Gene chip for detecting environmental pollutant DEHP (di-2-ethylhexyl phthalate), and preparation method and application thereof

A gene chip and environmental detection technology, which is applied in the field of environmental pollutant detection, can solve the problems of undisclosed gene chips, etc., and achieve the effects of sensitive detection results, rapid identification, and strong specificity

Active Publication Date: 2015-11-11
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, there is no relevant research report on the gene chip for detecting environmental pollutant DEHP and its preparation method and application at home and abroad.

Method used

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  • Gene chip for detecting environmental pollutant DEHP (di-2-ethylhexyl phthalate), and preparation method and application thereof
  • Gene chip for detecting environmental pollutant DEHP (di-2-ethylhexyl phthalate), and preparation method and application thereof
  • Gene chip for detecting environmental pollutant DEHP (di-2-ethylhexyl phthalate), and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0040]A kind of gene chip that is used to detect environmental pollutant DEHP, comprises the probe that spots on glass slide, and described probe comprises probe 1, probe 2 and probe 3, wherein

[0041] Probe 1 has the nucleotide sequence shown in SEQIDNO.1, and the fragment length is 125bp. The nucleotide sequence of the forward primer used in amplification probe 1 is: 5'-GGGACATATCCAATGGTGAC-3', and the reverse primer used in amplification probe 1 The nucleotide sequence of the primer is: 5'-GCCCTCTTGCGTTCTAGTTT-3';

[0042] Probe 2 has the nucleotide sequence shown in SEQIDNO.2, the fragment length is 200bp, the forward primer nucleotide sequence used for amplification probe 2 is: 5'-TGCCCGTGCTTACTATTGAC-3', the reverse primer used for amplification probe 2 The nucleotide sequence of the primer is: 5'-CCTTTCTTCCTCGTTATCCT-3';

[0043] Probe 3 has the nucleotide sequence shown in SEQIDNO.3, the fragment length is 179bp, the forward primer nucleotide sequence used for amplif...

specific Embodiment 2

[0044] 1. Discovery of DEHP biomarkers

[0045] Molecular markers for candidates were obtained through the previous analysis of differentially expressed genes and temporal expression changes of proteins in different tissues of Clam philippines.

[0046] 2. Gene chip preparation

[0047] a. Extraction of total RNA: Take 1.0 mL of clam body cavity fluid, centrifuge at 800 g for 5 min, collect blood cells, add 1.0 mL of RNAiso-plus reagent (purchased from Takara Company), oscillate and mix, and place at room temperature for 5 min, then add 0.2 mL of chloroform, Shake and mix well, let stand at room temperature for 10min, 4°C, 12000rpm, centrifuge for 15min, pipette the supernatant into a PE tube, add the same volume of isopropanol as the supernatant, mix well, let stand at room temperature for 5min, 4°C, 12000rpm , centrifuge for 10 min, remove the supernatant, add 1 mL of ethanol with a mass percentage concentration of 75% to the pellet, centrifuge at 4°C, 12000 rpm for 5 min, ...

specific Embodiment 3

[0059] 1. Preparation of low-concentration hybridization template

[0060] a, sample acquisition: laboratory conditions simulate DEHP stress Philippine clam, DEHP stress experiment is that Philippine clam is exposed to the seawater of the DEHP that final concentration is 0.4mg / L; In seawater; after 36 hours of stress, take 1.0 mL of clam coelom fluid, centrifuge at 800 g for 5 min, and collect blood cells;

[0061] b. Total RNA extraction: The obtained blood cells were immediately extracted and purified with the AxyPrep Total RNA Mini Kit;

[0062] c. Labeling of RNA samples and acquisition of cDNA: the control group was labeled with Cy3 fluorescence, and the treatment group was labeled with Cy5 fluorescence:

[0063] Total RNA 20ug make up 8μL

[0064] Oligo (dT) 18 primer 1 μL

[0065] External standard (no homology gene fragment) 1 μL

[0066] Denature at 65oC for 5 minutes, and place at room temperature for 10 minutes;

[0067] Then add 5×1ststrandbuffer4μL

[0068] ...

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Abstract

The invention discloses a gene chip for detecting an environmental pollutant DEHP (di-2-ethylhexyl phthalate), and a preparation method and application thereof. The gene chip is characterized by comprising a probe 1, a probe 2 and a probe 3 of which the nucleotide sequences are disclosed as SEQ ID NO.1-3. The preparation method comprises the following steps: total RNA (ribonucleic acid) extraction, cDNA (complementary deoxyribonucleic acid) synthesis, PCR (polymerase chain reaction) amplification, product segment recovery and chip preparation, wherein the PCR amplification primers have amino acid sequences disclosed as SEQ ID NO.4-9. The application method comprises the following steps: after the sample and gene chip are hybridized, if the gene expressions corresponding to the probe 2 and probe 3 decrease and the gene expression corresponding to the probe 1 does not change obviously, the gene chip is responsive and sensitive to 0.4 mg / L DEHP; and if the gene expressions corresponding to the probe 1 and probe 3 increase and the gene expression corresponding to the probe 2 does not change obviously, the gene chip is responsive and sensitive to 4 mg / L DEHP. The gene chip has the advantages of high specificity, high sensitivity and high detection speed.

Description

technical field [0001] The invention belongs to the field of environmental pollutant detection, and in particular relates to a gene chip for detecting environmental pollutant DEHP and its preparation method and application. Background technique [0002] Bis(2-ethylhexyl) phthalate (DEHP), as the most commonly used plasticizer at present, is an environmental hormone with strong stability and refractory characteristics. With the mass production and use of plastic products, while causing environmental "white pollution", DEHP also continues to enter the water environment. A large number of organic pollutants, including DEHP pollutants, enter the marine environment through various channels, and eventually migrate to marine organisms or accumulate in the bottom and suspended matter, causing great harm to the marine environment and marine organisms. The toxic effects of DEHP on marine organisms mainly include enrichment and degradation, genotoxicity of aquatic organisms, reproduct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C40B50/06C12Q1/68
Inventor 李成华陈晓聪金春华李晔张卫卫韩庆喜
Owner NINGBO UNIV
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