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Microfluidic protein chip for detecting genetically modified crops and kit of microfluidic protein chip

A protein chip and genetically modified technology, applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of unknown genetically modified products damaging trade reputation, health and safety threats, etc., to avoid the decline of probe activity, facilitate operation, and save testing effect of labor

Inactive Publication Date: 2014-09-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existence of unknown genetically modified products that cannot be detected and discovered in time has greatly damaged trade reputation and threatened people's health and safety

Method used

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  • Microfluidic protein chip for detecting genetically modified crops and kit of microfluidic protein chip
  • Microfluidic protein chip for detecting genetically modified crops and kit of microfluidic protein chip
  • Microfluidic protein chip for detecting genetically modified crops and kit of microfluidic protein chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1, a method for preparing a microfluidic protein chip for high-throughput detection of transgenic proteins, the following steps are performed in sequence:

[0080] Step 1: Based on μParaflo TM The microfluidic biochip synthesizes six single-stranded oligonucleotides of different sequences in situ, and the 3' end of the nucleic acid is connected to the bottom of the chip. Number the positions of each probe (single-stranded oligonucleotide) from A to F, repeat 6 positions for each probe, and number each position, for example, type A probes are counted as A1, A2, ... A5 , A6, other types of probes are similar. Therefore, there are a total of 36 probe sites on the chip. The sequence and number of each probe are as follows in Table 1:

[0081] Table 1. Sequences and numbers of probes

[0082]

[0083] Store the nucleic acid chip synthesized in this step at room temperature for long-term stable storage (the storage period can generally be as long as 2 years)...

Embodiment 2

[0128] Embodiment 2. A transgenic microfluidic protein chip detection kit, which is used to synthesize a transgenic microfluidic protein chip and perform transgene detection; the detection kit includes the following components:

[0129] ①. A microfluidic DNA chip containing 6 probes; prepared according to the first step of Example 1.

[0130] ②, 6 kinds of antibody-nucleic acid complex probes; prepared according to the second step of Example 1.

[0131] ③, 6 detection antibodies:

[0132] Including: biotin-labeled anti-Cry1Ac rabbit polyclonal antibody; biotin-labeled anti-Cry1c rabbit monoclonal antibody; anti-Cry1Ab mouse antibody; anti-Cry1F mouse antibody; anti-SBTI mouse antibody; anti-NPTⅡ mouse antibody.

[0133] The rabbit antibody can be purchased from Yikang (Hangzhou) Biotechnology Co., Ltd.; the mouse antibody can be purchased from Huaan (Hangzhou) Biotechnology Co., Ltd., for example.

[0134] 7. Blocking buffer (same as Example 1);

[0135] 8. Washing buffer (...

Embodiment 3

[0138] Embodiment 3, the method for using the transgenic microfluidic protein chip detection kit as described in embodiment 2, the following steps are carried out in sequence:

[0139] 1), instant generation of microfluidic protein chips (same as the third step in Example 1);

[0140] 2) Perform sample detection on the generated protein chip, the specific steps are as follows:

[0141] The transgenic detection sample is extracted and purified to obtain the sample detection solution. Add 50 μL of sample detection solution to the microfluidic system, set the flow rate to 20 μL / min, and incubate overnight (16 hours) at 4°C. After the reaction, the chip was washed with PBST buffer at 30° C. for 20 min to wash away unbound samples on the chip surface.

[0142] The preparation method of PBST buffer solution is: weigh 8g NaCl, 0.2g KCl, 1.44g NaCl 2 HPO 4 , 0.24g KHPO 4 , dissolved in 800mL distilled water, added 2mL Tween-20, adjusted the pH to 7.2, added distilled water to mak...

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Abstract

The invention discloses a microfluidic protein chip for high-through detecting genetically modified crops. The microfluidic protein chip comprises a nucleic acid chip which is synthesized on the basis of mu ParafloTM microfluidic protein chip and contains six nucleotide sequences and 6 types of antibody-oligonucleotide composite probes, thereby simultaneously detecting 6 transgenetic proteins of trans-bacillus thuringiensis Bt insecticidal crystal protein (Cry1Ac), trans-bacillus thuringiensis Bt insecticidal crystal protein (Cry1c), trans-bacillus thuringiensis Bt insecticidal crystal protein (Cry1F), trans-bacillus thuringiensis Bt insecticidal crystal protein (Cry1Ab), soybean trypsin (SBTI) and neomycin phosphate transferase II gene (NPTII). The invention further provides a transgenic assay kit which comprises a mu ParafloTM microfluidic deoxyribose nucleic acid (DNA) chip which contains six types of probes, six types of antibody-oligonucleotide composite probes, six types of detection antibodies and the like.

Description

technical field [0001] The invention relates to the technical field of transgenic component detection and microfluidic protein chips, in particular to a high-throughput microfluidic protein chip and related kits for detecting multiple transgenic components. Background technique [0002] Over the past decade, transgenic technology has been extensively developed. Researchers have used transgenic technology to breed herbicide-resistant, drought-resistant, salt-alkali-resistant, heavy metal-resistant and pest-resistant varieties with high nutritional value, which play an important role in increasing food production, reducing post-harvest losses and increasing the nutritional value of agricultural products. With the continuous research, absorption and popularization of transgenic crops, the diversity of genetically modified crops is becoming more and more abundant. Many countries and regions have successively approved the commercial production of genetically modified crops, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/573
Inventor 郑晓冬沙莎殷赟高晓莲周小川
Owner ZHEJIANG UNIV
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