Method for high-throughput detection of transgenic corn by virtue of micro-fluidic chip

A technology of transgenic corn and microfluidic chip, which is applied in the field of high-throughput screening, can solve the problems of cumbersome steps, high detection cost, and cumbersome operation

Inactive Publication Date: 2015-04-29
曹际娟 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ordinary PCR method has been gradually replaced by the real-time fluorescent PCR detection method due to its cumbersome operation, environmental pollution, and low sensitivity.
However, the single-channel TaqMan real-time

Method used

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  • Method for high-throughput detection of transgenic corn by virtue of micro-fluidic chip
  • Method for high-throughput detection of transgenic corn by virtue of micro-fluidic chip
  • Method for high-throughput detection of transgenic corn by virtue of micro-fluidic chip

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0099] Example 1.

[0100] (1) Microfluidic chip design

[0101] microfluidic chip Array Cards, TAC (Applied ThermoFisher Scientific Inc., Foster City, CA) is a real-time fluorescent quantitative PCR reaction plate with a 384-well plate structure. The injection-molded 384-well plate is divided into 16 columns, each two columns share a sample addition channel, and each channel contains 48 cavities with a volume of 1uL. The microwells are pre-embedded with DNA primer pairs designed for the detection target and the corresponding Probes (as in Table 1). All probes used in the experiments contained fluorescent labels, either FAM or VIC. The present invention is designed to simultaneously detect 6 samples and one negative control sample and one positive control sample.

[0102] In addition, the chip also includes detection primer probe sets for the maize endogenous genes hmgA and adh1.

[0103] Table 1: Specific Primer and Probe Sequences for Microfluidic Chips

[0104]...

Example Embodiment

[0118] Example 2. Microfluidic chip detection specificity and sensitivity test

[0119] (1) Specificity test

[0120] 18 transgenic standard products including T25, MON88017, MON87640, MON89034, MIR162, TC1507, NK603, MON863, MON810, GA21, DAS40278, BT176, BT11, 98140, 59122, 3272, MIR604, LY038, etc. Specificity of primers and probes. Non-GMO corn samples were also used for specific verification.

[0121] In the specificity test, the reaction conditions were: 50 °C for 2 min; 95 °C for 10 min; 95 °C for 15 sec, 60 °C for 1 min, 40 cycles.

[0122] (2) Sensitivity test

[0123] A series of different concentrations of 18 transgenic standards including T25, MON88017, MON87640, MON89034, MIR162, TC1507, NK603, MON863, MON810, GA21, DAS40278, BT176, BT11, 98140, 59122, 3272, MIR604, LY038, etc. were used for sensitivity test , to determine the minimum detection concentration of the TAC fluid chip.

[0124] (3) Test results

[0125] The test results show that the detection se...

Example Embodiment

[0130] Embodiment 3. Actual sample detection test

[0131] The method established in Example 1 was used to screen and detect corn strains on 16 samples including corn flour, corn kernels, puffed corn flour, and corn distiller's grains.

[0132] At the same time, the real-time fluorescent PCR method for transgene detection (SN / T1196-2012) was used to verify the results. All single real-time PCR reactions were performed on an AB7900HT real-time PCR instrument. Each response includes: Universal PCR Master Mix (2×) 12.5μL, upstream primer (10μmol / L) 0.5μL, downstream primer (10μmol / L) 0.5μL, probe (5μmol / L) 1μL, DNA template (40ng / μL~50ng / μL) ) 2μL, ROX DyeII 0.5μL, supplemented with ddH 2 0 to 25 μL. The reaction parameters of real-time fluorescent PCR were: pre-denaturation at 95°C for 30s; 95°C for 5s, 60°C for 34s, 40 cycles.

[0133] The detection results are shown in Table 3: it can be seen that the detection results of the TAC fluid chip method are completely consiste...

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Abstract

The invention discloses a method for high-throughput detection of transgenic corn by virtue of a micro-fluidic chip. The micro-fluidic chip which contains not less than 20 reaction pores in each column of fluid channels and has not less than 3 columns of the channels, wherein the reaction pores of each column of the fluid channels are respectively embedded with 20 groups of specific primer probe compositions as shown in SEQ ID NO.1-60 in advance. According to the method disclosed by the invention, a TaqMan Array Card micro-fluidic chip detection technology is applied to the high-throughput screening detection of a transgenic corn line; in a polymerase chain reaction (PCR) amplification process, the screening detection of 2 corn endogenous genes and the screening detection of 18 exogenous recombinant lines are simultaneously completed, and the detection sensitivity can reach up to 1%. Compared with a single detection, the method can be used for detecting the endogenous genes and the exogenous genes in one PCR amplification reaction, thereby effectively avoiding possible errors caused by misoperation in experiments; and therefore the high-throughput screening detection of multiple samples and multiple gene targets can be achieved.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a high-throughput screening method for corn containing transgenic components. Background technique [0002] Corn is one of the most important food crops in the world, and its yield per unit area ranks first. There are more than 350 kinds of corn pests in the world, among which the Lepidoptera corn borer is the most widely distributed and the most harmful. It is an important corn pest worldwide and seriously affects the yield and quality of corn. Over the past ten years, breakthroughs have been made in the cultivation of transgenic plants, and a number of new varieties have been introduced, which have played an obvious role in improving crop production and have also shown great potential. However, there is much debate about the safety of GM corn. In the face of the impact of biogenetic engineering technology on my country's economic interests and the possible risks ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2537/143C12Q2545/114C12Q2563/107
Inventor 曹际娟徐君怡
Owner 曹际娟
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