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Method of extracting target nucleic acid and performing PCR augmentation

A target nucleic acid and nucleic acid technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as laboriousness, achieve good repeatability, good specificity, and stable experimental results

Active Publication Date: 2009-11-25
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All of the above methods must be extracted and purified before performing PCR detection. It will inevitably be more difficult when processing a large number of clinical samples.

Method used

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  • Method of extracting target nucleic acid and performing PCR augmentation
  • Method of extracting target nucleic acid and performing PCR augmentation
  • Method of extracting target nucleic acid and performing PCR augmentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: The fluorescent quantitative PCR detection embodiment of HBV DNA

[0034] Aspirate 2 microliters of lysate (0.1 mM surfactant peptide dissolved in 80 mM KCl, 0.1% SDS, 0.5% ethanol) with a filter tip, 3 microliters of the sample to be tested (serum or plasma known to be positive for HBV DNA), Add it into the PCR reaction tube, pipette the tip of the pipette to mix evenly, and after standing for 5 minutes, suck up 45ul of the prepared PCR reaction solution with a filter tip, and perform real-time fluorescence quantitative PCR amplification on the Stratagene Mx3000P or ABI7300 PCR instrument .

[0035] The concentrations and ratios of the components in the PCR reaction solution are as follows:

[0036] Element

[0037] The PCR reaction procedure is:

[0038]

[0039] 1) With the negative and positive reference material in the HBV national standard product as the sample to be tested, adopt the method of the present invention to detect on the S...

Embodiment 2

[0046] Embodiment 2: The rapid fluorescent quantitative PCR detection embodiment of HBV DNA and corresponding internal standard

[0047] As in the method of Example 1, draw 2 microliters of lysate (the surfactant peptide of 0.1mM is dissolved in 80mM KCl, 0.1% SDS, 0.5% ethanol) with a filter tip suction nozzle, 3 microliters of the sample to be tested (known HBV DNA Positive serum or plasma), add to the PCR reaction tube, pipette tip to mix evenly, let stand for about 5min. According to the number of reactions, 0.3ul internal standard (10 4 copies / ul) the ratio of the internal standard into the PCR reaction solution. In this embodiment, low-concentration HBV DNA samples (concentrations of 500 IU / ml and 200 IU / ml) and a negative sample were selected as samples to be tested. Such as Figure 4 , the group with higher fluorescence signal is the amplification curve of the internal standard, and the group with lower fluorescence signal is the amplification curve of the sample to...

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Abstract

A method of extracting target nucleic acid and performing PCR augmentation comprises the following steps: adding a cracking liquid, a sample containing target nucleic acid and a reaction liquid which performs PCR augmentation into a PCR reaction tube and evenly mixing; placing in the PCR apparatus for performing PCR augmentation. The invention only needs simple instrument to rapidly and high efficiently extract target nucleic acid from the sample and to synchronously perform real-time fluorescence PCR augmentation detection; the invention has wide detection linear range, accurate quantification for samples of different concentrations, good specificity and good repetitiveness; the invention is extremely simple in operation and suitable for the PCR apparatus of different types; it can be widely applied to the fields of detection of pathogenic microorganism, medicolegal expertise, tissue and blood typing, genetic mutation detection.

Description

technical field [0001] The invention relates to a method for extracting target nucleic acid and performing PCR amplification detection. Background technique [0002] Since the advent of PCR technology in 1985, this technology has been applied to various information fields of life sciences. Because of its high sensitivity, strong specificity, easy operation, and short time required, it has been widely used in pathogenic microorganisms. detection. Since clinical specimens contain proteins, lipids and other substances that can interfere with the PCR reaction, nucleic acid extraction must be performed before performing the PCR reaction. The first step of PCR detection technology is the preparation of template DNA, that is, the extraction of DNA in the sample, which directly affects the result of PCR reaction. Extraction and purification of DNA from samples has long been a time-consuming, cumbersome process that severely slows down detection. [0003] Currently, the extraction...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 戴立忠罗艳熊晓燕
Owner SANSURE BIOTECH INC
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