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MiRNA-122 detection kit based on digital PCR platform and application thereof

A detection kit, miRNA-122 technology, applied in the biological field to reduce errors or errors, facilitate long-term stable preservation, and achieve good results

Inactive Publication Date: 2016-12-07
成都仕康美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching, there is no convenient and fast kit for miRNA-122 detection based on digital PCR platform

Method used

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  • MiRNA-122 detection kit based on digital PCR platform and application thereof
  • MiRNA-122 detection kit based on digital PCR platform and application thereof
  • MiRNA-122 detection kit based on digital PCR platform and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the composition of kit

[0024] A miRNA-122 detection kit based on a digital PCR platform, which includes 9 μl of reverse transcription buffer, 1 μl of reverse transcriptase, 17 μl of PCR buffer, 20 μl of ultrapure water, 3 μl of positive control and 3 μl of negative control. The transcription buffer comprises the reverse transcription primer whose nucleotide sequence is shown in SEQ ID No.1, and the sequence is:

[0025] 5'-GTCGTATCCAGTGCCACACGTCCTCGAGGCTTAAGCACTGGATACGACCAAACACC-3'; the PCR buffer includes a detection primer with a nucleotide sequence as shown in SEQ ID No.2 and a detection primer with a nucleotide sequence as shown in SEQ ID No.3. The sequence of the primer is 5'-ATGGCTGGAGTGTGACAATGG-3', and the sequence of the primer under detection is 5'-GTATCCAGTGCCACACGTCCTC-3';

[0026] The negative control is a synthetic nematode-derived miRNA-39 of about 850 copies / ul, the nucleotide sequence of the miRNA-39 is shown in SEQ ID No.4, and the seq...

Embodiment 2

[0031] Embodiment 2: reaction system and reaction conditions

[0032] 1. The miRNA-122 reverse transcription system is shown in Table 1:

[0033] Table 1: miRNA-122 reverse transcription system

[0034] Element

volume

reverse transcription buffer

9ul

reverse transcriptase

1ul

RNA sample (5-50ng total)

5ul

total

15ul

[0035] 2. miRNA-122 reverse transcription reaction conditions are shown in Table 2:

[0036] Table 2: miRNA-122 reverse transcription reaction conditions

[0037] step

time

temperature

1

30min

16℃

2

30min

42℃

3

5min

85℃

4

Forever

4℃

[0038] 3. The miRNA-122 detection reaction system is shown in Table 3:

[0039] Table 3: miRNA-122 detection reaction system

[0040] Element

volume

PCR buffer

17ul

RT samples

3ul

total

20ul

[0041] 4. The reaction conditions for miRNA-122 detection...

Embodiment 3

[0045] Example 3: Detection of the expression level of miRNA-122 in the plasma of patients after liver transplantation

[0046] 1. Sample collection and processing

[0047] 5ml of blood from liver transplant recipients was collected using common EDTA blood collection tubes, in total two. One was from a recipient one week after transplantation and the other was from a recipient about 6 months after transplantation. Transfer the blood to a new 15ml centrifuge tube, centrifuge at 2000g for 10min at 4°C, and draw the upper layer of plasma into a new 15ml centrifuge tube. Then centrifuge the plasma at 4000g for 20min at 4°C, and draw the upper layer of plasma into a new 15ml centrifuge tube. Total RNA was extracted using QIAGEN's miRNeasy Serum / Plasma Kit according to the operating instructions.

[0048] 2. Experimental grouping and detection

[0049] Design experimental group A: recipients one week after transplantation ( figure 1 Shown as A), experimental group B: recipients 6...

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Abstract

The invention discloses an miRNA-122 detection kit based on a digital PCR (Polymerase Chain Reaction) platform and application of the miRNA-122 to preparation of a kit for evaluating liver transplantation injury. Ingredients of a reverse transcription buffer solution and a PCR buffer solution of the kit provided by the invention are optimized, so that the long-period stable storage is facilitated; meanwhile, the sample loading workload can be simplified to the minimum, so that mistakes or errors possibly occurring during the sample loading can be reduced; and the obtaining of the stable and reliable detection result is facilitated. Compared with conventional common-use miRNA reverse transcription stem-loop primers and detection primers, the kit provided by the invention has the advantages that the basic group modification and optimization is performed on the reverse transcription stem-loop primer and detection primer sequence design; when the kit is used for digital PCR platform detection, the result is better and more stable; the miRNA-122 is used as an independent molecular marker; and through the clinic verification test, the application of the miRNA-122 detection kit as a molecular marker to the preparation of the kit for evaluating liver transplantation injury is realized through the development of the miRNA-122 detection kit.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a miRNA-122 detection kit based on a digital PCR platform and an application thereof. Background technique [0002] Liver transplantation is the most effective means to save the lives of various end-stage liver failure or damaged patients. After transplantation, invasive diagnostic methods such as puncture needles are often used clinically to detect liver graft damage and rejection, which is not only expensive, but also lacks sufficient sensitivity because changes in detection markers often occur in the middle and late stages of physiological lesions. It is also associated with a high risk of complications, making it difficult to use as a means of long-term monitoring of transplant rejection. [0003] MicroRNA (miRNA) is a non-coding small RNA molecule of about 22 nucleotides widely present in eukaryotes, which is crucial to cell development, differentiation, proliferati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/686C12Q2600/158C12Q2600/166C12Q2600/178C12Q2527/125C12Q2525/207C12Q2545/113
Inventor 魏亮卢俊杨洪吉
Owner 成都仕康美生物科技有限公司
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