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A kit and method for efficient and rapid detection and quantification of serum or plasma nucleic acid

A technology of a kit and a nucleic acid releasing agent, which is applied to kits and fields for efficient and rapid detection and quantification of serum or plasma nucleic acids to achieve accurate results, accurate detection results, and fast speed

Active Publication Date: 2022-08-02
SHENZHEN SHINEWAY HI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the nucleic acid detection kits on the market need to go through a complicated nucleic acid extraction and purification process, and then perform PCR detection.

Method used

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  • A kit and method for efficient and rapid detection and quantification of serum or plasma nucleic acid
  • A kit and method for efficient and rapid detection and quantification of serum or plasma nucleic acid
  • A kit and method for efficient and rapid detection and quantification of serum or plasma nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Evaluation of adding N-acetyl cystine (NAC) to nucleic acid releasing agent

[0061] (1) Preparation of nucleic acid release agent A (without NAC): composed of 600 mM KOH, 0.06% (w / v) LLS, 2% (v / v) Tween 20, and the solvent is deionized nuclease-free water.

[0062] (2) Preparation of nucleic acid release agent B (containing 0.5% NAC): 600 mM KOH, 0.06%-0.1% (w / v) LLS, 0.05%-2% (v / v) Tween 20, 0.5% of NAC, and the solvent is nuclease-free deionized water.

[0063] (3) Preparation of reaction solution: the reaction solution is formulated as follows: 10 mM KCl, 5 mM MgCl 2 , 2mM EDTA-2Na, 25mM Tris-acetic acid, 10mM (NH 4 ) 2 SO 4 , BSA with a mass concentration of 0.02%, glycerol with a volume concentration of 8%, 1M betaine, trehalose with a mass concentration of 12%, 300nM upstream and downstream primers, 300nM probes, 1.5mM dNTPs, mixed The enzyme is composed of Proclin300 with a mass concentration of 0.03%, pH 8.0, and the solvent is deionized water w...

Embodiment 2

[0067] Example 2 Concentration evaluation of LLS (lithium dodecyl sulfate) in nucleic acid release agent

[0068] (1) Preparation of nucleic acid release agent A (0.1% LLS): composed of 600 mM KOH, 0.1% (w / v) LLS (lithium dodecyl sulfate, 2% (v / v) Tween 20), solvent It is nuclease-free deionized water.

[0069] (2) Preparation of nucleic acid release agent B (0.3% LLS): composed of 600 mM KOH, 0.3% (w / v) LLS (lithium dodecyl sulfate), 2% (v / v) Tween 20, The solvent was nuclease-free deionized water.

[0070] (3) Preparation of reaction solution: as in Example 1.

[0071] (4) Method: Two PCR reaction tubes, one of which was added with 5 μL of nucleic acid release agent A, and the other with 5 μL of nucleic acid release agent B, and then 5 μL of HBV DNA positive serum was added to the two PCR reaction tubes, respectively. Repeatedly pipetting for 5-7 times, mix well, and let stand at room temperature for 2 minutes; then add 40 μL of reaction solution to the two PCR tubes resp...

Embodiment 3

[0075] Example 3 Evaluation of the concentration of KOH in the nucleic acid releasing agent (template is DNA)

[0076] (1) Preparation of 7 kinds of nucleic acid release agents with different KOH concentrations (KOH concentrations are 300mM\350mM\400mM\450mM\500mM\550mM\600mM): 300mM~600mM KOH, 0.06% (w / v) LLS, 2% (v / v) Tween 20 in nuclease-free deionized water.

[0077] (2) Preparation of reaction solution: as in Example 1.

[0078] (3) Method: Add 5 μL of 7 kinds of nucleic acid release agents with different KOH concentrations into 7 PCR reaction tubes respectively, and then add 5 μL of HBV DNA positive serum to the 7 PCR reaction tubes respectively, mix well, and let stand at room temperature for 2 minutes; then add 40 μL of the reaction solution to the 7 PCR tubes respectively; finally mix and centrifuge, place the 7 PCR reaction tubes on the fluorescence quantitative PCR amplifier, select the FAM fluorescence channel, and set the amplification program to carry out the re...

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Abstract

The present invention relates to a kit for efficient and rapid detection and quantification of serum or plasma nucleic acid and a detection method thereof. The kit includes a nucleic acid release agent, a reaction solution, a reference substance, a standard product, and an internal standard. The nucleic acid release agent is composed of KOH, lithium dodecyl sulfate or sodium dodecyl sulfate, Tween 20, and a solvent. It is nuclease-free deionized water. The present invention provides a method for efficient and rapid detection and quantification of hepatitis virus nucleic acid in serum or plasma, wherein the nucleic acid release agent can efficiently and rapidly release various forms of nucleic acid in serum (or plasma), and then directly add the reaction solution to carry out Detection, the formula is simple, no pollution to nucleic acid amplification, accurate detection results, can detect both DNA and RNA, more universal for nucleic acids of different complexities, easily solves most complex template amplification difficulties The problem.

Description

technical field [0001] The invention relates to biomedical clinical molecular diagnosis and animal epidemic prevention molecular diagnosis, and provides a kit and method thereof, which can efficiently and rapidly detect and quantify specific pathogenic microorganisms and other specific free nucleic acid molecules in human and animal serum (or plasma). technical background [0002] The screening and diagnosis of many pathogenic microorganisms is performed by detecting pathogenic microorganisms in serum or plasma. For example, the DNA viruses of human blood screening are mainly HAV, HBV, HCV, HIV, Treponema pallidum, Epstein-Barr virus, CMV, and neonatal intestinal tract. DNA virus, etc. In terms of animal epidemic prevention, such as ASFV, PDCoV, highly pathogenic porcine PRRS virus, porcine pseudorabies virus, etc., serum or plasma can be detected and quantified. Traditional immunological and microbiological methods to detect various pathogenic microorganisms have the disad...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/706C12Q1/707C12Q1/6851C12Q2531/113C12Q2527/125C12Q2563/107
Inventor 温维佳高一博杨成勇
Owner SHENZHEN SHINEWAY HI TECH CO LTD
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