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Mass spectrum method for quantifying nucleic acid based on DNA-polypeptide probe technology and application thereof

A technology of DNA probes and probes, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems affecting PCR amplification efficiency and response, deviation, and affecting signal collection, achieving high accuracy, High precision and high detection specificity

Active Publication Date: 2021-06-22
GUANGDONG HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

qRT-PCR requires fewer samples, but qRT-PCR technology usually requires pre-amplification of target RNA and further fluorophore labeling steps, and the background difference between samples will affect the amplification efficiency and response of PCR. Quenching affects signal collection and introduces bias
There is currently no quantitative method that can directly and specifically detect lncRNAs, therefore, it is imperative to develop new strategies that can provide quantitative results of lncRNAs in a sensitive and direct manner

Method used

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  • Mass spectrum method for quantifying nucleic acid based on DNA-polypeptide probe technology and application thereof
  • Mass spectrum method for quantifying nucleic acid based on DNA-polypeptide probe technology and application thereof
  • Mass spectrum method for quantifying nucleic acid based on DNA-polypeptide probe technology and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Construction of mass spectrometry method based on DNA-polypeptide probe technology quantitative nucleic acid

[0047] This embodiment takes HULC nucleic acid as an example, and provides a mass spectrometry method based on DNA-polypeptide probe technology to quantify HULC. Based on the nucleotide sequence of the nucleic acid to be tested, DNA probes are designed; characteristic polypeptides are screened out, and combined with DNA probe to construct a DNA-polypeptide probe; hybridize the DNA-polypeptide probe with the nucleic acid to be tested; perform proteolysis on the hybridized DNA-polypeptide probe, so that the characteristic polypeptide in the DNA-polypeptide probe is hydrolyzed into a reporter peptide ; Construct the LC-MS / MS detection method of the reporter peptide; use the LC-MS / MS detection method to detect the concentration of the reporter peptide; convert the concentration of the reporter peptide into the copy number of the nucleic acid to be teste...

Embodiment 2

[0220] Example 2 Application of the construction-based nucleic acid mass spectrometry quantitative method in the detection of HULC copy number in hepatocytes

[0221] In this embodiment, five different hepatocytes were used as samples to be tested, and the copy number of HULC was detected. The five kinds of liver cells used were normal liver cell L02, normal liver cell 7702, liver cancer cell SK-Hep1, liver cancer cell HepG 2 , Liver cancer cell 7721.

[0222] Refer to the construction method of the DNA-polypeptide probe in Example 1, the hybridization of the DNA-polypeptide probe and the nucleic acid to be tested, the enzymatic hydrolysis to obtain the characteristic polypeptide reporter peptide, and the LC-MS / MS of the characteristic polypeptide reporter peptide constructed based on Example 1 The detection method is to detect the copy number of HULC in the above five kinds of hepatocytes.

[0223] Wherein, the extraction process of total RNA in the above five kinds of live...

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Abstract

The invention belongs to the technical field of biological rapid detection, and particularly relates to a mass spectrum method for quantifying nucleic acid based on a DNA-polypeptide probe technology, wherein the method comprises the following steps: designing a DNA probe on the basis of the nucleotide sequence of to-be-detected nucleic acid; screening out a characteristic polypeptide, and constructing a DNA-polypeptide probe with the DNA probe; hybridizing the DNA-polypeptide probe with the to-be-detected nucleic acid; carrying out protease enzymolysis on the hybridized DNA-polypeptide probe, and hydrolyzing the characteristic polypeptide in the DNA-polypeptide probe into reporter peptide; constructing an LC-MS / MS detection method of the report peptide; detecting the concentration of the report peptide by using the constructed LC-MS / MS detection method; and converting the concentration of the report peptide into the copy number of the to-be-detected nucleic acid to realize quantitative detection of the to-be-detected nucleic acid. Based on the method, the quantification of nucleic acid is converted into the quantification of polypeptide, and the method has the advantages of high detection efficiency and strong specificity; in addition, the method is used for detecting the liver cancer marker HULC, so that risk assessment of the liver cancer is facilitated.

Description

technical field [0001] The invention belongs to the technical field of rapid biological detection, and in particular relates to a method and application of lncRNA mass spectrometry quantification based on DNA-polypeptide probe technology. Background technique [0002] Long non-coding RNA (Long non-coding RNA, lncRNA) is a non-coding RNA longer than 200 nucleotides. With the gradual deepening of research, the function of lncRNA has been gradually discovered by researchers, and it is closely related to the occurrence and development of tumors. HULC is the earliest lncRNA found in liver cancer. Studies have found that HULC is significantly elevated in patients with liver cancer, and can participate in the regulation of the molecular mechanism of liver cancer. It is expected to become a potential biomarker for the diagnosis of liver cancer. [0003] At present, the methods for quantitative detection of lncRNA mainly include fluorescent dye method, Northern blot analysis, microa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12Q1/6886
CPCC12Q1/682C12Q1/6886C12Q2600/178C12Q2521/537C12Q2525/203C12Q2565/627C12Q2563/131C12Q2537/16
Inventor 黄宪章张乔轩蔡志梁韩丽乔严君展敏欧阳芬王建兵柯培锋庄俊华
Owner GUANGDONG HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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