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Visual detection method for ultrafast amplification of copper and calcium-cleaved functional nucleic acids with universal isolation

An amplification system and fast technology, applied in the field of biosensors, can solve the problems of expensive, cumbersome sample processing, and lack of universal applicability

Active Publication Date: 2020-07-28
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Common detection methods for copper ions mainly include atomic spectroscopy, including atomic absorption spectroscopy, inductively coupled plasma emission spectrometry and fluorescence spectroscopy, visible spectrophotometry, flow injection chemiluminescence and differential potential stripping methods, etc., but these The detection methods all require expensive instruments and professionally trained operators, and the sample pretreatment is also relatively cumbersome, which is not universally applicable
The detection methods of calcium ions in water mainly include titration analysis and atomic photometry. This method generally requires professional technicians to collect samples to the laboratory for analysis, and has the same shortcomings as copper ion detection.

Method used

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  • Visual detection method for ultrafast amplification of copper and calcium-cleaved functional nucleic acids with universal isolation
  • Visual detection method for ultrafast amplification of copper and calcium-cleaved functional nucleic acids with universal isolation
  • Visual detection method for ultrafast amplification of copper and calcium-cleaved functional nucleic acids with universal isolation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Example 1 Copper and calcium-cutting functional nucleic acid visual detection method for universal partition ultrafast amplification

[0154] 1. Experimental materials

[0155] SYBR Gold nucleic acid dye, nucleic acid molecular weight standard ultra-low range DNA ladder, dNTP, ExTaq DNA polymerase, 10×Taq buffer, hemin, copper chloride, calcium chloride, 2,2-azino-bis(3 -Ethyl-benzothiazole-6-sulfonic acid) diamine salt (ABTS), H 2 o 2 , 4-Hydroxyethylpiperazineethanesulfonic acid (HEPES), MES, LiCl, Tris, potassium chloride, sodium chloride, magnesium chloride, disodium edetate, sulfuric acid, tetramethylbenzidine, urea, all Purchased from Thermo Scientific Life Technologies. All experimental water was from Milli-Q pure water system.

[0156] The sequence design is as follows (SEQ ID NO:1-13):

[0157]

[0158] Note: The ribozyme cuts the target product and the 3' end sequence of the amplified template is complementary;

[0159] The bold base sequence added to...

Embodiment 2

[0187] The specificity investigation of embodiment 2 detection method

[0188] According to the biosensor constructed in Example 1 (1), 10nM Cu 2+ , 10 μM Mg 2+ 、Cd 2+ , Ca 2+ , Zn 2 + 、Cr 3+ , Pb 2+ , Fe 2+ Added to the system for detection, the results show that the established Cu 2+ The biosensor has good specificity ( Figure 8 ).

[0189] According to the biosensor constructed in Example 1(2), 10nM Ca 2+ , 10 μM Mg 2+ 、Cd 2+ 、Cu 2+ , Zn 2 + 、Cr 3+ , Pb 2+ , Fe 2+ Added to the system for detection, the results show that the established Ca 2+ The biosensor has good specificity ( Figure 9 ).

[0190] According to the biosensor constructed in Example 1(3), 10nM Cu 2+ , 10nM Ca 2+ , 10 μM Mg 2+ 、Cd 2 + 、Cu 2+ , Zn 2+ 、Cr 3+ , Pb 2+ , Fe 2+ Added to the system for detection, the results show that the established Cu 2+ , Ca 2+ The biosensor has good specificity ( Figure 10 ).

Embodiment 3

[0191] Embodiment 3 standard addition experiment

[0192] Get high-purity water and detect with the biosensor constructed in embodiment 1, Cu 2+ , Ca 2+ No detection was carried out, and the standard addition experiment was carried out on it, and the results obtained by continuous measurement are shown in Table 1.

[0193] Table 1 The results of the standard recovery experiment

[0194]

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Abstract

The invention provides a universal-partition ultrafast-amplification cleavage type functional-nucleic acid visual detection method for copper and calcium. According to the invention, primers, a template and probes are ingeniously designed, so ultrafast amplification of the template can be realized in the presence of copper and calcium ions, and amplification products are allowed to form G-quadruplexes in a proper environment; and color development is performed by utilizing the peroxidase-like activity of the G-quadruplexes. Thus, the problem of difficulty in realizing visual detection of traditional PCR products is overcome, and rapid and visual detection of copper and calcium ions is realized. Moreover, the method provided in the invention has the characteristics of high specificity and high sensitivity to copper and cadmium ions, and is more objective and accurate in detection results.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to a method for visual detection of copper and calcium-cutting functional nucleic acids with universal partition ultrafast amplification. Background technique [0002] Copper is a transition element, chemical symbol Cu, atomic number 29, atomic weight 63.546, belonging to group IB. Pure copper is a soft metal, its surface is reddish orange with metallic luster when it is just cut, and its single substance is purplish red. Good ductility, high thermal conductivity and electrical conductivity. The amount of copper in the normal human body is 100-200 mg. In the human body, copper ions are mainly used as catalytic cofactors or structures for many enzymes and proteins, and are widely involved in many important metabolisms in the body. The process affects the generation of human blood, the formation of connective tissue, the central nervous system, the metabolism of cholesterol and gl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2521/337C12Q2525/186C12Q2565/607
Inventor 罗云波许文涛黄昆仑杜再慧田晶晶
Owner CHINA AGRI UNIV
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