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PCR quantitative detection method and reagent kit based on ERalpha activation effect environment incretion interferent

An endocrine disruptor and quantitative detection method technology, applied in the field of environmental endocrine disruptor detection, can solve the problems of combination limitation, unsatisfactory sensitivity and accuracy, and achieve the effect of small quantitative error, high cost and high-throughput monitoring

Inactive Publication Date: 2008-02-20
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Application Information

AI Technical Summary

Problems solved by technology

Kim et al. prepared a fluorescence-ER EDCs array slide screening technology, and used a microplate reader to measure the combined fluorescence intensity to quantify EDCs. However, due to the use of a wave plate solid phase, the combination is limited. This method detects in μM ~nM level, sensitivity and accuracy are not yet ideal

Method used

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  • PCR quantitative detection method and reagent kit based on ERalpha activation effect environment incretion interferent
  • PCR quantitative detection method and reagent kit based on ERalpha activation effect environment incretion interferent
  • PCR quantitative detection method and reagent kit based on ERalpha activation effect environment incretion interferent

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Experimental program
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Effect test

Embodiment 1

[0038] Implementation 1 Acquisition of recombinant human ERα (hERα) active protein, the schematic diagram is shown in Figure 3:

[0039] 1. Construction of baculovirus transfer vector: pSG5 and hERα cDNA were digested with BamH I and EcoR I, and the large fragment of the linearized vector was ligated with the 1875bp hERα DNA fragment to obtain the pSG5-hERα plasmid that preserved hERα DNA. BamH I and EcoR I digest pSG5-hERα plasmid and pFastBacl plasmid, and connect hERα to the large fragment of the vector to obtain the hERα baculovirus transfer vector pFast-hERα. The digestion reaction was carried out at 37°C, and the enzyme used for ligation was T4 DNA ligase.

[0040] 2. Construction of baculovirus expression vector and expression of hERα: transform pFast-hERα into Escherichia coli DH10BAC competent cells containing a baculovirus shuttle vector (bacmid, Bacmid), so that the hERα DNA of pFast-hERα and the Bacmid occurrence site Specific transposition, construction of recomb...

Embodiment 2

[0043] Implementation 2 Design of double-stranded DNA probes that can specifically bind to hERα

[0044] According to the estrogen response element (ERE), design a short fragment DNA probe of 89bp, its full sequence is: 5'-GTCTGGCCTGGCGTGGAAGTTTGGTCTGGCTCACTGCAGA GGTCAaggTGACC CACGTAGTCACTTGTCTAAAAGGGATGGTTTGTGTG-3' contains an estrogen response element (ERE) core sequence 5'-GGTCAnnnTGACC-3' (n is any nucleotide), and the nucleotides around the core sequence can be adjusted.

Embodiment 3

[0045] Implementation 3 Preparation of hERα monoclonal antibody

[0046] The peptide synthesizer synthesizes a polypeptide of 18 amino acids in the A / B region of hERα: Ala-Phe-Tyr-Arg-Pro-Asn-Ser-Asp-Asn-Arg-Arg-Gln-Gly-Gly-Arg-Glu-Arg- Leu was covalently linked to the carrier keyhole limpet hemocyanin (KLH), and Balb / c mice were immunized together with Freund's adjuvant, and splenocytes and mouse B cell tumor line Sp2 / 0 were collected Hybridoma strains were formed by hybridization and fusion, antigen-specific hybridoma strains were screened, cloned again, single clones were picked and cultured, and the culture fluid was separated and purified by saturated ammonium sulfate fractional precipitation to obtain McAbs that could specifically bind to hERα.

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Abstract

The utility model relates to an ER Alpha activation-based PCR quantitative detection method on environment incretion interferent. The method comprises: the acceptor allosteric dimerization takes place after the combination of trace amount of environment incretion interferent and an estrogen acceptor; identify and combine a DNA dual-chain probe, which comprises kernel series 5-GGTCAnnnTGACC-3 of estrogen reaction elements; generate a ligand-ER-DNA probe compound, which is absorbed and separated with monoclonal antibody; and then carry out real-time fluorescent quantitative PCR quantity augmentation; finally evaluate the EDCs biological effect in the sample. The utility model method, with simple and convenient operation plus high sensitivity, can reach even exceed the HPLC-MS / GS analytical chemistry method, and can be extensively used in testing application for food, environment and other fields. The utility model method also relates to reagent kits; and has advantages of simple, convenient and sensitive operation in detection range of 100pM to 1nM.

Description

technical field [0001] The invention relates to the field of detection of environmental endocrine disruptors, in particular to a PCR quantitative detection method and kit for environmental endocrine disruptors based on ERα activation effect. Background technique [0002] With the intensification of industrial pollution, the impact of environmental endocrine disruptors (EDCs, also known as environmental estrogens) on human health has increasingly attracted the attention of governments in various countries. EDCs refer to exogenous substances that interfere with the maintenance of homeostasis in organisms and regulate the production, release, metabolism, binding, excretion, and interaction of hormones in the development process. They have interference effects on human endocrine and immune systems. Benzene, dioxins, alkylphenols, phthalates, agricultural chemicals, natural or synthetic hormone drugs, etc. Since EDCs usually exist in the form of trace mixtures with great differe...

Claims

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Application Information

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IPC IPC(8): G01N21/00C12Q1/68
Inventor 邓国宏谭文婷王宇明章容陈健
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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