Method for detecting transgenic tomato by using in-situ synthetic microfluidic chip

A microfluidic chip and in-situ synthesis technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. It can solve the problems of not meeting the needs of food supervision, low efficiency, narrow detection range, etc.

Inactive Publication Date: 2013-09-11
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional GMO detection methods are often limited to a single gene, with narrow detection range and low efficiency
In the case of more and more types of exogenous genes introduced into agricultural products, the detection of a single item can no longer meet the needs of food supervision, and there is an urgent need to develop high-throughput rapid genetically modified detection technology

Method used

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  • Method for detecting transgenic tomato by using in-situ synthetic microfluidic chip
  • Method for detecting transgenic tomato by using in-situ synthetic microfluidic chip
  • Method for detecting transgenic tomato by using in-situ synthetic microfluidic chip

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Embodiment 1. A method for detecting transgenic tomato by in-situ synthesis microfluidic chip, using "Huafan No. 1" tomato as the sample to be tested, and performing the following steps in sequence:

[0066] 1) Design of chip probes for exogenous gene detection in transgenic tomato: design 8957 oligonucleotide probes as described in Table 1;

[0067] 2) Preparation of oligonucleotide chip: the schematic diagram of the arrangement of probes on the chip is as follows: Figure 4 shown. Perform fluorescence analysis on the synthesized chip on a chip scanner to detect the fluorescence background of the chip itself; when the background signal intensity of the chip is less than 20 and there is no obvious signal point, the chip is considered to meet the requirements.

[0068] Then use quality control cDNA (an artificially synthesized oligonucleotide complementary to the chip quality control probe) or a DNA fragment from a plasmid (using the plasmid PBI121 as a template, the 35s...

Embodiment 2

[0113] Example 2 Change the sample to be tested in Example 1 from "Huafan No. 1" to "Lichun ZN", and the rest are the same as in Example 1.

[0114] Remarks: In step 4), only method A (common PCR) and method B (multiple PCR) are used.

[0115] The end result is as figure 2 Shown:

[0116] Method A--Display common PCR labeling method ( figure 2 a), exogenous genes 35s, acc, nos and nptII were detected, and the number of detected probes accounted for 79% (49), 32% (16), and 66.7 of the probes designed for these genes, respectively % (30) and 43% (86), the average signal intensities of the top 10 probes detected by each gene were 20081, 19509, 7382 and 13305 respectively; the internal reference genes fru, apx, mcpi and lat52 were detected , the number of detected probes accounted for 16.9% (13), 19.6% (20), 54.2% (13) and 51.9% (28) of the probes designed for these genes, respectively. The average signal intensities of the first 10 detected probes were 6186, 11339, 4754 and...

Embodiment 3

[0124] Example 3 Change the sample to be tested in Example 1 from "Huafan No. 1" to "He He 903", and the rest are the same as in Example 1.

[0125] Remarks: Only method A (ordinary PCR) was used in step 4).

[0126] The end result is as image 3 Shown: The internal reference genes fru, apx, mcpi and lat52 were all detected, and the number of detected probes accounted for 16.9% (13), 15.6% (16), and 50 of the probes designed for these genes, respectively. % (12) and 42.6% (23), the average signal intensities of the first 10 probes detected by each gene were 5372, 8490, 4517, and 3102; no exogenous genes were detected, indicating "Cooperation 903" Tomato samples were transgenic negative samples.

[0127] Verification experiment 3:

[0128] The genomic DNA of the "Cooperation 903" tomato sample described in Example 3 was amplified by ordinary PCR, and the amplified band was detected by agarose gel electrophoresis;

[0129] The results obtained are: the result is the same as ...

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Abstract

The invention discloses a method for detecting a transgenic tomato by using an in-situ synthetic microfluidic chip. The method comprises the following steps of: 1) designing a chip probe for detecting an exogenous gene of the transgenic tomato; 2) preparing an oligonucleotide chip; 3) extracting genome deoxyribonucleic acid (DNA) in a tomato sample to be detected; 4) marking a specific segment of the genome or the entire genome by a fluorescent agent; 5) chip hybridization; 6) judging whether the sample belongs to the transgenic tomato variety. By adopting the method, an oligonucleotide probe is designed according to the sequence information of the known tomato exogenous gene which has been publically reported; the microfluidic gene chip is prepared by utilizing an in-situ synthesis method, so as to achieve simultaneous detection of existence of the exogenous genes related to insect resistance, antiviral action, herbicide resistance, delayed fruit, softening resistance, storage resistance and the like on the same chip. Thus, an effective method is provided for rapid detection and identification of the transgenic ingredients of the tomato and highly processed products; a foundation is provided for building and perfecting a transgenosis detection system and a supervisory system.

Description

technical field [0001] The invention relates to a method for detecting and identifying transgenic components in tomato, in particular to a method for detecting known exogenous genes in tomato by using an in-situ synthetic microfluidic chip. Background technique [0002] Tomato (Solanum lycopersicum) is one of the important crops of Solanaceae. Its fruit is rich in vitamins, carotene, and lycopene, and has high nutritional value and health benefits. It is an important fruit and vegetable product in the world. In the past ten years, the planting area and output of tomato in the world have increased by 38% and 42% respectively, and the industrialization trend is obvious. Since 1994, when the extended ripening and fresh-keeping transgenic tomato FLAVR SAVR bred by Calgene Corporation of the United States was approved to enter the market, various improved tomato varieties have been reported one after another, and some of them have been approved. It has become an irresistible tre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 冯俊丽王凤军梁彦君
Owner ZHEJIANG SCI-TECH UNIV
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