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Visual detection method and detection probe for simultaneously detecting two circulating miRNAs

A detection method and a detection probe technology, which are applied in the fields of molecular biology and chemiluminescence, can solve the problems of reducing detection efficiency and single detection target, and achieve the effects of improving detection efficiency, simplifying detection operation, and simplifying complexity

Inactive Publication Date: 2021-05-14
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection probes invented by most of the current research work can only detect a single target miRNA, and there is a problem of single target detection
In addition, the traditional chemiluminescence detection method usually requires expensive instruments to collect signals, and it also has high requirements on the operating skills of technicians, and the instrument can only detect many samples one by one, which reduces the detection efficiency

Method used

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  • Visual detection method and detection probe for simultaneously detecting two circulating miRNAs
  • Visual detection method and detection probe for simultaneously detecting two circulating miRNAs
  • Visual detection method and detection probe for simultaneously detecting two circulating miRNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] like figure 1 As shown, according to Embodiment 1 of the present invention, a method for simultaneously visually detecting multiple samples containing two miRNAs is proposed, including the following steps:

[0045] Step 1, preparing gold nanoparticles;

[0046] Step 2. Prepare gold nanoparticles-substrate DNA chain HP complex HP@AuNPs, and combine with enzyme chains A and B to form AuNPs-DNA walker detection probe;

[0047] Step 3, performing a signal amplification reaction with the target miRNA and the detection probe;

[0048] Step 4. Visual detection of target miRNA based on chemiluminescence.

[0049] Specific steps are as follows:

[0050] Step (1) preparing gold nanoparticles specifically includes the following steps:

[0051] Before the start of the experiment, all the glass instruments needed were freshly prepared with aqua regia (V HCl :V HNO3 = 3: 1) Soak overnight, then rinse repeatedly with a large amount of deionized water, put in an oven to dry for l...

Embodiment 2

[0068] Example 2: Specific detection of target miRNA

[0069] The sample containing 100 nM target miRNA or interfering miRNA and the blank sample were subjected to the same experimental operation according to steps (3)-(4) in Example 1. The mobile phone takes pictures to record the signal difference.

[0070] like Figure 5 As shown, the luminescence intensity of the interfering miRNA is comparable to that of the blank sample, and only when the target miR-499 exists will a significantly enhanced blue light signal be produced. This result indicates that the invented detection probe has good specificity for the detection of the target miR-499.

[0071] like Image 6 As shown, the luminescence intensity of the interfering miRNA is comparable to that of the blank sample, and only when the target miR-133a exists will a significantly enhanced green light signal be produced. This result indicates that the invented detection probe has good specificity for the detection of the targ...

Embodiment 3

[0074] Example 3: Simultaneous visual detection of multiple samples containing miR-133a and miR-499.

[0075] Mix different concentrations of miR-499 and miR-133a in 10% healthy human serum, where sample numbers 1, 2, 3, 4 and 5 represent the combined concentration of miR-499 and miR-133a at 100 fM (100 fM) , 100pM (100fM), 100nM (100fM), 100nM (100pM), 100nM (100nM).

[0076] according to figure 1 Described method, all samples are carried out identical experiment according to the step (3) among the example 1. The difference is that each of the above-mentioned samples to be tested is divided into two parts, which are respectively added to the two detection probes for amplification reaction.

[0077] After all the reactions were completed, the mixture was centrifuged at 8000rpm for 10min to remove free DNA strands and unbound heme in the supernatant, and then the gold nanoprobe pellet for detecting miR-499 was redispersed in a solution containing 1mM luminol In 10 mM TE, the...

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Abstract

The invention provides a visual detection method and a detection probe for simultaneously detecting two circulating miRNAs. The method comprises the following steps: step 1, preparing gold nanoparticles; step 2, preparing a gold nanoparticle-substrate DNA chain HP compound HP (at) AuNPs, and combining the compound HP (at) AuNPs with enzyme chains A and B to form an AuNPs-DNA walker detection probe; step 3, performing signal amplification reaction on the target miRNA and a detection probe; and step 4, carrying out visual detection on the target miRNA based on chemiluminescence. The detection probe provided by the invention can realize simultaneous quantitative detection of a plurality of samples containing two different circulating miRNAs, and detection results are not interfered with each other. According to the detection method provided by the invention, a chemiluminescence signal is recorded in a mobile phone photographing manner, expensive instruments are not needed, the cost is reduced, the detection operation is simplified, and immediate detection of miRNA can be realized.

Description

technical field [0001] The invention belongs to the fields of molecular biology and chemiluminescence, and specifically relates to a probe capable of sensitively detecting two circulating miRNAs simultaneously and a visual detection method. Background technique [0002] microRNA (miRNA) is a class of endogenous non-coding small molecules with a length of about 18-22 nucleotides, which play an important role in regulating gene expression in many cells. Studies have shown that miRNAs in body fluids such as serum and plasma have good stability against degradation against RNases, repeated freezing and thawing, and extreme pH. [0003] At present, the methods commonly used to detect miRNA mainly include qRT-PCR, RNA blot, colorimetric method, fluorescence method, chemiluminescence method and SERS method, etc. These methods have their own advantages, among which chemiluminescence assay has attracted much attention due to its advantages of no excitation light, high detection sensi...

Claims

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Application Information

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IPC IPC(8): C12Q1/682C12Q1/6806C12N15/11
CPCC12Q1/682C12Q1/6806C12Q2525/207C12Q2563/137C12Q2563/155C12Q2521/345C12Q2525/301
Inventor 李涛徐杰石琳
Owner UNIV OF SCI & TECH OF CHINA
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