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Transgenic cotton detection chip, kit and use

A detection kit and detection chip technology, applied in the biological field, to achieve the effect of sensitive detection results and low homology

Inactive Publication Date: 2011-06-29
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, there are few reports at home and abroad on the rapid and high-throughput detection of transgenic cotton using chip technology

Method used

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  • Transgenic cotton detection chip, kit and use
  • Transgenic cotton detection chip, kit and use
  • Transgenic cotton detection chip, kit and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The inventors of the present invention realized the simultaneous and rapid detection of 8 kinds of transgenic cottons through the visual chip technology for the first time.

[0050] The main detection instruments used:

[0051] Micropipettes (10 μL, 100 μL, 1000 μL Eppendorf), fluorescent quantitative PCR instrument (ABI 7700 Applied Biosystems, USA)), high-speed desktop centrifuge (Pico17Thermo), high-speed pulverizer (IKA-WEARKE GERMANY), nucleic acid and protein analyzer (DYY -6C Beijing Liuyi Instrument Factory), the ultraviolet gel imager is the Gene Genius system produced by Syngene; the AD3200 chip spotting instrument is produced by BioDot.

[0052] Detection of the main reagents:

[0053] The sequences of primers and probes were designed by our laboratory and synthesized by Beijing Aoke Biotechnology Co., Ltd. (see Table 1 for the specific sequences). Taq enzyme, dNTPs, 10×PCR Buffer, ethidium bromide, DNA Ladder Marker (Takara); TaqMan Universal PCR Master Mi...

Embodiment 2

[0080] Simultaneous Detection of Eight Kinds of Transgenic Cotton Using Multiplex PCR

[0081] In this experiment, by adjusting the sequence length and TM value, the annealing temperature of the eight sets of primer sequences was kept at about 60°C, and the hybridization temperature of the eight probes was kept at about 50°C, so as to ensure the simultaneous amplification of the multiplex of the eight transgenic cotton samples. The conditions for PCR and hybridization were the same.

[0082] Firstly, the 8 kinds of samples were detected respectively by conventional PCR method to determine the specificity of each primer sequence (results see image 3 ). The eight sets of primers were set at 5 concentration gradients (0.1 μmol / L, 0.2 μmol / L, 0.4 μmol / L, 0.8 μmol / L, 1.0 μmol / L), and cross-mixed sequentially for multiplex PCR amplification to determine the concentration of multiplex PCR. The optimal concentration of each set of primers in the reaction system.

[0083] The resul...

Embodiment 3

[0085] Taking sample Mon 1445 as an example, determine the spotting concentration of the hybridization probe. Spot the Probe-Mon 1445 (SEQ ID NO.17) probe solution with a concentration of 1 μmol / L, 5 μmol / L, and 10 μmol / L sequentially on the chip, and perform a hybridization test according to the conditions described in Example 1. The result is as Figure 4 As shown, the hybridization signal intensity of the three probes is basically the same, that is, when the probe concentration is 1 μmol / L, it has reached saturation, and on this basis, increasing the probe concentration has little effect on the hybridization signal strength.

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PUM

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Abstract

The invention relates to a transgenic cotton detection chip, which comprises a substrate and a nucleic acid probe positioned on the substrate, wherein the nucleic acid probe may be one or more of nucleic acid probes having the sequences SEQ ID No.3, SEQ ID No.5, SEQ ID No.8, SEQ ID No.11, SEQ ID No.14, SEQ ID No.17, SEQ ID No.20 and SEQ ID No.23. The invention also relates to a kit having the transgenic cotton detection chip, a method for detecting transgenic cotton and the use of the transgenic cotton detection chip and the kit in the detection of the transgenic cotton. The transgenic cottondetection chip can be used to detect the transgenic cotton simply, quickly, specifically and sensitively with high throughput.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a transgenic cotton detection chip, a kit and an application. Background technique [0002] Genetically modified organisms refer to the use of biotechnology to transfer foreign genes to other species to modify their genetic characteristics, so as to obtain new biological varieties with traits and nutritional qualities required by humans. [0003] Development Status of Transgenic Cotton [0004] Cotton is an important economic crop, and 1.8 billion people in the world live by growing cotton. Since the birth of the first transgenic cotton in the United States in 1987, research on cotton genetic engineering has rapidly spread around the world. At present, more than 50 genetically modified cotton varieties have passed the national and provincial examination and approval in my country, and the cumulative promotion has exceeded 6 million hectares. So far, genetica...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 陈颖邓婷婷韩建勋黄文胜白素兰
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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