Cadmium ion detection method based on T3 DNA ligase
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A detection method and technology for cadmium ions, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve the problems of large amount of products and non-specific binding.
Active Publication Date: 2021-09-07
NANJING NORMAL UNIVERSITY
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Although this amplification method is very convenient and efficient, the RCA system will cause non-specific and non-specific binding due to the large amount of product in practical applications.
This is a big challenge for its practical application
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[0026] Buffer 2 was specifically prepared as follows: 50mM Tris-HCl, 10mM MgCl2, 10mM (NH4)2SO4, 4mM DTT (pH7.5@25°C).
[0027] Preparation of SYBR Green I dye solution: first prepare solution 3, specifically: 10mM Tris-HCl, 10mM EDTA, pH=8.0), and then dilute SYBR Green I dye and solution 3 at a volume ratio of 1:100.
[0028] Table 1 Template sequences used in the T3 DNA ligase-based cadmium ion detection method
[0029] template sequence 5'P-ACTGTTGTGGTATTATTTTTGGTTGTGCAGT-OH 3' Primer sequence 5'-GGTTAATACCAC-3'
[0030] (1) Take 5 μL of sample solutions with different concentrations of cadmium ions and incubate with 5 μL of template sequences. Then add 1 μL of T3 DNA ligase, 20 μL of 2×T3 DNA ligase reaction buffer 1, and 9 μL of deionized water. Incubation was carried out for 20-40min.
[0031] (2) Take 10 μL o...
Embodiment 2
[0035] Sensitivity experiment
[0036] Sample solutions with cadmium ion concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100nM were selected for verification. The results are as follows: figure 2 shown. It is determined that the detection linear range is 0-100nM, and the detection limit reaches 0.08ppb.
Embodiment 3
[0038] selective experiment
[0039]Repeat the steps in the implementation example 1, other conditions remain unchanged, select lead ion, nickel ion, cobalt ion to detect fluorescence as interfering elements, obtain the selectivity result figure of this method, under the same conditions, detect the fluorescence of other ions and cadmium ions Value such as image 3 As shown, it can be seen from the figure that the method of the present invention has good selectivity to the target molecule.
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Abstract
The invention discloses a T3 DNA ligase-based cadmium ion detection method, which comprises the following steps: designing a DNA template, and when a target object cadmium ion exists, reducing the distance between two ends of a chain by the target object, so that a 3'end and a 5 'end are complementarily paired to form a blunt end; under the action of T3 DNA ligase, cyclizing the template; adding a section of oligonucleotide chain complementarily paired with the annular template as a primer for subsequent amplification reaction; under the action of adding phi 29 DNA polymerase, starting a rolling circle amplification reaction to obtain a longer nucleotide chain; adding a fluorescent dye SYBR Green I into the obtained product, inserting the dye into a DNA chain, detecting the sample by using an ultraviolet spectrophotometer under the excitation wavelength of 517-537 nm, and then measuring the fluorescence intensity under the excitation wave of 487-507 nm; detecting the cadmium ions by using a Real-Time PCR method, and the accuracy of the method disclosed by the invention is verified.
Description
technical field [0001] The invention relates to an analysis and detection method, in particular to a T3 DNA ligase-based cadmium ion detection method. Background technique [0002] Due to the discharge of industrial wastewater, some heavy metal elements are discharged into rivers, lakes or oceans. Heavy metal elements enter the organism along the food chain and cannot be biodegraded, but gradually accumulate in the organism. When a certain amount is reached, it will affect the normal physiological activities of the organism and endanger the health of the organism. In view of the harmful effects of cadmium ions, the US Environmental Protection Agency (EPA) has set a maximum cadmium pollution value of 5ppb in drinking water. Therefore, it is a crucial task to realize the highly sensitive detection of cadmium ions. Common detection techniques for cadmium ions include atomic absorption spectrometry, atomic fluorescence spectrometry, and inductively coupled plasma spectrometry....
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