MiRNA-106b detection kit based on digital PCR platform

A detection kit and digital technology, applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., to achieve the effects of improving stability, reducing errors, and improving reverse transcription efficiency

Inactive Publication Date: 2021-04-23
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV +1
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] MicroRNA has been analyzed in more and more studies of Alzheimer's disease, but there is no report on the quantitative detection of MCI using a group of miRNAs (miRNA-106b) in plasma miRNA, so as to realize the detection of Alzheimer's disease. Prevention and Early Diagnosis of Mer's Disease

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • MiRNA-106b detection kit based on digital PCR platform
  • MiRNA-106b detection kit based on digital PCR platform
  • MiRNA-106b detection kit based on digital PCR platform

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment approach

[0035] Droplet digital PCR (ddPCR) is an absolute quantitative technology for nucleic acid molecules. Its principle is: after the standard PCR reaction system is generated by droplets, each droplet contains or does not contain one or more copies of the target molecule (DNA template ), to achieve "single-molecule template PCR amplification". After PCR cycles, the microdroplets containing nucleic acid molecular templates will give fluorescent signals, and the microdroplets without templates will have no fluorescent signals; finally, according to the principle of Poisson distribution and the positive Droplet ratio, using MicroDrop TMQuantDrop, the supporting data analysis software for the digital PCR instrument, can calculate the concentration or copy number of the target molecule to be detected; digital PCR can directly calculate the copy number of the target sequence, so accurate absolute quantitative detection can be performed without relying on control samples and standard cur...

Embodiment 1

[0037] Example 1: A method for preparing a reverse transcription buffer for detecting miRNA-106b based on a digital PCR platform

[0038] (1) Pre-denaturation of sample total RNA before reverse transcription, prepare reaction solution: 3 μl of nuclease-free water, 2 μl of total RNA template, 0.5 μl of 1 ug / μl reverse transcription primer, as shown in Table 1:

[0039] Table 1

[0040] total RNA 2μl reverse transcription primer 0.5μl nuclease free water 3μl total capacity 5.5μl

[0041] (2) Fully mix the reaction solution in step (1), centrifuge briefly at 70°C for 5 minutes; incubate on ice for 5 minutes;

[0042] (3) Prepare reverse transcription buffer: 0.65 μl of 10mM dNTPs, 2.5 μl of 5X Buffer, 0.5 μl of reverse transcriptase, 3.35 μl of nuclease-free water, as shown in Table 2:

[0043] Table 2

[0044] 5X Buffer 2.5μl dNTPs 0.65μl reverse transcriptase 0.5μl nuclease free water 3.35μl total cap...

Embodiment 2

[0046] Example 2: A method for preparing a PCR reaction solution based on a digital PCR platform for detecting miRNA-106b

[0047] (1) PCR reaction master mix: MicroDrop from Guangdong Yongnuo Medical Technology Co., Ltd. TM Digital PCR reaction master mix (including dUTP / UDG) for digital PCR instrument;

[0048] (2) Primers: the upstream primer and the downstream primer are shown in SEQ ID NO.2 and SEQ ID NO.3, wherein the concentration of each primer is prepared as 10 μM, and the final concentration is 200 nM;

[0049] (3) The PCR reaction solution is as shown in Table 1: 10 μl of PCR reaction master mix, 0.4 μl of 10 μM upstream primer, 0.4 μl of 10 μM downstream primer, 5.2 μl of nuclease-free water, and the total volume of one reaction is 16 μl, as shown in Table 3;

[0050] table 3

[0051]

[0052]

[0053] (4) Extraction of total RNA: Use QIAGEN's miRNeasy Serum / Plasma Kit to extract total RNA from the sample according to the kit instructions;

[0054] (5) PCR...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to the technical field of biology, in particular to a miRNA-106b detection kit based on a digital PCR (Polymerase Chain Reaction) platform, which mainly comprises a PCR reaction solution, detection primers, a reverse transcription buffer, a reverse transcription primer and a positive control; the reverse transcription primer is characterized in that a nucleotide sequence as shown in SEQ ID NO.1 is taken as a reverse transcription primer Pri-RT. The detection primers are characterized in that a nucleotide sequence shown as SEQ ID NO.2 is used as an upstream primer Pri-F1, and a nucleotide sequence shown as SEQ ID NO.3 is used as a downstream primer Pri-R1. The reverse transcription buffer and the PCR buffer applied to the kit increase the sample loading amount of RNA of a reverse transcription system and the sample loading amount of a PCR reaction system, so that the detection stability of the kit is improved, and errors occurring during sample loading are reduced. A digital PCR technology is adopted for detection, higher sensitivity, specificity and accuracy are achieved, the detection sensitivity and accuracy can be improved through the reaction solution or the kit provided by the invention, and an effective method is provided for clinical prognosis observation and detection of trace lesion samples.

Description

technical field [0001] The invention relates to a biotechnology field, in particular to a miRNA-106b detection kit based on a digital PCR platform. Background technique [0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system with cognitive and behavioral dysfunction, accounting for about 50-60% of dementia cases in the elderly. Among them, senile dementia is the most common form of AD, which has the characteristics of insidious onset and progressive aggravation, and cognitive and social dysfunction will gradually appear. In order to identify and intervene AD early, many scholars put forward the concept of mild cognitive impairment (MCI) on this basis, which is used to define those elderly people who have mild memory or cognitive impairment but do not meet the diagnostic criteria of AD. . MCI patients are a high-risk population for AD. Studies have found that the annual conversion rate is 3-10% in community settings and 10-15% in specialize...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/6883C12Q1/686C12Q2600/178C12Q2600/158
Inventor 王志罗景燕何继谦郑耿鸿周加鑫
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap