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Transgenic agricultural product detection kit

A detection kit and transgenic technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve problems such as infection and inability to exclude viruses.

Inactive Publication Date: 2005-06-08
上海中科伍佰豪生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even if the above-mentioned genes are detected in the sample to be tested, the possibility of viral infection cannot be ruled out

Method used

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  • Transgenic agricultural product detection kit
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  • Transgenic agricultural product detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Oligonucleotide primers and hybridization membrane design and synthesis

[0119] According to the promoter, terminator and marker gene of the transgene involved in the transgenic product, the cauliflower mosaic virus 35S promoter (P35S), the nopaline synthase terminator (TNOS), and the nopaline synthase promoter (PNOS) were selected. ), neomycin phosphotransferase gene (NPTII), β-glucuronidase gene (GUS), green fluorescent protein gene (GFP), 5-enolpyruvyl oxalate-3-phosphate synthase gene (EPSPS, Wild-type gene and modified gene CP4), cowpea trypsin CpTI, antibacterial peptide Shiva gene. According to the nucleotide sequences of these gene promoters, terminators, and marker genes, the specific primers and specific probes shown in Table 1A-B and Table 2 were designed and synthesized, respectively.

[0120] name

[0121] name

[0122] Note: X in Table 1 means with biotin label.

[0123] Table 1 lists the sequences of the chimeric primers and their r...

Embodiment 2

[0128] Preparation of genetically modified product detection kit

[0129] A. Detection kit composition and storage

[0130] Primer set (I)

250mL

-20℃

Primer set (II)

250mL

-20℃

MPCR Mixture

20mL×200 sticks

-20℃

Taq enzyme

150mL

-20℃

Control DNA

100mL

-20℃

mineral oil

5mL

room temperature

[0131] Wherein, the primers in the primer set (I) are directed against cauliflower mosaic virus 35S promoter (P35S), nopaline synthase terminator (TNOS), nopaline synthase promoter (PNOS), neomycin phosphotransferase gene (NPTII), β-glucuronidase gene (GUS) and green fluorescent protein gene (GFP); The primers in the primer set (II) are aimed at 5-enol type pyruvyl oxalate-3-phosphate synthase gene ( EPSPS wild-type gene and modified gene CP4), cowpea trypsin gene CpTI, antibacterial peptide Shiva gene.

[0132] Hybridization membrane (in hybridization bag)

100 pieces

room t...

Embodiment 3

[0146] Detection of genetically modified products

[0147] 1. DNA extraction of genetically modified agricultural products:

[0148] Soak soybeans in water for 4-8 hours to make them soft; soak in 70% ethanol for 1 minute; fully soak in sterile distilled water twice, each time for 5 minutes; put a soybean into a 1.5ml sterile centrifuge tube, add 400mL 2 ×CTAB extract [CTAB (W / V) 2%; Tris-HCL (pH8.0) 100mmol / l; EDTA 20mmol / l; NaCL 1.4mol / l; mercaptoethanol 2%], use a special glass grinder or Grind soybeans as much as possible with ordinary glass rods; keep warm in 65°C water bath for 30 minutes; add 400mL chloroform, mix well, 14,000rpm, 10min; take the supernatant into another 1.5ml centrifuge tube; add 1.5 times, that is, 600uL CTAB precipitation buffer solution [CTAB 1%, Tris-HCl (pH8.0) 50mmol / l, EDTA 10mmol / l, mercaptoethanol 1%]; place at room temperature for one hour; 12,000rpm, 5min, discard the supernatant; add 400mL 1.25M NaCL to dissolve the precipitate ; Repeated...

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Abstract

The invention relates to a detection method for genetically modified agricultural products, which comprises the following steps: (a) extracting the DNA of the genetically modified product; (b) performing PCR amplification on the extracted DNA with 3-10 pairs of primers, and the primers are respectively Specific amplification is selected from the amplified product of the following group of genes: P35S; TNOS; PNOS; NPTII; GUS; GFP; EPSPS wild-type gene; EPSPS modification gene CP4; The amplified product in step (b) is hybridized with the hybridization membrane labeled with a specific probe; (d) detecting the hybridization result. The invention also provides a corresponding detection kit and related primers and probe sequences. The invention can detect genetically modified products efficiently, accurately and simply, and can be used in customs, commodity inspection and other fields.

Description

technical field [0001] The present invention relates to product detection technology, more specifically, the present invention relates to the detection method of transgenic agricultural product, detection kit and corresponding probe sequence, MPCR primer sequence and so on. Background technique [0002] Genetically modified crops can speed up photosynthesis or improve the ability of crops to resist disease, salt and drought, so as to increase crop yield. However, we cannot fail to see the possible negative impact of genetically modified crops on biodiversity in rural areas. Genetically modified crops are like a double-edged sword. While making full use of them, we must always be vigilant against them. Especially for crops imported from abroad, it is more necessary to know whether they are genetically modified products. [0003] Because genetically modified crops are difficult or even impossible to distinguish from the outside, they need to be tested at the genetic level, wh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 龚毅杨胜利陶震
Owner 上海中科伍佰豪生物工程有限公司