative of the presence of the D-Genome
Three T durum accessions from the John Innes Centre germplasm collection that had been included as more diverse examples of the species did however produce an amplified product. This indicated that the D-genome was present in these materials Further analysis of these accessions by Acid-PAGE showed that in every case that the putative pure T durum cultivar that had been included in the germplasm collection as a T durum cultivar was either a T aestivum or a mixture of the two species.
3 The analyses of the nine amphiploid lines showed that the specific amplified product (indicative of the presence of a D-genome) was obtained only from the three primary octaploid lines (H chilense X T aestivum), HT4, HT73 and HT 1 16. The primary and secondary hexaploids (H chύese X T turgidum) all failed to produce a specific amplified product
4 The results obtained from the analyses of other Tnticum and related species (lines 1 -37, appendix 13) proved to be extremely interesting both in terms of the specificity and strength of the detection of the D-Genome, with the majority of the D-Genomes being detected There were however, a few non-conforming results One of the most suφπsing results involved T palecolchicum which gave a weakly positive result despite being tetraploid and having only the A and B Genomes This species however is unusual tn that it has only ever been found growing with hexaploid wheat, it is therefore assumed to be a regression product and hence the reason for the positive result It was also noticeable that while the species carrying the D-Genome produce a reasonably strongly amplified product the amphiploid lines and substitution lines produce weakly amplified products. Langdon ( 1A) ID andAe vavilovi & Ae μtvenahs gave negative or weakly amplification. In the case of Langdon ( 1 A) 1 D it is possible that the I D chromosome carries very few copies of the Dgas44 sequence and therefore not detectable using our assay
Overall, the results of this analysis are in very good agreement with the expectation on the basis of the genomic constitution of the various lines or species Example 6
Detection using Southern Blotting
DNA extracted from pasta as descnbed above, was digested to completion with the following combinations of endonuc leases were used .-Taql, EcoRl/Bam l, EcoRV/Sstl,
Bam HI IBgl II DNA extracted from T durum plant leaves as described above in
Example 2 was also prepared in this way to act as positive controls.
A probe was prepared by PCR from the Dgas44 sequence using the following as pnmer sequences
AGCAACTCCCCCTTAGCGGAGCAC (SEQ ID NO 8)
GGTGACTCCATGCAGTCAAATCTG (SEQ ID NO 20)
SEQ ID No 12 compnses a forward pπmer based upon base 5 to 29 of the Dgas44 sequence and SEQ ID no 20 comprises a reverse primer based upon bases 408 to 431 of the Dgas 44 sequence.
The thus produced probe was radiolabelled using inseπs with j3P by the method of Feinberg and Vogelstein. ( 1983) Anal. Biochem 132, 13-16
Hybridisation was performed with a probe made by PCR from plasmids containing the Dgas44 sequence using the method of Southern, E.M (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis J.Mol.Biol. 98. 503- 517.
The positive controls were very radioactive compared to the pasta dilutions, giving in some instances overexposure of the film in certain areas. The pasta samples also gave a signal but this was weaker Example 7 Dot blotting
A dot blot assay was performed DNA extracted from pure T durum was used as a positive control as well as DNA extracted from the 56° pasta prepared as descnbed above
A nitrocellulose membrane was cut to 9 x 12cm and washed in a buffer comprising 175.2g sodium chloride, and 88 2g sodium citrate per litre, adjusted to pH 7 0 with NaOH (SSC) for 10-15 min. and drained well.
The membrane was placed on a sealing gasket which was fixed into dot blotting apparatus which was attached to a vacuum lOOμl 20x SSC was loaded to each well and then drained with a full vacuum for approx 5 min Subsequently DNA at concentrations of 1, 2, 5 and lOμg/well (in 60μl 04 M NaOH solution) was loaded to each well After gentle vacuuming for 20 min until wells look empty and dry
The membrane was removed and rinsed in 2x SSC for 15 min After blotting dry, the membrane was subjected to hybridisation as described above in Example 5 and the results are shown in Figure 7
This shows that increasing contamination makes a difference which is detectable on hybridisation and these differences are visible to the naked eye This may be quantified using densitometry
Example 8
PCR using different pnmers
Pπmers of SEQ ID Nos 8 and 20 were used in a touchdown PCR reaction at conditions 68-58°C (Don R.H et al , (1991 ) Nucleic Acids Research 19, 4008) to amplify DNA prepared as descnbed in Example 2 above The reagents used in a standard 50μl reaction per tube was as follows:
Water 30μl (or 40μl if no cresol red used) Cresol red 1 Oμl ( 1 Omg/ml dissolved in 60% sucrose) 1 Ox PCR buffer 5μl (Mg +, 15mM) DNTP's 2μl (2.5mM) Forward pπmer 0.5μl (20μM) Reverse pπmer 0.5μl (20μM) NP40 0.5μl (5%) Tween-20 0.5μl (5%)
1 μl DNA (20ng/ul)
For single copy sequences 0.2μl Taq (5U/μl) ( For repeated sequences 0.1 μl Taq was used)
Although the results were of variable quality and there were problems with very feint and diffuse bands (probably due to the high copy number of this sequence in the genome giving rise to a large amount of PCR products, not all of precisely the same length and possibly also some degree of length polymoφhism among the genomic copies of this sequence). These problems can be minimised to a degree by appropriate modification of the PCR conditions.
With these primers, a temperature of 60°C was found to be optimum and increasing the buffer/Mg"* concentration to 2.0mM was suitable. The concentration of Taq polymerase which gave shaφer bands was 0.25U, with a primer concentration of xθ.5. A representative result is shown in Figure 8.
This indicates that contamination at levels as low as 1% may be detected with this method. Example 8
Quantitative PCR using TAQMAN™ Probe
PCR reactions were earned out using pπmers of SEQ ID Nos 4 and 5 as the forward and reverse primers respectively and in addition a TAQMAN ™ probe of SEQ ID No 10.
' TTG GGA GGC ATG GTG AAA GTT GGT GAT3' (SEQ ID NO 10) Other conditions were as follows.
1. Cycler Conditions
STEP ! 1 Cycle 50°C 2 min
STEP 2 1 Cycle 95°C 10mm 5 STEP 3 45 Cycles 95°C 15sec
Figure imgf000039_0001
(All temperature changes at 1°C / Sec) 0
2. PCR REACTION MIXTURE CONSTITUENTS (25μl total per tube) (final tube concentrations given)
1 OX Buffer (a PE- Applied Biosystems propπetry mix of unknown ^ composition)
Magnesium Chloride 5mM dATP 200μM dGTP 200μM dCTP 200μM 0 dUTP 400μM
Forward Pnmer 300nM
Reverse Primer 300nM
Taqman Probe 200nM
AmpliTaq DNA polymerase 0.02U/μl 5 Uracil N-glycosidase O.OlU/μl water
Inclusion of this probe allowed quantification of the T aestivum content of the sample. 0 Development of the fluorescent signal from the TAQMAN ™ probe was monitored in the conventional manner. Examples of the results are illustrated in Figures 1 1 to 13. These figures show the fluorescence detected in the PCR reaction as a function of the PCR cycle number. The fluorescence detected is in proportion to the amount of specific target sequence amplified in the reaction. The software used allows the determination of the cycle threshold value (Ct) which is the cycle or part of it when the detected fluorescence differs from the 0% standard by 6 standard deviations.
They show the decrease in Ct as the adulteration level increases; i.e. the number of target sequences present in these samples is increasing, which in turn least to a fluorescent signal of equal intensity at an earlier cycle time.
The Ct value was then plotted against the known adulteration level within the standards and a seπes of calibration curves plotted (Figure 14). By combining this data, a calibration curve against which pastas of unknown heating history can be measured was derived (Figure 15).
Example 9
Amplication of the PSR 28 intron using Touchdown PCR
Samples prepared as described in Example 1 were subjected to a touchdown PCR reaction as follows:
Forward primer 3 y AATTGG G GCCIT GGC TTC TAT TTC ATG3 (SEQ ID No. 12)
Reverse primer 5 CAC CTA (SEQ ID No. 13)
STEP
1 94°C 3min
2 94°C 30sec
3 62°C lmin
Figure imgf000041_0001
5 94°C 30sec
6 60°C lmin
7 72°C l min
(steps 5-7 a total of 3 times)
8 94°C 30sec
9 58°C lmin
10 72°C lmin
(steps 8-10 a total of 3 times)
1 1 94°C 30sec
12 56°C l min
13 72°C lmin
(steps 1 1-13 a total of 3 times)
14 94°C 30sec
15 54°C lmin
16 72°C lmin
(steps 1 1-13 a total of 3 times)
17 94°C 30sec
18 54°C lmin
19 72°C lmin
(steps 17-19 a total of 3 times)
20 94°C 30sec
21 54°C lmin
22 72°C lmin
(steps 20-22 a total of 3 times)
23 94°C 30sec
24 52°C lmin
Figure imgf000042_0001
(steps 23-25 a total of 24 times)
26 10°C 20h
(all temperature changes were at 0.5°C / sec)
REACTION MIXTURE (for 50ul reactions)
μl water 12
Cresol red 10
10X PCR Buffer (Boehnnger) 5 (15mM Mg2+) dNTP's 5 (2mM)
Forward primer 5 (2μM)
Reverse pnmer 5 (2μM)
NP40 2.5 (1%)
Tween-20 2.5 (1%)
DNA 3 (20μg/μl)
Taq (Boehnnger Manheim) 0.2 (5U/μl)
Amplication of the target sequence was achieved.
Example 10 A TAQMAN™ Assay for PSR 128
Using similar conditions to those descnbed in Example 9 above, samples prepared as described in Example 1 were subjected to quantitative PCR using the following reagents.
Forward primer s AAG GAG CTC GCC AAC GG3 (SEQ ID No 14)
Reverse Primer AAC CGA GGG TCC AGA AGA GAC G3' (SEQ ID No 15)
TAQMAN™ probe: 5 GCT AGA TGG CTG GCT TCT ATT TCA TGA TCC A3 (SEQ ID No 19) Calibration curves similar to those described in Example 8 were prepared (Figures 16 and 17). Reliable and accurate quantification of the amount of T. aestivum was possible using this method.
SEQUENCE LISTING
GENERAL INFORMATION :
( i ) APPLICANT :
(A) NAME: The Minister of Agriculture, Fisheries and
Food in Her Britannic Majesty's Government of the United Kingdom and Northern Ireland
(B) STREET: Whitehall Place
(C) CITY: London
(E) COUNTRY: Great Britain
(F) POSTAL CODE (ZIP): SW1A 2HH
(A) NAME: Wiseman, Gordon
(B) STREET: 44 Vicarage Road
(C) CITY: Winslow
(D) STATE: Bucks
(E) COUNTRY: Great Britain
(F) POSTAL CODE (ZIP): MK18 3BH
(A) NAME: Bryan, Glenn James
(B) STREET: Brewlands Farmhouse
(C) CITY: Inchture
(D) STATE: Perthshire
(E) COUNTRY: Great Britain
(F) POSTAL CODE (ZIP): PH14 9RN
(A) NAME: Gale, Michael Dennis
(B) STREET: 9 Mount Pleasant
(C) CITY: Norwich
(E) COUNTRY: Great Britain
(F) POSTAL CODE (ZIP): NR2 2DG
(ii) TITLE OF INVENTION: Detection Means
(iii) NUMBER OF SEQUENCES: 20
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2296 base pairs
(B) TYPE: nucleic acid <C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:
CTGCAGCAAC TCCCCCTTAG CGGAGCACTT GGCCAACACG GTCGGTCTCC CCGGCTCAAT 60
GTACTCCGTC CGGGTGATTA CCACGTCCGG GTCGTCTGCT GGTGGAGCTG TAGCGGAGGC 120
TCCGGGTTAA ATGAAGCTTC GGTCGTTGCC TTAGTAGTTG GGGCGGTGGC TTCAGGTGCC 180
GTGTCCATTG GAGTGGCGGC TTCGGGGGCG GAGGCAGCTG GCGGTTCTTG GACTGTCACC 240
GCCGGCTCAG GCAAATCCGG CACTCCGGCC GACTTGGTCT TCTTCACCCT CTTGGTGAGC 300
TTTGCTTGGG CACTGAAAGA ACAACAAAAG TTAGTACAAA GCATATGAGT AAAGATCAGA 360
ACAACATGAG ATGATTGTTG TTACCCGGGC GCGGTCTTGA AAGCCGGCAG ATTTGACTGC 420
ATGGAGTCAC CCGAAGAGGG GGAGGTCTCC TGATAATTGG AGTCAGAAGA GTTGAGTGGC 480
TGACGGATAA CACCCGCCAA CGGTGAAAAG GGTACAAGGG GAAATAACCT CAGTTCGGCG 540
TTTCCTTGTT GCGTCGGGTA ACCCGGACGG AGAGGTCGGT GTCCTTGTTG GTCCGGGTGT 600
GACGCCGGCT TTCGTGAACC TGTCGCTTCA AAGAGAACAA TTGAGGGTCT TGATGAGCTA 660
AAGGATGTGA AAAGCTTACT TTCCGGGTTA CCCTTCCAGA CTTTCAGCTG TGGCAAGGGC 720
TCCGAGTCGG AGGAAAGTGA CATTACCTCT TCAACCACCG GGTTACTGGC GCCGGTGTCA 780
TCCTGTCAAT GAGCAAGTGT TGATAAGGCG GACAACAACA AACAACAAAT GCAGCAAGAA 840
TTTAAAGGCT TAAGGGTTAC CTCTGACTCG GAGCTGTNGC TTAATGTGCA TCAGCTCCGA 900
AGCAGTAGGT CTGCTTCCGT TTTTGCGAGG GGCGGCTTTC TTGGCGGCTT TCTTTGCCTT 960 CCTGGCTTTC TTGGCCGCCT CATGGTCATA CTTGACCCGC CAGAACTTAT CATCAGCCTG 1020
GCAAAAATAC AATGAGGAAT TATTAAAACA GGTTTAGATG AAGGTTATAT GCAGACGAAG 1080
ATAAACAGTT AAAGTCAGCG GCTTACGCTG GGGCTGGATT GGTCTTGCAG AAGGGGACTA 1140
GCCCGGTCCT CCACAGTCTG GCGAGGGCTC TCGTTTAAAA GAGCCTTGGT CTCCTCCTCT 1200
GCAACGTCTT CTGGAAGATC ATTGCGCTGT GCCTCTTGGG GTCATCCTTT CACCCGGTGT 1260
ACTCACACAT TAAGCCGGGG ACGGCGGCTC AATGGGATCA CCCGCCATGA GATCCAGACC 1320
CGGACCAGGT CGATTCCATT CAGACCGTTG CCAGGAGGGC CTTGATTTTG TTGATAGTAG 1380
GAAGCAGAGG CTGGTGCTCC GCTGGAGTCA GCTTGTCCGA CAGTGGGTGA GTCGGCTCTA 1440
GACGTGTTGG GCGAAAGCCG GGCAGCGGGC TTTCATCAGC CGGGGACGTG TCTTGGCAGT 1500
AGAACCAAGT CATGTTCCAG TCCTTGGGGT GGCTCGGCGG CTCTACGTAA GGGAAAAGAC 1560
AGTCCCTACG CCGCTGGATG GAAATGCCAC CTAACTCCAG ACTCGGCCCG TTGGCGCACT 1620
CATTCTGGCG GTTTAAGTAA AACAGCTCTC TGAAGAGCAG CAAACTAGGC TCTTCTCCAA 1680
GGTACACTTC GCAAAAGACT TGGAAATTGC AGATGTTGGA TACGGAGTTG GGTCCTATAT 1740
CTTGAGGCCG CAAGTCAAAG AAGTTGAGCA CGTCCCTGAA AAACTTTGAG CCGGGTGGTG 1800
CGAAGCCCCG GCTCATATGA TCTGCGAAAA TCACTACCTC CCCGTCCTTT GGCTGTGGTC 1860
TCCCTTCTGA CGGGTCAGGG GCACGGTAGG ACATGATTTC CTTCTTAGGC AAATATCCGG 1920
ACTTCACAAA ATTGGCTAGG GTCTCATTGG TGACGTTGAC CTCATCCAGT TGCAAGTGAT 1980
GGGAGCCTTG GGAGGCATGG TGAAAGTTGG TGATCTATGA CAAAAAGGAA AATGTCCGGG 2040
TTAATTATAA GCCGGAAATC TACAGGTCAA AACTAAGGTG GCGGCTTATG AAGGGGACTA 2100
ATGATATATA CTCAGGTACA GTGGGTTACT TAAGCCGCAG GGGCATTCAG AATAAGTTGA 2160
TGATCTACGG CCTTATCACA GTTAAGCCGG AGGAATCTAC AGCGCGTGGT TTCTTTAAAC 2220
CGGAATTATA AGCCGCCAAG ATTCAATGGT TGCAGTTTTT TACTAAGTGT GGTAAAACAG 2280
GTTTCACATA CTGCAG 2296 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GTCCTATATC TTGAGGCCGC AAGTCAAAGA AGTTGAGCAC GTCCCTGAAA AACTTTGAGC 60
CGGGTGGTGC GAAGCCCCGG CTCATATGAT CTGCGAAAAT CACTACCTCC CCGTCCTTTG 120
GCTGTGGTCT CCCTTCTGAC GGGTCAGGGG CACGGTAGGA CATGATTTCC TTCTTAGGCA 180
AATATCCGGA CTTCACAAAA TTGGCTAGGG TCTCATTGGT GACGTTGACC TCATCCAGTT 240
GCAAGTGATG GGAGCCTTGG GAGGCATGGT GAAAGTTGGT GATCTATGAC AAAAAGGAAA 300
ATGTCCGGGT TAATTATAAG CCGGAAATCT ACAGGTCAAA ACTAAGGTGG CGGCTTATGA 360
AGGGGACTAA TGATATATAC TCAGGTACAG TGGGTTACTT AAGCCGCAGG GGCATTCAG 419
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 287 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CTTCTGACGG GTCAGGGGCA CGGTAGGACA TGATTTCCTT CTTAGGCAAA TATCCGGACT 60
TCACAAAATT GGCTAGGGTC TCATTGGTGA CGTTGACCTC ATCCAGTTGC AAGTGATGGG 120 AGCCTTGGGA GGCATGGTGA AAGTTGGTGA TCTATGACAA AAAGGAAAAT GTCCGGGTTA 180
ATTATAAGCC GGAAATCTAC AGGTCAAAAC TAAGGTGGCG GCTTATGAAG GGGACTAATG 240
ATATATACTC AGGTACAGTG GGTTACTTAA GCCGCAGGGG CATTCAG 287
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CTTCTGACGG GTCAGGGGCΛ C 21
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CTGAATGCCC CTGCGGCTTA AG 22
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GTCCTATATC TTGAGGCCGC AAG 23 (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AACCCACTGT ACCTGAGTAT ATATC 25
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
AGCAACTCCC CCTTAGCGGA GCAC 24
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CAGCTCCGAG TCAGAGGTAA CC 22 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
TTGGGAGGCA TGGTGAAAGT TGGTGAT 27
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO-11:
CCTCGAGAAC AAGGAGAAGG AGCTCGCCAA CGGTACCTCC ATAGTCTCTC TTCTTCTTCT 60
TCTTTTTTCA AATATCCAGC TAGATGGCTG GCTTCTATTT CATGATCCAT CGTCTCTTCT 120
GGACCCTCGG TTCCTAATCG GACGGCTCTT GTTAATTTTT GGATTTTTTT TTCATTCATT 180
CATTCAGGTA GACTGGCGAT 200
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
ATGGCTGGCT TCTATTTCAT G 21
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CACCTACTCC TCCACACTTT G 21
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
AAGGAGCTCG CCAACGG 17
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
AACCGAGGGT CCAGAAGAGA CG 22 (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GCTAGATGGC TGGCTTCTAT TTCATGATCC A 31
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GCGGCTTAAG CTGTAGATTT CCGGCT 26
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GCGGCTTAAG TTCATAAGCC GCCACC 26 ( 2 ) INFORMATION FOR SEQ ID NO : 19 :
( i ) SEQUENCE CHARACTERI STICS :
( A ) LENGTH : 549 base pairs
( B ) TYPE : nucleic acid
( C ) STRANDEDNESS : double
( D ) TOPOLOGY : l inear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
CCTCGAGAAC AAGGAGAAGG AGCTCGCCAA CGGTACCTCC ATAGTCTCTC TTCTTCTTCT 60
TCTTTTTTCA AATATCCAGC TAGATGGCTG GCTTCTATTT CATGATCCAT CGTCTCTTCT 120
GGACCCTCGG TTCCTAATCG GACGGCTCTT GTTAATTTTT GGATTTTTTT TTCATTCATT 180
CATTCAGGTA GACTGGCGAT GCTGGCGTTC CCGGGGTTCC TGGTGCAGCA CAACGTGACC 240
GGCAAGGGCC CGTTCGAGAA CCTGCAGCAG CACCTGGCCG ACCCATGGCA CACCACCATC 300
ATCCAGACCA TCTCCGGCCA GTAAATCCGT CGATTTCACC GACCGGGGAG GTTTCAGGTG 360
GTCTGAATTG TTATTTTGGG GTACTGAGGA TGTACAAAGT GTGGAGGAGT AGGTGGACAG 420
TGCAAAAATG TGTTGTAATC TTAAAGCTCC GGTGGTGAAA CTTTCTTCTT GTGGATTTCG 480
CCATGGCTTC CGATGGAGGC TGGACTTGTA ATATCACCCA TGCGTGTTAT TCTAAACATG 540
GCAAAACTA 549
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:20:
GGTGACTCCA TGCAGTCAAA TCTG 24

Claims

Claims
1. A method for detecting the presence of wheat having a D-genome in a processed food product, which method comprises extracting DNA from said product and detecting the presence of a nucleotide sequence which is characteristic of the D-genome.
2. A method according to claim 1 wherein said food product comprises flour, semolina, or pasta products.
3. A method according to claim 1 or claim 2 wherein the said nucleotide sequence is amplified prior to or during detection.
4. A method according to claim 3 wherein the amplification is carried out using the polymerase chain reaction (PCR).
5. A method according to any one of the preceding claims wherein the nucleotide sequence which is detected is present as a single copy in the D-genome.
6. A method according to claim 3 wherein the nucleotide sequence which is detected is a D-genome specific derivative of the PSR 128 sequence shown in Figure 9.
7. A method according to claim 6 wherein a derivative of the PSR 128 sequence is amplified using a pair of primers selected from either
5 ATG GCT GGC TTC TAT TTC ATG3' (SEQ ID No. 12) and ? CAC CTA CTC CTC CAC ACT TTG (SEQ ID No. 13); or
5 AAG GAG CTC GCC AAC GG3' (SEQ ID No 14)
5'AAC CGA GGG TCC AGA AGA GAC G3 (SEQ ID No 15).
8 A method according to claim 7 wherein the pair of primers used are
' AAG GAG CTC GCC AAC GG3 (SEQ IDNo 14) and ' AAC CGA GGG TCC AGA AGAGAC G3 (SEQ IDNo 15)
9 A method according to any one of claims 1 to 4 wherein the nucleotide sequence detected comprises or is a D-genome specific derivative of the Dgas44
10 A method according to claim 9 wherein a characterising portion of the sequence shown in Figure 2 is amplified and detected
1 1 A method according to claim 10 wherein the sequence amplified comprises the sequence of Figure 3.
12 A method according to claim 10 or claim 1 1 wherein the amplification is carried out using forward primers selected from either
CTTCTGACGGGTCAGGGGCAC (SEQ ID NO 4)
or GTCCTATATCTTGAGGCCGCAAG (SEQ ID NO 6)
and reverse primers selected from either
CTGAATGCCCCTGCGGCTTAAG (SEQ ID NO 5)
or AACCCACTGTACCTGAGTATATATC (SEQ ID NO 7)
13 A method according to claim 12 wherein the forward pnmer compnses
CTTCTGACGGGTCAGGGGCAC (SEQ ID NO 4) and the reverse primer compnses
CTGAATGCCCCTGCGGCTTAAG (SEQ ID NO 5)
14 A method according to any one of claims 1 to 3 wherein the nucleotide sequence is detected by Southern or Dot blotting using a radiolabelled probe deπved from the said characteristic sequence.
15 A method according to any one of the preceding claims wherein the amount of said wheat present in the product is quantified
16 A method according to claim 15 wherein a second nucleotide sequence of said DNA is also amplified, said second sequence being found on the A and B and D genomes, the second nucleotide sequence is also detected and used as an internal standard
17 A method according to claim 16 wherein said second nucleotide sequence is deπved from the Dgas44 sequence
18 \ method according to claim 17 wherein said second nucleotide sequence comprises all or a substantial non-D-genome specific portion of a region of the Dgas44 sequence defined by base 5 to 876 of sequence of Figure 1
19 A method according to claim 15 wherein the quantification is effected by amplifying said target sequence by the using a pair of pπmers in the presence of a known concentration of a further sequence which competes with the said sequence for a pπmer, but which is distinguishable from said sequence duπng detection, and comparing signals produced by said further sequence and said target sequence
20. A method according to claim 15 wherein the quantification is effected using fluorescence based analysis
21. A method according to claim 20 wherein the amplification reaction is effected in the presence of a probe which includes a quencher moiety and a fluorescence reporter, said probe being arranged to be selectively cleaved duπng the amplification reaction so as to produce a flurorescent signal.
22. A method according to claim 21 wherein the amplification of a region of the PSR128 intron is effected using as pπmers
5 AAG GAG CTC GCC AAC GG3' (SEQ ID No 14) and
5 AAC CGA GGG TCC AGA AGA GAC G1 (SEQ ID No 15) ; and the probe comprises a sequence
5 GCT AGA TGG CTG GCT TCT ATT TCA TGA TCC A3 (SEQ ID No 16)
23. A method according to claim 21 wherein the amplification of a region of Dgas44 is carried out using the pπmers defined in claim 13, and the probe comprises a sequence * TTG GGA GGC ATG GTG AAA GTT GGT GAT (SEQ ID NO 10)
24. A nucleotide sequence which comprises or hybridises with all or part of the D- genome nucleotide sequence shown in Figure 8 (SEQ ID No 1 1 )
25. A nucleotide probe or primer which is specific for the nucleotide sequence of claim 24.
26. A pair of amplification pπmers which compπse either
CTTCTGACGGGTCAGGGGCAC (SEQ ID NO 4) or GTCCTATATCTTGAGGCCGCAAG (SEQ ED NO 6)
and either CTGAATGCCCCTGCGGCTTAAG (SEQ ID NO 5) or AACCCACTGTACCTGAGTATATATC (SEQ ID NO 7)
27 A pair of pπmers according to claim 26 which compπse SEQ ID NOS 4 and 5.
28 λ pair of amplification pπmers which are selected from either s ATG GCT GGC TTC TAT TTC ATG3 (SEQ ID No. 12) and " CAC CTA CTC CTC CAC ACT TTG3 (SEQ ID No 13); or
' AAG GAG CTC GCC AAC GG3 (SEQ ID No 14) and
' AAC CGA GGG TCC AGA AGA GAC G3 (SEQ ID No 15)
29 A kit for detecting the presence of wheat having a D-genome in a processed food product, said kit compnses either a labelled probe which hybπdises with a sequence which is specific for the D-genome. or means for amplifying such a sequence.
30 A kit according to claim 29 wherein the said sequence which is specific for the D- genome is or is deπved from the Dgas44 sequence or from the PSR 128 intron sequence.
31 A kit according to claim 29 or claim 30 which compnses a pair of pπmers according to any one of claims 26 to 28
32 Λ kit according to anv one of claims 27 to 30 which further comprises other reagents required to effect the polymerase chain reaction
32 Λ. kit according to any one of claims 27 to 31 which further compnses means for extracting DNA from said processed food product
33. A method for identifying the presence of a D-genome in a crop sample, which method compnses extracting DNA from said sample, and detecting the presence of a nucleotide sequence which comprises or hybridises with all or part of the nucleotide sequence shown in Figure 2
34. A method according to any one of claims 29 to 33 wherein the detection is effected by amplifying said sequence using PCR.
Abstract
A method for detecting the presence of wheat having a D-genome in for example a processed food product is described The method comprises extracting DNA from said product and detecting the presence of a nucleotide sequence which is characteristic of the D-genome and in particular is derived from the Dgas44 sequence or from the PSR 128 intron The sequence is suitably detected using PCR
A particular sequence within each of the Dgas44 and the single copy PSR128 intron has been identified as being a suitable target for detection
The method is useful in detecting contamination of flour, semolina, or pasta products which are made from T durum by bread wheat
Reagents useful in the method as well as kits for conducting the method are also described and claimed
PCT/GB1997/001988 1996-07-25 1997-07-23 Detection of d genome in wheat species WO1998004737A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
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GB9615679.9 1996-07-25

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EP1473364A1 (en) * 2002-02-15 2004-11-03 Nisshin Seifun Group Inc. Method of testing food
EP1482058A1 (en) * 2002-02-15 2004-12-01 Nisshin Seifun Group Inc. Method of testing wheat
EP1736543A1 (en) * 2004-04-09 2006-12-27 Nisshin Seifun Group Inc. Method of detecting and quantifying wheat endogenous dna sequence
US8173400B2 (en) 2006-05-15 2012-05-08 Nisshin Seifun Group Inc. Method of detecting or quantitating endogenous wheat DNA and method of determining contamination rate of genetically modified wheat in test sample
US9273362B2 (en) 2009-12-21 2016-03-01 Nisshin Seifun Group Inc. Method for detecting and quantifying wheat endogenous gene
RU2615449C1 (en) * 2015-12-14 2017-04-04 Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) Biological dna marker for determining impurities of soft wheat flour in flour durum wheat and its processing products
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