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Urease based microRNA detection kit

A detection kit, the technology of the kit, which is applied in the determination/inspection of microorganisms, the measurement of color/spectral properties, and the material analysis by observing the influence on chemical indicators, can solve the problems of nucleic acid molecule detection that have not been seen before, To achieve the effect of simplified operation, convenient detection and good specificity

Inactive Publication Date: 2020-07-31
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the principle of the mismatch reaction between DNA bases and heavy metal ions has not been used for the detection of nucleic acid molecules.

Method used

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  • Urease based microRNA detection kit
  • Urease based microRNA detection kit
  • Urease based microRNA detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 prepares double-stranded DNA solution

[0049] In this embodiment, a solution of double-stranded DNA is prepared, and the double-stranded DNA plays a role of molecular recognition in the process of detecting miRNA.

[0050] First, the substrate strand and the mismatched strand are synthesized, and their sequences are shown in Table 1.

[0051] The toehold structure refers to the single-stranded part of the double-stranded DNA that cannot be paired at the end. In Table 1, the underlined part of the substrate strand / mismatched strand sequence is the toehold structure, and the sequence numbers of the strands are all defined by the length of the toehold. Substrate strands and mismatched strands with the same sequence number are paired and used in conjunction with each other, for example, the substrate strand with sequence number 3nt (sSub-3nt) is used in conjunction with the mismatched strand with sequence number 3nt (mSub-3nt).

[0052] The bases of the toeh...

Embodiment 2

[0059] Embodiment 2 draws standard curve

[0060] In the present embodiment, the standard solution curve is drawn, and the steps are as follows:

[0061] ① Take 30 μL of the double-stranded DNA solution containing the C-C structure prepared in Example 1, add 10 μL of let-7a solution with a concentration of 0 nM to it, and place it at room temperature for 30 minutes to carry out the displacement reaction. Then 6 μL of urease with a concentration of 100 nM was added, mixed with 4 μL of ultrapure water to maintain a total volume of 50 μL, and then incubated at room temperature for 30 min to obtain a reaction mixture.

[0062] ② Take 10 μL of 2.5 mM phenol red solution and 10 μL of 5 M urea aqueous solution at room temperature and add them to 30 μL of ultrapure water to obtain 50 μL of urea-phenol red mixed solution.

[0063] ③ Mix the solutions prepared in ① and ②, react at room temperature for 20 minutes, measure the ultraviolet absorption of the reaction mixture at a wavelengt...

Embodiment 3

[0066] Embodiment 3 clinical detection

[0067] In this example, the concentration of let-7a in cancer tissues and paracancerous tissues from 5 lung cancer patients was detected, and the significant difference was analyzed. The steps are as follows:

[0068] (1) The cancer tissues and paracancerous tissues of 5 lung cancer patients were collected, and recorded as 1#~5# in turn, the cancer tissues were recorded as C, and the paracancerous tissues were recorded as A, and the total RNA of 1#~5# tissues was extracted, Make RNA aqueous solution.

[0069] (2) Let-7a detection in RNA aqueous solution

[0070] ①Take two parts (30 μL) of the double-stranded DNA solution containing the C-C structure prepared in Example 1, add 1#A and 1#C sample solutions to it in turn, and place it at room temperature for 30 minutes to carry out the displacement reaction. Then 6 μL of urease with a concentration of 100 nM was added, mixed with 4 μL of ultrapure water to maintain a total volume of 50 μ...

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Abstract

The invention discloses a microRNA let-7a detection kit, and belongs to the field of molecular detection. The kit comprises a molecular recognition reagent and a signal conversion reagent, wherein themolecular recognition reagent is a DNA double-strand molecule embedded with silver ions, the DNA double-strand molecule is formed by complementarily pairing bases of a substrate chain and a mismatched chain, each of the substrate chain and the mismatched chain has the cytosine base C, and the bases are paired with each other under the action of the silver ions; and the signal conversion reagent is composed of urease, urea and phenol red. When let-7a exists in a sample, the let-7a and the mismatched chain are competitively combined with the substrate chain, the mismatched chain is replaced, the silver ions are dissociated, the urease activity is inhibited, and a urea-phenol red solution system always keeps yellow; and if no let-7a exists, the urea-phenol red solution system is red. The kitprovided by the invention has high stability, sensitivity and convenience.

Description

technical field [0001] The invention belongs to the field of molecular detection. Background technique [0002] Cancer, also known as malignant tumor, is a key factor threatening the health of the world and a major cause of death caused by diseases worldwide. microRNAs are small non-coding RNA gene products with a sequence length of 18nt to 24nt. MicroRNAs often regulate protein levels by interacting with the 3' untranslated regions of target messenger RNAs (mRNAs) and are often aberrantly expressed in cancer tissues. [0003] The microRNAs of the let-7 family can down-regulate the expression of oncogenic proteins such as RAS, HMGA2, and MYC, so they are generally considered as tumor suppressor genes. The expression in non-small cell lung cancer (NSCLC) tumor tissues is much lower than that in normal tissues. It is often used as the target detection substance of lung cancer biosensor. [0004] The length of mature microRNA is relatively short, coupled with its sequence ho...

Claims

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Application Information

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IPC IPC(8): C12Q1/58C12Q1/34G01N21/78G01N21/31
CPCC12Q1/58C12Q1/34G01N21/78G01N21/31C12Q2334/70Y02A50/30
Inventor 林锋邓锐杰张婷曾珍朱云柯刘成武梅建东
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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