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A method for detecting serum miRNAs in cancer patients based on short nucleotide chain ligation

A short nucleotide and chain connection technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of complex operation, hindering clinical application, high cost of detection specificity and detection throughput, and achieve operational Easy to use, accurate and reliable detection results, high sensitivity and specificity

Active Publication Date: 2020-10-30
深圳市展行生物有限公司
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Problems solved by technology

These two methods have defects such as high cost and complicated operation in detection specificity and detection throughput, which hinder their clinical application.

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  • A method for detecting serum miRNAs in cancer patients based on short nucleotide chain ligation
  • A method for detecting serum miRNAs in cancer patients based on short nucleotide chain ligation
  • A method for detecting serum miRNAs in cancer patients based on short nucleotide chain ligation

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specific Embodiment 1

[0034]A method for detecting serum miRNA in cancer patients based on short nucleotide chain ligation, the establishment and optimization of high-throughput SOL-based technology for detecting serum miRNA: pretreatment of serum samples, and capture sequence, nucleic acid probe 1, nucleic acid Probe 2, signal amplification sequence hybridization, and then use SA-HRP to catalyze the chemiluminescent substrate solution to generate strong fluorescence, and determine the expression level of target miRNA in serum by detecting the fluorescence intensity; systematically optimize serum processing time, nucleic acid hybridization temperature, signal Amplify the number of Biotin on the sequence and other conditions, and establish a new method for miRNA detection with strong specificity, accurate and reliable results, and easy operation. miRNA; at the same time, using this miRNA detection method to detect miR-16 and miR-21 in the serum samples of 20 cases of healthy controls and 34 cases of ...

specific Embodiment 2

[0036] A method based on short nucleotide chain ligation to detect serum miRNA in cancer patients, using SOL technology to detect specific miRNA markers in serum for early diagnosis of lung cancer: using a new method of SOL detection combined with well-explored serum pretreatment methods, Two miRNAs, miR-16 and miR-21, were detected in the serum samples of 20 healthy controls and 34 NSCLC patients of different stages, ages and genders; miR-16 with relatively stable expression was used as an internal reference substance, and miR-21, which was specifically highly expressed in the serum of NSCLC patients, was used as a miRNA marker. Fluorescent quantitative PCR was used to detect miR-16 and miR-21 in the collected serum samples, and the Ct value detected by miR-21 was normalized with the Ct value of the internal reference miR-16. The multiples are expressed by 2-ΔCt, see Table 1-2.

[0037] Table 1 Real-time quantitative PCR detection of Ct values ​​of miRNAs in the serum of hea...

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Abstract

The invention discloses a method used for detecting serum miRNA of patients with cancer based on short nucleotide chain connection. According to the method, specific recognition and cutting of unpaired single strand nucleic acids and double-stranded nucleic acids with mismatched bases are carried out, mismatching and partial complementation of hybridization of a nucleic acid probe with target miRNA are detected, fluorescence is generated via enzymatic reaction of horse radish peroxidase combined on the nucleic acid probe with a chemiluminescent substrate, rapid amplification of detection signals is realized, and the target miRNA content is determined based on fluorescence intensity. The detection results are accurate; operation is simple and convenient; cost is low; detection throughput is high; and nucleic acid extraction or amplification is not needed.

Description

technical field [0001] The invention relates to the technical field of cancer medical diagnosis, in particular to a method for detecting serum miRNA of cancer patients based on short nucleotide chain connection. Background technique [0002] MicroRNA (miRNA) is a class of evolutionarily conserved endogenous non-protein-coding RNA molecules, about 20 to 24 nucleotides in length, capable of recognizing specific target messenger RNA (messagerRNA, mRNA) and negatively expressing it at the post-transcriptional level. Regulate genes. miRNAs negatively regulate target genes by cleaving or inhibiting specific mRNAs, and are widely involved in the regulation of life activities such as individual development, cell apoptosis, proliferation and differentiation. Disruption of miRNAs can affect tumorigenesis if miRNAs target tumor suppressor genes or oncogenes. Since more than half of miRNA genes are located in tumor-associated genomic regions or vulnerable sites, abnormally expressed m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/682C12Q1/6834C12Q1/6886
CPCC12Q1/682C12Q1/6834C12Q2525/207C12Q2563/131
Inventor 李富荣凌凯李聪蒋太峰卫志坚戴蘡璎
Owner 深圳市展行生物有限公司
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