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Kit and detection method based on ultrasensitive detection device of peripheral blood free nucleotide miRNAs

A sensitive detection and nucleotide technology, applied in the field of biological detection, can solve the problems of ordinary PCR, high technical difficulty, difficult to popularize application, short miRNA sequence, etc., and achieves the effects of strong specificity, direct curative effect and prognosis, and high sensitivity.

Active Publication Date: 2019-06-07
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the main detection methods of miRNA in peripheral blood include cloning sequencing, Real-Time PCR, Northern blot hybridization, chips, etc.; trace amounts of miRNA in peripheral blood usually need to be detected by amplification, but due to the short miRNA sequence, ordinary PCR is difficult to carry out, although At present, there are real-time PCR based on stem-loop and polyA tailing, but these special amplification techniques are difficult and difficult to be widely used

Method used

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  • Kit and detection method based on ultrasensitive detection device of peripheral blood free nucleotide miRNAs
  • Kit and detection method based on ultrasensitive detection device of peripheral blood free nucleotide miRNAs
  • Kit and detection method based on ultrasensitive detection device of peripheral blood free nucleotide miRNAs

Examples

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Effect test

Embodiment 1

[0035] Example 1, a peripheral blood free nucleotide miRNAs ultrasensitive detection device

[0036] A peripheral blood free nucleotide miRNAs ultrasensitive detection device, the structure is as follows image 3 As shown in middle A, it includes a microarray detection pool 1 and a clutch-type external magnetic plate 2 that matches the bottom structure of the detection pool; wherein, the microarray is arranged in N*N, such as 6×6, and N is greater than or equal to 2; the detection pool consists of The diameter from the top to the bottom gradually decreases, and the bottom of the preferred detection cell is "V"-shaped, and the "V"-shaped microarray detection cell is more conducive to the enrichment of fluorescence and improves the detection sensitivity of the probe.

[0037] During the detection, after the pre-mixed reaction solution and the biological sample to be tested are reacted on the microarray substrate, the adsorption of the nanomagnetic microspheres by an external mag...

Embodiment 2

[0038] Embodiment 2, the method for detecting peripheral blood free nucleotide miRNAs

[0039] The method for detecting free nucleotide miRNA-150 in the peripheral blood of patients with non-small cell lung cancer (NSCLC) using the detection device in Example 1 comprises the following steps:

[0040] (1) Drop premix solution A and premix solution B into the detection pool of the detection device. Premix solution A is a double-loop fluorescent hairpin probe that specifically binds to miRNA-150. The probe sequence is as follows:

[0041] D1: FAM-cgcgattctcccaacccttgtaccagtgatcgcgtgagcacatgaaatacactggagaatcg-BHQ1 (SEQ ID NO. 1);

[0042] D2: FAM-atgcgctctcccaacccttgtaccagtggcgcatcgattctccagtgtacaatcccacagtg-NH2 (SEQ ID NO. 2);

[0043] D3: cactgtgggattgtatttcatgtgctca-BHQ1 (SEQ ID NO. 3);

[0044] The mixed reaction solution B is TNE buffer, which is composed of Tris, EDTA and NaCl, and the pH of the buffer is 8.0.

[0045] The above probe sequences are complementary paired to...

Embodiment 3

[0052] Example 3, Sensitivity Verification of Detecting Peripheral Blood Free Nucleotide miRNAs

[0053] Using miRNA-150 after gradient dilution as a template, verify the sensitivity of the kit and detection method based on the peripheral blood free nucleotide miRNAs ultrasensitive detection device of the present invention, the specific steps are as follows:

[0054] (1) Oligo miRNA-150 (synthesized by TaKaRa Company) was serially diluted to 1μM, 1nM, 10pM and 1pM as a sensitivity verification experiment sample;

[0055] (2) Use the device in Example 1 to detect, drop the premixed solution A and the premixed solution B into the detection pool of the detection device, the premixed solution A is the double-circle link fluorescent phosphor that specifically binds to miRNA-150 Clip probe, the probe sequence is as follows:

[0056] D1: FAM-cgcgattctcccaacccttgtaccagtgatcgcgtgagcacatgaaatacactggagaatcg-BHQ1 (SEQ ID NO. 1);

[0057] D2: FAM-atgcgctctcccaacccttgtaccagtggcgcatcgattct...

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Abstract

The invention relates to a kit of a peripheral blood free-nucleotide miRNAs-based ultrasensitive detection device and a detection method of the kit. The kit comprises 1) a microarray detection pool; 2) an external magnetic field; 3) premixed reaction solutions A and 4) premixed reaction solutions B. During detection of multiple targets to be detected, the premixed reaction solutions A for respectively and correspondingly detecting the targets to be detected are configured in parallel and are respectively marked as a premixed reaction solution A1 and a premixed reaction solution A2. The kit iscombined with biomolecular diagnosis, nano-magnetic microsphere enrichment and biochip technologies to develop a novel technique capable of directly and accurately detecting peripheral blood miRNAs related to occurrence, development and drug resistance of a malignant tumor without amplification at high sensitivity and high flux. The kit has an important value in the aspects of noninvasive detection of medical tumors, personalized medicine application, survival after chemotherapy and the like.

Description

technical field [0001] The invention belongs to the field of biological detection, relates to a test kit based on a peripheral blood free nucleotide miRNAs ultrasensitive detection device, and also relates to a detection method using the test kit. Background technique [0002] MicroRNA (miRNA) is a kind of endogenous short single-stranded RNA with a length of about 21-25bp, which regulates cell growth, apoptosis and signal transduction by regulating genes. miRNA polymorphism is closely related to drug metabolism and drug resistance, abnormal expression of miRNA plays an important role in predicting tumor occurrence, development and drug sensitivity; currently there are stable miRNA in peripheral blood, which can be used as a highly sensitive and specific Biomarkers. [0003] At present, the main detection methods of miRNA in peripheral blood include cloning sequencing, Real-Time PCR, Northern blot hybridization, chips, etc.; trace amounts of miRNA in peripheral blood usuall...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6837
CPCC12Q1/6837C12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/178C12Q2563/107
Inventor 张阳府伟灵田晖艳陈庆海
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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