Methods for detecting genetically modified organisms (GMO)
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IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/6806
C12Q-001/686
C12Q-001/06
C12Q-001/6876
C40B-030/04
G01N-033/543
G01N-033/68
C12Q-001/6881
출원번호
15998965
(2017-02-17)
등록번호
11060126
(2021-07-13)
국제출원번호
PCT/CA2017/050210
(2017-02-17)
국제공개번호
WO2017/139898
(2017-08-24)
발명자
/ 주소
Akimova, Olga
Deboer, Anna
Li, Lianghong
Overgaard, Steven
Wlodek, Mike
출원인 / 주소
NUTRASOURCE PHARMACEUTICAL AND NUTRACEUTICAL SERVICES INC.
대리인 / 주소
Bereskin & Parr LLP/S.E.N.C.R.L., s.r.l.
인용정보
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초록▼
The present application relates to the detection of material from genetically engineered Genetically Modified Organisms (GMO) using a panel of GMO proteins and GMO gene elements. In particular, the present application discloses an improved method for detecting the presence or absence of a panel of G
The present application relates to the detection of material from genetically engineered Genetically Modified Organisms (GMO) using a panel of GMO proteins and GMO gene elements. In particular, the present application discloses an improved method for detecting the presence or absence of a panel of GMO proteins and gene elements in a sample.
대표청구항▼
1. A method for testing a sample for the presence of Genetically Modified Organism (GMO) material comprising: (i) homogenizing the sample by one of: (a) if the sample is a solid, grinding the solid to a powder,(b) if the sample is a semi-solid, freezing the sample and grinding frozen sample to a pow
1. A method for testing a sample for the presence of Genetically Modified Organism (GMO) material comprising: (i) homogenizing the sample by one of: (a) if the sample is a solid, grinding the solid to a powder,(b) if the sample is a semi-solid, freezing the sample and grinding frozen sample to a powder, and(c) if the sample is a liquid, agitating the sample if necessary for homogenization;(ii) extracting DNA from a portion of the sample obtained in (i);(iii) extracting protein from a portion of the sample obtained in (i);(iv) confirming a presence of DNA in the extract obtained in (ii) and if DNA is present, performing a PCR amplification on the extract to detect the presence of GMO gene elements in the extract, wherein the GMO gene elements comprise P35S promoter from cauliflower mosaic virus (CaMV), NOS terminator from A. tumefaciens and P34S promoter from figwort mosaic virus (FMV); and(v) confirming a presence of protein in the extract obtained in (iii) and if protein is present, performing an immunoassay on the extract to detect the presence of GMO proteins in the extract, wherein the GMO proteins comprise Cry1Ab, Cry2Ab, Cry34Ab1, Cry1Ac, Cry3Bb, Cry1F, VIP3a, NPTII, PMI, CP4 EPSPS and PAT,wherein the immunoassay comprises detecting a plurality of protein standards to obtain a standard curve and the method quantifies an amount of the GMO proteins as a weight of GMO protein per weight of the sample (w/w) using the standard curve. 2. The method of claim 1, wherein the plurality of protein standards comprises a dilution series of three or more dilutions for each GMO protein. 3. The method of claim 1, wherein the protein standards and the presence the GMO proteins in the sample are detected concurrently. 4. The method of claim 1, wherein the immunoassay is an Enzyme-Linked Immunosorbent Assay (ELISA). 5. The method of claim 4, wherein the ELISA comprises separate incubations with a capture antibody and a detection antibody. 6. The method of claim 1, wherein the immunoassay has a detection limit of at least 4 parts per billion (ppb) for one or more of the GMO proteins. 7. The method of claim 1, wherein the immunoassay has a detection limit of at least at least 10 ppb for one or more of the GMO proteins. 8. The method of claim 1, further comprising detecting a level of total protein in the sample. 9. The method of claim 8, wherein detecting the level of total protein in the sample comprises a Bradford assay and/or SDS-PAGE. 10. The method of claim 1, wherein if valid results are not obtained in the PCR amplification of step (iv) the method comprises: adjusting the extraction of DNA from the sample in step (ii) or adjusting the PCR amplification of step (iv); andretesting the sample for the presence of the GMO gene elements,wherein the adjusting the extraction of DNA from the sample comprises extracting DNA from >100 mg of the sample, eluting DNA in a smaller volume of water to provide a more concentrated sample, and/or performing a second DNA elution. 11. The method of claim 10, wherein if valid results are not obtained in the immunoassay of steps (v): adjusting the extraction of protein from the sample in step (iii) or adjusting the immunoassay of step (v); andretesting the sample for the presence or absence of the GMO proteins,wherein the adjusting the immunoassay assay comprises adjusting the pH to an optimal range and/or removing heavy metals, optionally using dialysis. 12. The method of claim 1, wherein the plurality of protein standards comprises a dilution series of five or more dilutions for each GMO protein. 13. The method of 1, wherein the plurality of protein standards and the presence of the GMO proteins in the sample are detected on the same plate. 14. The method of claim 1, wherein, when the sample is a raw material the GMO gene elements are detected using RT-PCR and when the sample is a baked good or a processed food, the GMO gene elements are detected using ddPCR. 15. The method of claim 1, further comprising generating a summary indicative of the presence GMO material in the sample based on results from steps (iv) and (v). 16. The method of claim 1, wherein the sample is a food product or an ingredient in the food product and the method further comprises labelling the food product based on results from steps (iv) and (v). 17. The method of claim 16, wherein if the method does not detect GMO material the food product the product is labeled as GMO-free. 18. The method of claim 1, further comprising treating the sample to remove PCR inhibitors prior to (iv).
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