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

A technology for quantitative detection of endocrine disruptors, applied in the field of detection of environmental endocrine disruptors, can solve problems such as binding limitations, unsatisfactory sensitivity and accuracy, and achieve the effects of small quantitative errors, high cost, and high-throughput monitoring

Inactive Publication Date: 2010-03-24
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Abstract
  • Description
  • Claims
  • 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
Comparison scheme
Effect test

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 GGTCAagg TGACCCACGTAGTCACTTGTCTAAAAGGGATGGTTTGTGTG-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 of the preparation of 3hERα monoclonal antibody

[0046] The peptide synthesizer synthesizes a 18-amino acid polypeptide in the hERαA / B region: Ala-Phe-Tyr-Arg-Pro-Asn-Ser-Asp-Asn-Arg-Arg-Gln-Gly-Gly-Arg-Glu-Arg-Leu , covalently linked to the carrier keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH), and Freund's adjuvant together to immunize Balb / c mice, and the splenocytes were hybridized with the mouse B cell tumor line Sp2 / 0 Hybridoma strains were formed by 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.

Embodiment 4

[0047] Implementation of 4 fluorescent quantitative PCR detection probe MGB-TaqMan and corresponding primers. FAM is a reporter group, MGB is a quencher group, and its sequence is:

[0048] MGB-TaqMan: FAM-TGGTCTGGCTCACTGC-MGB

[0049] Upstream primer FP: 5'-TGGCCTGGCGTGGAAGT-3'

[0050] Downstream primer RP: 5'-ACAAACCATCCCTTTTGACAAGTG-3'

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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 placeafter 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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Patent Type & Authority Patents(China)
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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