Nest polymerase chain reaction (PCR) detecting method of transgenic crop cauliflower mosaic virus

Abstract

A nest polymerase chain reaction (PCR) detecting method of a transgenic crop cauliflower mosaic virus relates to the field of molecular biology. The method designs two specificity outer primers and adopts a CaMV35S promoter published by an announcement NO. 953-6-2007 of a ministry of agriculture to detect a primer serving as an inner primer according to a conserved sequence of a cauliflower mosaic virus CaMV35S promoter, and the group of primers can detect the cauliflower mosaic virus CaMV35S promoter in different transgenic crops and has the characteristic of a broad spectrum. The detecting method has the advantages of being efficient, sensitive, accurate and the like, can avoid a 'false negative' result, and can effectively solve the technical problem of detecting transgenic crops. The nest PCR detecting method can provide technical support for agricultural transgenic organism safety management directly, can conduct effective monitoring on research, production and sales of transgenic crop products, and has important meaning on guaranteeing health of people, environment safety and biodiversity.

Description

technical field

[0001] The invention relates to the field of molecular biology, in particular to a method for detecting cauliflower mottle virus CaMV35S promoter in transgenic crops by using nested PCR technology. Background technique

[0002] At present, the global commercial production of genetically modified crops is developing rapidly. Since the first commercial planting in 1996, genetically modified crops have been quickly accepted by farmers due to their advantages such as greatly reducing field labor, reducing production costs, and mitigating environmental degradation, and the planting area has continued to expand. While genetically modified crops have brought huge economic benefits to people, their potential ecological safety and food safety issues have also attracted increasing attention. Now transgenic technology is still in the process of development, the international biosafety assessment system is not yet mature, the existing knowledge is not enough to assess t...

Examples

Embodiment 1

[0038] In this example, the nested PCR detection method was used to detect whether the soybean sample A contained the CaMV35S promoter of the transgenic component cauliflower mottle virus. The operation process is as follows:

[0039] (1) DNA extraction Weigh 0.1 g of soybean sample A, and use the CTAB method for DNA extraction.

[0040] (2) PCR amplification The first round of PCR amplification, the 20μl reaction system includes: 10mmol / L Tris-HCl, 50mmol / L KCl, 1.5mmol / L MgCl2, 0.15μmol / L dNTP, 0.5μmol / L 35S EF and 35S ER Primer, 1 unit of Taq DNA polymerase, 50ng DNA template; the reaction program is as follows: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 40 s, annealing at 58°C for 40 s, extension at 72°C for 45 s, 35 cycles; extension at 72°C for 5 min, 4°C save.

[0041] In the second-round PCR reaction system, 2 μL of the first-round PCR amplification product was added as a template, and the reaction program was: pre-denaturation at 94°C for 10 minut...

Embodiment 2

[0045] In this example, the nested PCR detection method was used to detect whether the corn sample B contained the cauliflower mottle virus CaMV35S promoter of the transgenic component. The operation process is as follows:

[0046] (1) DNA extraction Weigh 0.1 g of corn sample B, and use the CTAB method for DNA extraction.

[0047] (2) PCR amplification The first round of PCR amplification, the 20μl reaction system includes: 10mmol / L Tris-HCl, 50mmol / L KCl, 1.5mmol / L MgCl2, 0.15μmol / L dNTP, 0.5μmol / L 35S EF and 35S ER Primer, 1 unit of Taq DNA polymerase, 50ng DNA template; the reaction program is as follows: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 40 s, annealing at 58°C for 40 s, extension at 72°C for 45 s, 35 cycles; extension at 72°C for 5 min, 4°C save.

[0048] In the second-round PCR reaction system, 2 μL of the first-round PCR amplification product was added as a template, and the reaction program was: pre-denaturation at 94°C for 10 minutes, de...

technical field

[0001] The invention relates to the field of molecular biology, in particular to a method for detecting cauliflower mottle virus CaMV35S promoter in transgenic crops by using nested PCR technology. Background technique

[0002] At present, the global commercial production of genetically modified crops is developing rapidly. Since the first commercial planting in 1996, genetically modified crops have been quickly accepted by farmers due to their advantages such as greatly reducing field labor, reducing production costs, and mitigating environmental degradation, and the planting area has continued to expand. While genetically modified crops have brought huge economic benefits to people, their potential ecological safety and food safety issues have also attracted increasing attention. Now transgenic technology is still in the process of development, the international biosafety assessment system is not yet mature, the existing knowledge is not enough to assess t...

Examples

Embodiment 1

[0038] In this example, the nested PCR detection method was used to detect whether the soybean sample A contained the CaMV35S promoter of the transgenic component cauliflower mottle virus. The operation process is as follows:

[0039] (1) DNA extraction Weigh 0.1 g of soybean sample A, and use the CTAB method for DNA extraction.

[0040] (2) PCR amplification The first round of PCR amplification, the 20μl reaction system includes: 10mmol / L Tris-HCl, 50mmol / L KCl, 1.5mmol / L MgCl2, 0.15μmol / L dNTP, 0.5μmol / L 35S EF and 35S ER Primer, 1 unit of Taq DNA polymerase, 50ng DNA template; the reaction program is as follows: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 40 s, annealing at 58°C for 40 s, extension at 72°C for 45 s, 35 cycles; extension at 72°C for 5 min, 4°C save.

[0041] In the second-round PCR reaction system, 2 μL of the first-round PCR amplification product was added as a template, and the reaction program was: pre-denaturation at 94°C for 10 minut...

Embodiment 2

[0045] In this example, the nested PCR detection method was used to detect whether the corn sample B contained the cauliflower mottle virus CaMV35S promoter of the transgenic component. The operation process is as follows:

[0046] (1) DNA extraction Weigh 0.1 g of corn sample B, and use the CTAB method for DNA extraction.

[0047] (2) PCR amplification The first round of PCR amplification, the 20μl reaction system includes: 10mmol / L Tris-HCl, 50mmol / L KCl, 1.5mmol / L MgCl2, 0.15μmol / L dNTP, 0.5μmol / L 35S EF and 35S ER Primer, 1 unit of Taq DNA polymerase, 50ng DNA template; the reaction program is as follows: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 40 s, annealing at 58°C for 40 s, extension at 72°C for 45 s, 35 cycles; extension at 72°C for 5 min, 4°C save.

[0048] In the second-round PCR reaction system, 2 μL of the first-round PCR amplification product was added as a template, and the reaction program was: pre-denaturation at 94°C for 10 minutes, de...

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