CRISPRCas12a system-based miRNA-21 detection method and kit

A miRNA-21 and detection method technology, applied in the field of biological detection, can solve problems such as time-consuming, complex data analysis, poor primer versatility, etc., and achieve the effects of improving detection sensitivity, simplifying detection process, and improving specificity

Pending Publication Date: 2022-05-27
重庆创芯生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, the above methods still face some shortcomings: for example, the design of stem-loop primers is relatively complicated, and the design of primers with different sequences will lead to large differences in detection performance, and the generality of the primers is poor, and the cost of large-scale detection is high; Simple, but the specificity and sensitivity of its detection are significantly lower than the stem-loop method
The above problems limit the further application of qRT-PCR-based miRNA detection methods in clinical translation
Sequencing technology has significantly improved the detection performance of miRNA by virtue of its high throughput and high accuracy, but this method is time-consuming, costly, and requires complex data analysis

Method used

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  • CRISPRCas12a system-based miRNA-21 detection method and kit
  • CRISPRCas12a system-based miRNA-21 detection method and kit
  • CRISPRCas12a system-based miRNA-21 detection method and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0025] A miRNA-21 detection method based on CRISPRCas12a system, including the following steps:

[0026] Step 1: Synthesize a pair of specific incision primers according to the sequence of the miRNA-21 target to be measured, and edit the corresponding guide RNA sequence according to the CRISPR gene.

[0027] Designing the corresponding forward and reverse incision primers for miRNA targets requires that the primer design contain at least three parts, namely the strand displacement fixation region, the incision enzyme recognition region, and the target recognition region from the 5' to 3' ends, respectively, such as Figure 1 as shown. The stable combination of the primer fixation region and the complementary chain is the basis for the efficient implementation of double chain replacement, and it is also the key to improving the detection sensitivity, if the TM value of the fixed region is too low, it will lead to the primer detaching from the complementary chain, and the amplificati...

Embodiment 2

[0057]One kit comprises: primers synthesized according to the method of Example 1, guide RNA, nonspecific fluorescent reporter molecules, DNA / RNA polymerase, incisionase, Cas12a nuclease, dNTP, negative control DEPC-H 2 O as well as reaction buffer. The reaction buffer is a mixed solution of potassium acetate (25-75 mM), magnesium acetate (5-20 mM), bovine serum albumin (50-150 μg / ml), Tris-acetic acid (10-30 mM). The Ph of the mixed solution is 7.5-8.5.

[0058] Among them, the non-specific fluorescent reporter molecules are modified with quenching groups and fluorophores at both ends. The types of quenched groups are Dabcyl, BHQ-1, QYS-7, BHQ-2, and the fluorophores are Pacific Blue, Oregon Green, Bodipy FL-X, FAM, TET, Bodipy R6G-X, JOE, HEX, Cy3, Cy3.5, Cy5.5, Rhodamine Red-X, TAMRA, Texas Red-X, Any one of the ROX.

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Abstract

The invention discloses a miRNA-21 detection method based on a CRISPRCas12a (clustered regularly interspaced short palindromic repeats associated protein 12a) system. The miRNA-21 detection method comprises the following steps: synthesizing a forward nick primer, a reverse nick primer and corresponding guide RNA (Ribonucleic Acid); carrying out isothermal amplification on the forward incision primer and the reverse incision primer; a product obtained after isothermal amplification and the guide RNA are detected through a CRISPRCas12a system, and a miRNA-21 detection result is obtained. The invention belongs to the technical field of biological detection, and can realize high-sensitivity and high-specificity detection of miRNA-21 in a short time.

Description

Technical field [0001] The present invention relates to a miRNA-21 detection method and kit based on the CRISPRCas12a system, which belongs to the field of biological detection technology. Background [0002] MicroRNA (miRNA) is an endogenous, non-coding RNA molecule that plays an important role in various biological processes such as cell differentiation, proliferation, death, organ development, metabolism and so on as an important regulator after gene transcription. Recent studies have shown that circulating miRNAs in the peripheral blood environment can be present in microcapsules (exosomes and exfoliated vesicles) and apoptotic bodies or conjugated to high concentrations of lipoproteins, with high stability, miRNA is one of the most potential biomarkers in the field of liquid biopsy compared with protein markers, circulating cells and circulating DNA. [0003] Although miRNA as a biomarker has great application prospects, the widespread popularity of miRNA detection in clinic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2521/327C12Q2563/107Y02A50/30
Inventor 左晨李俊杰兰华林张力心赵朝辉钟越杨燕斌谢国明
Owner 重庆创芯生物科技有限公司
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