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

[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...

[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...