Testing method and kit for secondary circulation amplification of microRNA (Ribose Nucleic Acid)

Abstract

The invention provides a testing method and a kit for secondary circulation amplification of microRNA (Ribose Nucleic Acid). According to the detection method provided by the invention, a secondary circulation amplification reaction can be finished by only a single operation, the sensitivity is high, and nine RNA chains in a 15-microliter reaction system can be detected; the design is simple, the compounding and modifying of nanometer materials are not related; the noise is low, so that the sensitivity, specificity and generality are increased; compared with a conventional Northern blot assay method and a conventional microarray assay method, the required sample amount is little, and the specificity is excellent; compared with a real-time quantitative PRC (Polymerase Chain Reaction), the method is a thermostatic reaction, so that the test can be realized on a normal fluorescence instrument, the use range of the method is enlarged, and the sequence design is simple and convenient; and the miRNA in a level of a single breast cancer cell can be detected. The technique disclosed by the invention can be used for designing diagnostic kits and devices for tumor.

Description

technical field

[0001] The invention designs a single-molecule-level secondary cycle amplification technology to realize rapid and one-step detection of microRNAs (miRNAs) and a kit thereof. Background technique

[0002] MiRNAs are a large family of highly conserved endogenous non-coding RNAs with a length of about 18-22 nucleotides, which widely exist in eukaryotes. Because miRNAs can bind to target messenger RNA through base complementarity, resulting in its degradation and inhibition of protein synthesis, thereby regulating the level of post-transcriptional gene expression. miRNAs have a very wide range of gene regulation and expression functions, and play an important role in the growth, early development, cell differentiation, and apoptosis of organisms. The expression in normal tissues and tumor tissues is significantly different, and they are involved in the occurrence of tumors ,develop.

[0003] Northern blot and gene chip are the most widely used miRNAs detection...

Examples

Embodiment 1

[0055] Example 1 uses miR-21 as a test object to further illustrate the present invention.

[0056] The enzymes and buffers used in this example include: NEB buffer 2, Bst large fragment DNA polymerase, lambda exonuclease, Nb.BbvCI nicking endonuclease, etc. were all purchased from NEB Company; molecular beacon probes, BSA, 8-base primers, miRNAs, DEPC water, dNTP, RNase inhibitor, etc. were purchased from Dalian Biotech.

[0057] The instrument used for fluorescence detection is a fluorescence spectrophotometer (Hitachi F-4500, Japan). Fluorescence spectrum measurement conditions: both the excitation and emission narrow peak widths are 5nm, the voltage PMT is 700v, the excitation wavelength is 490nm, and the emission wavelength scanning range is 500-600nm; a 250μL quartz cuvette is used for measurement.

[0058] The specific detection method is:

[0059] Step 1: Detection standard curve at 37°C: take 5-8 1.5ml centrifuge tubes, add molecular beacon probes with a final conce...

Embodiment 2

[0062] Example 2 uses the extract of breast cancer cells as the test substance to further illustrate the present invention.

[0063] Step 1: detection of breast cancer cells. Use mirVana first TM The kit extracts miRNA as the sample to be detected.

[0064] Step 2: Mix the sample to be tested with a final concentration of 500nM molecular beacon probe, 2μM 8-base primer, 400μM dNTPs, 16U Bst polymerase, 15U Nb.BbvCI nicking endonuclease, 12.5Ulambda exonuclease, Add BSA and RRI to a 1.5ml EP centrifuge tube. Detection was performed using a fluorometer. The breast cancer cell detection standard curve of cell number and fluorescence intensity can be obtained. (See Figure 4 ). The instrument setup parameters remain unchanged.

[0065] Step 3: We verified the extraction results of 1000 breast cancer cells. Using the above steps, we finally obtained a fluorescence value of 135. By comparing with the detection standard curve of breast cancer cells, it was found that 135 just...

technical field

[0001] The invention designs a single-molecule-level secondary cycle amplification technology to realize rapid and one-step detection of microRNAs (miRNAs) and a kit thereof. Background technique

[0002] MiRNAs are a large family of highly conserved endogenous non-coding RNAs with a length of about 18-22 nucleotides, which widely exist in eukaryotes. Because miRNAs can bind to target messenger RNA through base complementarity, resulting in its degradation and inhibition of protein synthesis, thereby regulating the level of post-transcriptional gene expression. miRNAs have a very wide range of gene regulation and expression functions, and play an important role in the growth, early development, cell differentiation, and apoptosis of organisms. The expression in normal tissues and tumor tissues is significantly different, and they are involved in the occurrence of tumors ,develop.

[0003] Northern blot and gene chip are the most widely used miRNAs detection...

Examples

Embodiment 1

[0055] Example 1 uses miR-21 as a test object to further illustrate the present invention.

[0056] The enzymes and buffers used in this example include: NEB buffer 2, Bst large fragment DNA polymerase, lambda exonuclease, Nb.BbvCI nicking endonuclease, etc. were all purchased from NEB Company; molecular beacon probes, BSA, 8-base primers, miRNAs, DEPC water, dNTP, RNase inhibitor, etc. were purchased from Dalian Biotech.

[0057] The instrument used for fluorescence detection is a fluorescence spectrophotometer (Hitachi F-4500, Japan). Fluorescence spectrum measurement conditions: both the excitation and emission narrow peak widths are 5nm, the voltage PMT is 700v, the excitation wavelength is 490nm, and the emission wavelength scanning range is 500-600nm; a 250μL quartz cuvette is used for measurement.

[0058] The specific detection method is:

[0059] Step 1: Detection standard curve at 37°C: take 5-8 1.5ml centrifuge tubes, add molecular beacon probes with a final conce...

Embodiment 2

[0062] Example 2 uses the extract of breast cancer cells as the test substance to further illustrate the present invention.

[0063] Step 1: detection of breast cancer cells. Use mirVana first TM The kit extracts miRNA as the sample to be detected.

[0064] Step 2: Mix the sample to be tested with a final concentration of 500nM molecular beacon probe, 2μM 8-base primer, 400μM dNTPs, 16U Bst polymerase, 15U Nb.BbvCI nicking endonuclease, 12.5Ulambda exonuclease, Add BSA and RRI to a 1.5ml EP centrifuge tube. Detection was performed using a fluorometer. The breast cancer cell detection standard curve of cell number and fluorescence intensity can be obtained. (See Figure 4 ). The instrument setup parameters remain unchanged.

[0065] Step 3: We verified the extraction results of 1000 breast cancer cells. Using the above steps, we finally obtained a fluorescence value of 135. By comparing with the detection standard curve of breast cancer cells, it was found that 135 just...

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