CN102534046A - Nest polymerase chain reaction (PCR) detecting method of transgenic crop cauliflower mosaic virus - Google Patents

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

A nested PCR detection method for cauliflower mottle virus in transgenic crops

technical field

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

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 the benefits and risks of genetically modified crops. In view of this, most countries in the world have formulated corresponding laws and regulations to supervise genetically modified crops and products, such as EU countries, Japan, South Korea, etc., have formulated mandatory labeling systems; The Regulations on the Safety Management of Agricultural GMOs and the Measures for the Administration of Labeling of Agricultural GMOs have launched the regulatory mechanism for agricultural GMOs. The Chinese government stipulates that all genetically modified crops listed in the labeling management catalog and used for sale should be labeled. Therefore, effective detection of genetically modified crops and products must be carried out. the

The detection of genetically modified crops and products mainly uses protein detection methods and DNA detection methods. Among the DNA detection methods, the polymerase chain reaction (PCR) is mainly used, which is divided into qualitative PCR detection and quantitative PCR detection. The nested PCR detection is a qualitative PCR detection, which is a new technology developed on the basis of ordinary PCR. A single PCR reaction is used to detect a gene. Nested PCR has been widely used in many detection fields due to its specificity and sensitivity. Generally speaking, nested PCR has a high degree of specificity, and its results generally do not need to be verified by other methods.

When using genetic engineering technology to integrate a foreign gene into the plant genome, the foreign gene must be equipped with a promoter. The cauliflower mottle virus CaMV35S promoter is a broad-spectrum exogenous promoter in transgenic crops, such as corn, cotton, soybean, tomato, etc. About 85-90% of transgenic crops use this promoter. The conserved sequence of CaMV35S promoter is quite stable, which is the best target for transgene detection. The detection of CaMV35S promoter by nested PCR can efficiently, sensitively and accurately detect whether there are transgenic components in crops and products.

Contents of the invention

The purpose of the present invention is to provide a nested PCR detection method for cauliflower mottle virus in transgenic crops, which is used to detect the CaMV35S promoter of cauliflower mottle virus in transgenic crops. The "false negative" results can effectively solve the technical problems in the detection of genetically modified crops.

The purpose of the present invention is achieved through the following technical solutions:

A nested PCR detection method for cauliflower mottle virus of transgenic crops, the method comprising: using Primer Premier V5.0 software to design nested PCR primers, and using Oligo V6.22 software to screen out primers with minimal mutual interference;

The primers and amplified fragment sizes used in the amplification are:

Primers used in the first round of PCR

Primer name: 35S EF sequence (5'→3') ATTCCATTGCCCAGCTATCTGTCA

                35S ER sequence (5'→3') TGTGGTGTTTGTGGCTCTGTCCTAA

Amplified fragment size: 454bp

Primers used in the second round of PCR

Primer name: 35S IF sequence (5'→3')GCTCCTACAAATGCCATCATTGC

      35S IR sequence (5'→3') GATAGTGGGATTGTGCGTCATCCC

Amplified fragment size: 195bp;

The first round of PCR reaction system and reaction program is as follows: add 2 μL (50 ng) of DNA template to the PCR reaction system; cycle; extend at 72°C for 5 min, and store at 4°C;

The second-round PCR reaction system and reaction program are as follows: add 2uL of the first-round PCR amplification product to the second-round PCR reaction system; Extend at ℃ for 45s, 35 cycles; extend at 72℃ for 5 min, store at 4℃ for testing;

The two rounds of PCR amplification products are detected by agarose gel electrophoresis, and the samples are analyzed.

The nested PCR detection method of a kind of transgenic crop cauliflower mottle virus, the described agarose gel concentration is 2%, the concentration of ethidium bromide in the gel is 0.5 μ g/mL, and the electrophoresis condition is 10V/cm voltage , electrophoresis time 1h.

In the nested PCR detection method for cauliflower mottle virus of a kind of transgenic crop, the sample analysis is as follows: the size of the fragment amplified by the second round of PCR is consistent with the size of the expected fragment, indicating that the CaMV35S promoter of cauliflower mottle virus is detected in the sample, namely positive; otherwise, the cauliflower mottle virus CaMV35S promoter was not detected, that is, negative.

In the nested PCR detection method for cauliflower mottle virus of transgenic crops, whether a DNA fragment with the same size as the expected fragment can be observed in the first round of PCR products is not used as a judgment result; only the second round of PCR products The observation results are taken as the final judgment result.

Advantage and effect of the present invention are:

1. The detection method of the present invention has the advantages of high efficiency, sensitivity, accuracy, etc., can avoid "false negative" results, and can effectively solve the technical problems of detection of genetically modified crops.

2. The present invention designs two specific outer primers according to the conserved sequence of the cauliflower mottle virus CaMV35S promoter, and quotes the CaMV35S promoter detection primer published in No. 953 Announcement-6-2007 of the Ministry of Agriculture as an internal primer, and this group of primers can be Detection of the cauliflower mottle virus CaMV35S promoter in different transgenic crops with a broad spectrum.

3. The combination of the present invention and the CaMV35S promoter detection primers published in Announcement No. 953-6-2007 of the Ministry of Agriculture makes the invention more normative and authoritative.

4. The invention can directly provide technical support for the safety management of agricultural genetically modified organisms, and can be widely used in the research, production application and monitoring of agricultural genetically modified organisms. This invention can effectively supervise the research, production and sales of genetically modified crop products, and is of great significance to the protection of people's health, environmental safety and biodiversity.

Description of drawings

Fig. 1 is the result figure of the first round of PCR amplification of the present invention;

Fig. 2 is a graph showing the results of the second round of PCR amplification of the present invention.

Detailed ways

The present invention will be described in detail below.

Detection method of the present invention is:

(1) DNA extraction Weigh 0.1g sample, and use CTAB method for DNA extraction.

(2) PCR amplification The first round of PCR amplification, 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 Taq DNA polymerase, 50ng DNA template; the reaction program is as follows: 94°C pre-denaturation for 10 min, 94°C denaturation for 40 s, 58°C annealing for 40 s, 72°C extension for 45 s, 35 cycles; 72°C extension for 5 min, 4°C save.

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, denaturation at 94°C for 40 seconds, annealing at 56°C for 40 seconds, extension at 72°C for 45 seconds, and 35 cycles; Extend at 72°C for 5 min, and store at 4°C until testing.

(3) Electrophoresis detection Two rounds of PCR amplification products were detected by agarose gel electrophoresis: the concentration of agarose gel was 2%, the concentration of ethidium bromide was 0.5 μg/mL, the electrophoresis condition was 10V/cm voltage, and the electrophoresis time was 1h .

(4) Result analysis The results of the first round of PCR amplification are shown in Figure 1, no DNA bands were observed around 454 bp, and cannot be judged; the results of the second round of PCR amplification are shown in Figure 2, observed around 195 bp A DNA band was found, which was consistent with the expected fragment size, indicating that the cauliflower mottle virus CaMV35S promoter was detected in the sample, which was positive.

Example 1

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:

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

(2) PCR amplification The first round of PCR amplification, 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 Taq DNA polymerase, 50ng DNA template; the reaction program is as follows: 94°C pre-denaturation for 10 min, 94°C denaturation for 40 s, 58°C annealing for 40 s, 72°C extension for 45 s, 35 cycles; 72°C extension for 5 min, 4°C save.

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, denaturation at 94°C for 40 seconds, annealing at 56°C for 40 seconds, extension at 72°C for 45 seconds, and 35 cycles; Extend at 72°C for 5 min, and store at 4°C until testing.

(3) Electrophoresis detection Two rounds of PCR amplification products were detected by agarose gel electrophoresis: the concentration of agarose gel was 2%, the concentration of ethidium bromide was 0.5 μg/mL, the electrophoresis condition was 10V/cm voltage, and the electrophoresis time was 1h .

(4) Result analysis No DNA band was observed around 454 bp in the first round of PCR amplification results, which cannot be judged; no DNA band was observed around 195 bp in the second round of PCR amplification results, indicating that the soybean sample The cauliflower mottle virus CaMV35S promoter was not detected in A and was negative.

Example 2

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:

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

(2) PCR amplification The first round of PCR amplification, 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 Taq DNA polymerase, 50ng DNA template; the reaction program is as follows: 94°C pre-denaturation for 10 min, 94°C denaturation for 40 s, 58°C annealing for 40 s, 72°C extension for 45 s, 35 cycles; 72°C extension for 5 min, 4°C save.

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, denaturation at 94°C for 40 seconds, annealing at 56°C for 40 seconds, extension at 72°C for 45 seconds, and 35 cycles; Extend at 72°C for 5 min, and store at 4°C until testing.

(3) Electrophoresis detection Two rounds of PCR amplification products were detected by agarose gel electrophoresis: the concentration of agarose gel was 2%, the concentration of ethidium bromide was 0.5 μg/mL, the electrophoresis condition was 10V/cm voltage, and the electrophoresis time was 1h .

(4) Result analysis No DNA band was observed around 454 bp in the first round of PCR amplification results, which cannot be judged; a DNA band was observed around 195 bp in the second round of PCR amplification results, which was consistent with the expected fragment size , indicating that the cauliflower mottle virus CaMV35S promoter was detected in corn sample B, which was positive.

Claims (4)

1. A nested PCR detection method of transgenic crop cauliflower mottle virus, is characterized in that, the method comprises: utilize Primer Premier V5.0 software to carry out the primer design of nested PCR, screen out mutual interference with Oligo V6.22 software Minimal Primer; The primers and amplified fragment sizes used in the amplification are: Primers used in the first round of PCR Primer name: 35S EF sequence (5'→3') ATTCCATTGCCCAGCTATCTGTCA       35S ER sequence (5'→3') TGTGGTGTTTGTGGCTCTGTCCTAA Amplified fragment size: 454bp; Primers used in the second round of PCR Primer name: 35S IF sequence (5'→3')GCTCCTACAAATGCCATCATTGC       35S IR sequence (5'→3') GATAGTGGGATTGTGCGTCATCCC Amplified fragment size: 195bp; The first round of PCR reaction system and reaction program is as follows: add 2 μL (50 ng) of DNA template to the PCR reaction system; cycle; extend at 72°C for 5 min, and store at 4°C; The second-round PCR reaction system and reaction program are as follows: add 2uL of the first-round PCR amplification product to the second-round PCR reaction system; Extend at ℃ for 45s, 35 cycles; extend at 72℃ for 5 min, store at 4℃ for testing; The two rounds of PCR amplification products are detected by agarose gel electrophoresis, and the samples are analyzed. 2. the nested PCR detection method of a kind of transgenic crop cauliflower mottle virus according to claim 1, is characterized in that, described agarose gel concentration is 2%, and ethidium bromide concentration is 0.5 μ g in the glue /mL, the electrophoresis condition is 10V/cm voltage, and the electrophoresis time is 1h. 3. the nested PCR detection method of a kind of transgenic crop cauliflower mottle virus according to claim 1, is characterized in that, sample analysis is: second round of PCR amplification fragment size is consistent with expected fragment size, shows that detection in sample If the cauliflower mottle virus CaMV35S promoter is found, it is positive; otherwise, if the cauliflower mottle virus CaMV35S promoter is not detected, it is negative. 4. the nested PCR detection method of a kind of transgenic crop cauliflower mottle virus according to claim 1, it is characterized in that whether the first round of PCR product can observe the dna fragment consistent with expected fragment size, all do not judge Result; and only the observation result of the second round of PCR products is used as the final judgment result.

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