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Method for detecting lead ions based on combination of fluorescence quantitative PCR (polymerase chain reaction) and nuclease GR-5

A GR-5, fluorescence quantitative technology, applied in the field of chemistry, can solve the problem of the lead ion detection method needs to be improved, and achieve the effect of high selectivity, high sensitivity, and improving the detection limit

Inactive Publication Date: 2014-04-02
CHANGSHU INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the current lead ion detection method still needs to be improved.

Method used

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  • Method for detecting lead ions based on combination of fluorescence quantitative PCR (polymerase chain reaction) and nuclease GR-5
  • Method for detecting lead ions based on combination of fluorescence quantitative PCR (polymerase chain reaction) and nuclease GR-5
  • Method for detecting lead ions based on combination of fluorescence quantitative PCR (polymerase chain reaction) and nuclease GR-5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Design and synthesis of corresponding oligonucleotide fragments;

[0030] The inventors designed a nuclease GR-5 that is dependent on lead ions, and modified it with biotinylation at its 5' segment. Complementary DNA is designed, and finally upstream and downstream primers for real-time fluorescent quantitative PCR are designed according to the sequence. Sequences were prepared by a DNA synthesizer.

[0031] Nuclease GR-5:

[0032] 5'-biotin-GGCTACGAGGGAAATGCGGTAATCATCTCTGAAGTAGCGCCGCCGTAGTG-3' (SEQ ID NO: 1).

[0033] Complementary DNA:

[0034] 5'-AATCTGGTTTAGCTACGCCTTCCCCGTGGCGATGTTTCTTAGCGCCTTACCACTrAGGAAGAGATGATT-3' (SEQ ID NO: 2)

[0035] Upstream primer: 5'-AATCTGGTTTAGCTACGCCTTC-3' (SEQ ID NO: 3)

[0036] Downstream primer: 5'-GTAAGGCGCTAAGAAACATCG-3' (SEQ ID NO: 4)

Embodiment 2

[0037] The hybridization of embodiment 2 DNA and the immobilization;

[0038]First, add 20 μL of 0.8% glutaraldehyde solution to the PCR tube to treat at 37 °C for 5 h, and then wash it with ultrapure water three times.

[0039] Next, treat again with 20 μL of 0.01 M carbonate buffer dissolved in streptavidin at 12.5 ng / mL for 2 h at 37 °C. Wash with PBST (10mM PBS, pH 7.2, 0.05% Tween-20) immediately after treatment.

[0040] Next, 20 μL of the mixture of nuclease GR-5 and complementary DNA was added to PCR tubes respectively, wherein the concentrations of nuclease GR-5 and complementary DNA were both 100 nM, and reacted at 37° C. for 40 minutes.

[0041] Finally, with sodium citrate buffer (750mM NaCl, 75mM C 6 h 5 Na 3 o 7 ) to wash 3 times. to remove unfixed DNA.

Embodiment 3

[0042] Embodiment 3 establishment of standard curve

[0043] Add 20 μL of lead ions to the PCR tubes obtained in Example 2 in order to make the final concentrations 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, and react at 37 ° C for 40 minutes, and finally use citric acid Wash 3 times with sodium buffer to remove unreacted DNA. Finally, 10 μL of PCR mixture, 2 μL of upstream and downstream primers, and 6 μL of water were sequentially added to the PCR tube for real-time fluorescent quantitative PCR.

[0044] Establishment of lead ion standard curve: Use real-time fluorescent quantitative PCR instrument to measure the cycle number of the amplification curve under different lead ion concentrations, and draw the standard curve of lead ion concentration according to the measured cycle number of the amplification curve under different lead ion concentrations.

[0045] The inventor found that: the linear equation of the standard curve is y=0.91748x+6.90575, y is the cycle number of th...

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Abstract

The invention provides a method for detecting lead ions based on combination of fluorescence quantitative PCR (polymerase chain reaction) and nuclease GR-5. The method comprises steps of (1) immobilizing nuclease GR-5 and complementary DNA (deoxyribonucleic acid) onto the inner surface of a PCR reaction vessel; (2) adding a sample to be tested into the PCR reaction vessel obtained in step (1), wherein when lead ions exist, in the presence of the nuclease GR-5, the complementary DNA breaks at the ribonucleotide so as to generate a short nucleic acid chain; (3) washing the PCR reaction vessel with citrate buffer solution so as to remove the nucleic acid molecules which are not immobilized; (4) adding an amplification primer into the PCR reaction vessel so as to carry out real-time fluorescence quantitative PCR on the immobilized complementary DNA; (5) confirming the concentration of the lead ions in the sample based on the Ct value of the real-time fluorescence quantitative PCR. According to the method, a brand-new biosensor with high selectivity and high sensitivity is provided and can be used for simply and rapidly detecting the concentration of lead ions in the sample.

Description

technical field [0001] The invention relates to the field of chemistry, in particular to a method for determining the concentration of lead ions in a sample, and more specifically to a method for detecting lead ions based on the combination of fluorescent quantitative PCR and nuclease GR-5. Background technique [0002] Lead and its compounds are one of the highly toxic global environmental pollutants. There is also a large amount of lead in automobile exhaust, smoke from plastic burning, paint, inferior children's toys, lead-acid batteries, industrial electroplating, and waste water from smelting. Popcorn in food, preserved eggs, and food packaged in tin foil also contain lead , the lead will eventually accumulate in water and cannot be degraded, which is extremely harmful to environmental organisms, especially to children. Its main toxic effects are anemia, nervous dysfunction, kidney damage, and reproductive system damage. Due to people's high attention to lead pollution...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
CPCC12Q1/686C12Q2545/114C12Q2521/327
Inventor 朱颖越邓大庆王立梅朱益波齐斌
Owner CHANGSHU INSTITUTE OF TECHNOLOGY
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