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Method for quantitatively assaying serum miRNA by utilizing RNase ONE nuclease and chemiluminescence technology

A technology of chemiluminescence and ribonuclease, applied in chemiluminescence/bioluminescence, analysis through chemical reaction of materials, preparation of test samples, etc., can solve problems such as inability to meet clinical detection requirements and complicated operation, and achieve Meet clinical detection needs, high sensitivity, and prolong the effect of detection time

Inactive Publication Date: 2018-06-12
SHANTOU UNIV MEDICAL COLLEGE +3
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to overcome the defects that the existing fluorescence quantitative PCR technology is complicated to operate and cannot meet the needs of clinical detection, and to provide a method for quantitative detection of serum miRNA markers using RNase ONE ribonuclease and chemiluminescence technology

Method used

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  • Method for quantitatively assaying serum miRNA by utilizing RNase ONE nuclease and chemiluminescence technology
  • Method for quantitatively assaying serum miRNA by utilizing RNase ONE nuclease and chemiluminescence technology
  • Method for quantitatively assaying serum miRNA by utilizing RNase ONE nuclease and chemiluminescence technology

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Embodiment 1～8

[0053] Examples 1-8 provide a method for quantitative detection of serum miRNA markers using RNase ONE ribonuclease and chemiluminescence technology. Such as figure 1 As shown, the method is as follows:

[0054] (1) Pretreatment of serum samples

[0055] The Tween 20 (10%) solution was diluted with TE buffer (pH 8.0) to a final concentration of 1%, and then Proteinase K was added to a final concentration of 200 μg / ml to prepare serum treatment solution. After the serum sample is thawed, vortex 5 to 10 times. The prepared treatment solution and serum samples were respectively placed in a constant temperature mixer at 37° C. for 30 minutes with slight shaking and mixing. The serum sample and the treatment solution were mixed at a volume ratio of 1:1, and reacted at 60° C. at a speed of 300 rpm for 60 to 90 minutes. After processing, the serum solution should be used for the next reaction immediately, or stored in a -80°C refrigerator.

[0056] (2) Amide reaction ligation of...

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Abstract

The invention relates to a method for quantitatively assaying a serum miRNA (microRNA) marker by utilizing RNase ONE ribonuclease and the chemiluminescence technology. According to the method, a serumsample is first pretreated, a captured sequence is fixed on an ELISA (enzyme-linked immune-sorbent assay) plate, a DNA-RNA hybrid probe and target miRNA in the serum are then hybridized in sequence,RNase ONE ribonuclease is then utilized to identify and cut base mismatch caused by heteroduplex hybridization or point mutation and partial complementation caused by unpaired base insertion / deletion,and finally, a signal amplification sequence and the chemiluminescence technology are utilized to assay the content of target miRNA in the serum. The method provided by the invention has high sensitivity and high specificity, and can meet the requirement of clinical assay.

Description

technical field [0001] The invention belongs to the detection method and technical field of blood free nucleic acid markers, and more specifically relates to a method for quantitatively detecting serum miRNA using RNase ONE nuclease and chemiluminescence technology. Background technique [0002] miRNA (microRNA) is a class of endogenous, non-coding single-stranded RNA molecules with a length of 19 to 24 nucleotides. It mainly combines with the messenger RNA (messager RNA, mRNA) that guides protein synthesis through complementary base pairing, resulting in its translation being blocked or degraded, thereby participating in the regulation of individual development, cell apoptosis, proliferation and differentiation and other life. Since more than half of miRNAs are located in vulnerable sites associated with tumors in the genome, abnormally expressed miRNAs are closely related to the occurrence, staging, metastasis and recurrence of various tumors. [0003] Because miRNAs have...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N1/38
CPCG01N1/38G01N21/76
Inventor 凌凯姜红岩
Owner SHANTOU UNIV MEDICAL COLLEGE
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