Method for quickly and quantitatively detecting target DNA

A quantitative detection and target technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of many operation steps, influence of target DNA, long time consumption, etc.

Active Publication Date: 2020-11-24
杭州百殷生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Traditional detection methods have many operating steps, which easily lead to a longer detection time, and it is difficult to quickly and quantitatively determine the DNA of bacteria or viruses

Method used

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  • Method for quickly and quantitatively detecting target DNA
  • Method for quickly and quantitatively detecting target DNA
  • Method for quickly and quantitatively detecting target DNA

Examples

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preparation example Construction

[0071] Cell fusion and preparation of primary antibody: Aseptically prepare mouse splenocyte suspension, mix with mouse myeloma cell SP2 / 0 at a ratio of 10:1, fuse with PEG, screen with HAT medium for 7 days, then replace with HT Culture medium, when there are obvious cell clusters in the wells, screen with the method consistent with the detection of serum titer, and the screened positive wells are subjected to 3 times of limited dilution to prepare monoclonal cell lines and expand them After culture, mouse ascites was prepared and purified with ProteinA to obtain the primary antibody.

[0072] Validation of the primary antibody:

[0073]Verification method 1: Use 0.1 μg / ml of different nucleic acids (DNA-RNA hybrids, double-stranded DNA, single-stranded DNA, single-stranded RNA) to duplicate 2 wells, coat at 4°C overnight, and block with gelatin at 37°C for 2 hours , react with different antibody concentrations (1μg / ml, 100ng / ml, 10ng / ml, 0) for 1 hour at room temperature (2...

Embodiment 1

[0103] Embodiment 1: A method for rapid quantitative detection of target DNA, comprising the steps of:

[0104] Step 1, denaturation: react the sodium hydroxide solution with the cervical exfoliated cell sample at 65°C for 30 minutes, and the concentration of the sodium hydroxide solution is 1.75mol / L, and the volume ratio of the sodium hydroxide solution to the sample to be tested is 1:2, crush the cells of the sample to be tested, denature the protein, degrade the RNA, and decompose the DNA into single-stranded DNA to obtain a denatured sample; step 2, hybridization: the denatured sample obtained in step 1 Mix with the HPV16 RNA probe (full-length single-stranded nucleic acid probe for target DNA, 8000bp in length) stored in the nucleic acid preservation solution with a pH value of 3.5-4.0, the volume of the HPV16 RNA probe and the denatured sample The ratio is 1:3, hybridize for 45 minutes under the condition of pH value 7.0-7.4 and temperature 65°C, so that the HPV16 RNA p...

Embodiment 2

[0112] Embodiment 2: A method for rapid quantitative detection of target DNA, the difference from Example 1 is that the HPV16 RNA probe consists of 4 sections of full-length discontinuous HPV16 RNA probes, and the full-length discontinuous HPV16 RNA probes The length is 2000bp.

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Abstract

The invention discloses a method for quickly and quantitatively detecting target DNA. The method comprises the following steps of 1, performing denaturation: breaking cells in a sampled sample to be detected, degrading RNA, and denaturing double-stranded DNA into single-stranded DNA to obtain a denatured sample; 2, performing hybridization: mixing the denatured sample with the pH value of higher than 7 with a single-stranded RNA probe stored in a nucleic acid preservation solution with the pH value of 3.5-4.0, and performing hybridizing for 45 minutes at 65 DEG C according to a base complementary pairing principle to form a DNA-RNA hybrid; 3, performing capture: fixing a first antibody on a carrier, and capturing the DNA-RNA hybrid for 60 minutes at 42 DEG C by using the first antibody; and 4, performing detection: combining the DNA-RNA hybrid with a second antibody labeled by alkaline phosphatase and stored in a protein preservation solution at 42 DEG C, performing a reaction for 30 minutes, performing washing, performing standing for 10 minutes at the room temperature under a dark condition, detecting the relative light quantum number, and rapidly and quantitatively detecting thetarget DNA within 3 hours. The method has the advantages of being rapid and quantitative in detection and more accurate in detection result.

Description

technical field [0001] The invention relates to the field of quantitative detection of target DNA, more specifically, it relates to a method for rapid quantitative detection of target DNA. Background technique [0002] The detection of target DNA is of great significance for judging the content of bacteria or viruses carried in a sample. [0003] The traditional detection method has many operation steps, which easily leads to a long detection time, and it is difficult to quickly and quantitatively determine the DNA content of bacteria or viruses carried in the sample, and the detection process involves the amplification of target DNA, which is easy As a result, the amount of target DNA is affected, which ultimately leads to inaccurate detection results. [0004] Therefore, a method for rapid and accurate quantitative detection of target DNA has far-reaching significance. Contents of the invention [0005] In view of the deficiencies in the prior art, the first object of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/682
CPCC12Q1/682
Inventor 胡杰锋姚尚华李德强许安庆
Owner 杭州百殷生物科技有限公司
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