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Kit using nanometer magnetic beads for purifying nucleic acid

A technology of nano-magnetic beads and kits, applied in inorganic chemistry, organic chemistry, silicon compounds, etc., can solve problems such as incomplete purification and gaps

Inactive Publication Date: 2010-03-31
上海裕隆生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these two methods are mainly used in the extraction of animal and plant tissues, not specifically for the detection and analysis of human body fluids for clinical testing.
And its main steps are: virus particle precipitation → adsorption of virus particles → cleavage and release of nucleic acid, which is not completely purified, and there is a gap with the clinical requirements for targeted processing of human body fluid samples

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: kit composition and preparation

[0035] 1. Lysate

[0036] Our company prepares: 8mol / L GuHCl, 50mmol / L Tris-HCl, Triton X-100 with a volume fraction of 2%, 25mmol / L EDTA, 0.3mol / L NaCl, and the pH value of the lysate is 6.4.

[0037] 2. Magnetic bead buffer

[0038] The inner core is Fe prepared by liquid deposition method 3 o 4 , the outer shell is carboxy-modified SiO 2 Aqueous solutions of monodisperse nano-phase superparamagnetic particles.

[0039] 1) Nano Fe 3 o 4 preparation of

[0040] Each 50mL of 0.2mol / L ferrous salt (FeSO 7H 2 O) and 0.3mol / L ferric salt (FeCl 3 ·6H 2 O) The solution is mixed in a 500mL three-necked flask, vigorously stirred (200-400r·min -1) at the same time turn on the ultrasonic generator, keep the water bath at constant temperature (40°C), add 0.5mol / L ammonia water as a precipitating agent into the dropping funnel, and add it dropwise to the reaction system under a nitrogen atmosphere to make the pH value of t...

Embodiment 2

[0056] Example 2: Using the kit to extract serum nucleic acid from patients with hepatitis B

[0057] 1. Allow the lysate to equilibrate at room temperature for 30 minutes or preheat it in a water bath at 55°C for 5 minutes before use.

[0058] 2. Add 900 μl of lysate, 45 μl of magnetic bead buffer, and 900 μl of serum (replenish to 900 μl with balance solution if insufficient) in sequence in a 2ml centrifuge tube, shake and mix, and mix every 5 minutes for a total of 15 minutes.

[0059] 3. Rely on magnetism to absorb and discard liquid.

[0060] 4. Add 900 μl of washing buffer 1, shake and mix for 2 minutes. Rely on magnetism to absorb and discard liquid.

[0061] 5. Add 900 μl of washing buffer 2, shake and mix for 2 minutes. Rely on magnetism to absorb and discard liquid.

[0062] 6. Add 900 μl of washing buffer 3, shake and mix for 2 minutes. Rely on magnetism to absorb and discard liquid.

[0063] 7. Uncap the 2ml centrifuge tube and place it in a metal bath at 55°...

Embodiment 3

[0066] Example 3: Using the kit to extract serum nucleic acid from patients with hepatitis C

[0067] 1. Allow the lysate to equilibrate at room temperature for 30 minutes or preheat it in a water bath at 55°C for 5 minutes before use.

[0068] 2. Add 900 μl of lysate, 45 μl of magnetic bead buffer, and 900 μl of serum (replenish to 900 μl with balance solution if insufficient) in sequence in a 2ml centrifuge tube, shake and mix, and mix every 5 minutes for a total of 15 minutes.

[0069] 3. Rely on magnetism to absorb and discard liquid.

[0070] 4. Add 900 μl of washing buffer 1, shake and mix for 2 minutes. Rely on magnetism to absorb and discard liquid.

[0071] 5. Add 900 μl of washing buffer 2, shake and mix for 2 minutes. Rely on magnetism to absorb and discard liquid.

[0072] 6. Add 900 μl of washing buffer 3, shake and mix for 2 minutes. Rely on magnetism to absorb and discard liquid.

[0073] 7. Uncap the 2ml centrifuge tube and place it in a metal bath at 55°...

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Abstract

The invention provides a kit using nanometer magnetic beads for purifying nucleic acid, particularly can purify nucleic acid of virus and Gram negative bacteria from human plasma or serum or urine andcan quickly and agilely purify nucleic acid on a large scale. The kit is composed of lysis solution, magnetic bead buffer solution 1, washing buffer solution 2, washing buffer solution 3, elution buffer solution and equilibrium liquid. The invention has high nucleic acid extracting speed and high sensitivity, has the potential of automatic upgrading compared with the traditional nucleic acid purifying method and makes large scale integration and homogeneity for extracting nucleic acid feasible.

Description

technical field [0001] The present invention relates to nucleic acid (comprising DNA and RNA) purification product, specifically, relate to a kind of kit that uses nano-magnetic beads to purify virus and Gram-negative bacterial nucleic acid from human body fluid (such as blood plasma or serum or urine) . Background technique [0002] Nucleic acid is a kind of biological information macromolecular compound. It has been more than 100 years since it was first isolated from cells. It was not until the middle of the last century that scientists' understanding of the structure and function of nucleic acid achieved a leap forward. [0003] Nucleic acid is divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and nucleotide is the basic structural unit of nucleic acid. Both DNA and RNA exist in plants and animals, but a virus contains only one nucleic acid, DNA or RNA. [0004] In 1953, Watson and Crick proposed the DNA double helix structure model, which greatly pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C07H1/06B01J20/10
Inventor 穆海东汪宁梅黎飒
Owner 上海裕隆生物科技有限公司
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