GB2405405A

GB2405405A - Standards in PCR

Info

Publication number: GB2405405A
Application number: GB0320329A
Authority: GB
Inventors: Peter Bowler; Katrina O'hara
Original assignee: RHM TECH Ltd
Current assignee: RHM TECH Ltd
Priority date: 2003-08-29
Filing date: 2003-08-29
Publication date: 2005-03-02
Also published as: GB0320329D0; WO2005021785A1

Abstract

The use of an individual single variety as a microsample as a standard in a DNA based method of gene quantification. Preferably the microsample is a single seed or specimen of a vegetable variety or a monoculture of an organism or the tissue of an animal species. The DNA based method may be copy number PCR (polymerase chain reaction amplification). The invention is exemplified by the use of single grain of Aestivum as a standard to detect the presence of wheat flour mixed with durum flour or alternatively suggested is the detection of mustard seed contaminated with rape seed.

Description

STANDARDS IN PCR

This invention relates to techniques for establishing standards for the characterization/quantification of genetically distinct varieties.

Molecular methods are able to provide a useful and selective means for the quantification of one biological material present in admixture with other closely related biological materials. This is possible due to the presence of unique DNA sequences in all species. If the sequences have been characterized, then they can be detected selectively and quantified. Currently the method of choice involves use of the polymerase chain reaction (PCR) in which targeted sequences of DNA are amplified. The amount can be quantified by means of a modified PCR technique, as described below.

Various chemistries are available for the quantification, but all are based upon binding of a modified DNA sequence (a probe) to the target strands, which fluoresces only when degraded or modified as the target sequences are amplified. This fluorescence is detected as a threshold cycle value (Ct), the threshold being the point at which sample fluorescence can be detected above the inherent background noise during amplification.

Quantification is based upon measurement of a specific DNA sequence present only in the "adulterating" sequence of interest and comparing its fluorescent signal with one similarly obtained from a "marker gene" present in both the "adulterating" and "adulterated" materials. There are two commonly used techniques for achieving this. The first involves using a range of comparative standards. The Ct value for the sequence of interest is subtracted from that for the marker gene and the difference is compared to similar data obtained from a series of standards. This method is known as the Delta Ct (AC:) approach.

The second uses a single standard (in contrast to the range used above) I which is itself diluted to produce a range of target DNA concentrations. The Ct values obtained for each of the "adulterating" and marker gene sequences are then compared with the equivalents for the diluted standard from which the number of copies of DNA present may be determined for the unknown sample. The ratio of these copy numbers for "adulterant" and marker gene sequences gives the proportion of adulterant. This is known as the copy number approach.

Currently, quantification using PCR requires the use of prepared standards as a calibrant. These standards may be those produced commercially, e.g. for Roundup Ready Soya (RRS), 0.1 - 5% RRS in normal soya used in the Ct method, or a single prepared standard used over a range of dilutions used for the copy number approach.

Currently, both approaches rely upon blends of 0% to 100% of materials of interest to produce a range of standards for use. Such standards are prepared and distributed by specialist organizations, such as IRMM in Europe, and are known as Certified Reference Materials.

The major disadvantage of this approach is that by blending two biological materials, errors and uncertainties arise due to the inherent variability of the biological materials themselves. The absolute levels of components contained in such biological materials will be affected by their growth environment, their variety and a number of other factors and hence the levels of specific components will vary. Consequently, the relative amounts of materials of interest may not be in the same ratios as the weights of components, even in accurately weighed blends.

The present invention has been devised in association with a desire to develop a pasta standard for PCR. Such a standard is necessary in order to establish clear limitation on the quantities of breadmaking or other flour that may be incorporated into a mix with durum flour for the manufacture of pasta products, and for monitoring compliance with regulations governing those limitations. As outlined below, however, far broader implications have since been envisaged and recognised.

In accordance with the invention, therefore, there is now provided use of an individual single variety microsample as an absolute standard in a DNA based method of gene quantification.

By an individual single variety microsample, there is to be understood in the context of the present description a single seed or like specimen of a vegetable variety; or a microsample from a monoculture of an organism such as bacteria; or a microsample of tissue from an animal species, which will be genetically unique to its variety yet capable of representing a standard for the genetic content of the variety. The microsample should be tested for the presence of a sequence of interest (the "adulterant") and, if that sequence is present, then that microsample by definition represents an absolute standard and may be used with total confidence as the standard for copy number approach PCR or other DNA-based quantification techniques.

As stated above, the invention has been derived from a consideration of the problems associated with the preparation of pasta standards for PCR, I namely the effects of variety, grain filling and other variables, all of which affect the ratios of DNA which can be extracted in standard blends in aestivum and durum.

Working with the copy number approach to PCR it has been possible to use nominally 100% product as a standard successfully for quantifications. It is apparent that this concept can be applied generically to PCR and to other DNA-based amplification techniques, where both the specific gene or sequence to be measured (the "adulterant") and the normalising or marker gene are present in the material to be quantified, i.e. the standard is 100% of the "adulterating" sequence. If these genes are present as single copies or in known numbers (rather than variable) then such materials can provide absolute standards for PCR (for example) using the copy number method for quantification.

The basis of the invention is therefore the use of only one material as a source of both "adulterant" and "normalising" genes with fixed copy number ratios, whereby the ratio between these is absolute, i.e. it cannot vary as in blended materials.

To ensure unequivocally that the standard is absolute, a single seed or part of a single organism should be used. The seed should first be tested for the presence of the "adulterating" sequence. If that sequence is present, then the seed by definition represents an absolute standard, and can be used as the absolute standard for the copy number approach PCR or other appropriate methodologies. The use of single seeds, or single organism material, obviates the need for conventional certified standards as the material may be self certified by a qualitative check for the presence of the "adulterating" sequence.

The following is presented as a practical example of the overall concept embodied by the invention.

Any aestivum grain could be used as a standard for the quantification of durum adulterated by aestivum. In this grain, there will be in the D genome (present only in aestivum) a specific sequence that can be measured in order to determine aestivum content, and the normalising An genomes present in both aestivum and durum. Hence, any single aestivum grain of any variety or quality may be used as an absolute standard. In the copy number approach to PCR, DNA is extracted and diluted serially to provide relative amounts of each of these DNA sequences, and samples may be measured for composition against each of the D and A/B genome sequences. From the dilution of the standard which is equivalent to the A/B and D genomes concentration in the sample, the percentage of aestivum can be readily calculated. The benefit of this is that ranges of standards containing both aestivum and durum are not required.

This technique can be applied to any material containing both genes of interest and the normalising genes and so may be applied to other situations.

For example, mustard seed can be quantitatively tested to determine any contamination with rape seed or the seed of other brassicas. The invention is potentially applicable also to the establishment of absolute standards for use in the quantitative testing of any materials for genetically modified varieties, which will allow for the more accurate monitoring of genetically modified materials in foods and their raw materials.

By means of the present invention, it is now possible to undertake the preparation of appropriate absolute standards and to make those standards available for commercial, industrial and legal testing and monitoring purposes. I

Claims

CLAIMS: 1. Use of an individual single variety microsample as an absolute

standard in a DNA-based method of gene quantification.
2. Use according to claim 1, wherein the microsample is a single seed or like specimen of a vegetable variety.
3. Use according to claim 1, wherein the microsample is from a monoculture of an organism.
4. Use according to claim 1, wherein the microsample is from tissue of an animal species.
5. Use according to any one of claims 1 to 4, wherein the DNA based method is copy number PCR.