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One-step rapid and efficient viral nucleic acid extraction method

A viral nucleic acid, rapid technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc., can solve problems such as weak virus specificity selection, affecting virus recovery rate, and many operation steps, and achieve high concentration. And the effect of stability, reduction of human influence, and fewer operation steps

Pending Publication Date: 2020-05-22
CHANGZHOU SMART LIFESCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technical solution does not realize the true "one-step and one-tube method" for extracting viral nucleic acids. According to the content recorded in the instructions, magnetic beads I and magnetic beads II need to be carried out in different containers, and the operation steps are relatively complicated. Many, affecting the recovery rate of the virus, the consumption of consumables is large and the cost is high during automatic operation; and the magnetic beads I used are gel magnetic beads, which are relatively weak for virus specific selection, and are not suitable for the nucleic acid extraction of some special viruses

Method used

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  • One-step rapid and efficient viral nucleic acid extraction method
  • One-step rapid and efficient viral nucleic acid extraction method
  • One-step rapid and efficient viral nucleic acid extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of virus S protein antibody immunomagnetic beads:

[0031] (1) Take 1ml Carboxyl-activated Magpoly Beads in a centrifuge tube, place the centrifuge tube on the magnetic separator for 1min, after the solution becomes clear, use a pipette to draw the supernatant and discard it; Note: Do not Remove the magnetic beads, the same below;

[0032] (2) Add 1ml, 50mM MES, pH 5.0, mix well, put the centrifuge tube on the magnetic separator for 1min, when the solution becomes clear, use a pipette to absorb the supernatant and discard it;

[0033] (3) Add 1ml (10mg / ml) EDC solution, mix well, and incubate at room temperature for 30min. Make sure that the magnetic beads are fully mixed, otherwise the activation efficiency will be affected;

[0034] (4) Place the centrifuge tube on the magnetic separator for 1 min. After the solution becomes clear, suck up the supernatant and discard it. Wash twice with pre-cooled deionized water and activation solution, as fast as poss...

Embodiment 2

[0047] Preparation of virus N protein antibody immunomagnetic beads:

[0048] (1) Take 1ml of amino-activated magnetic beads into a centrifuge tube, place the centrifuge tube on a magnetic separator for 1 min, and when the solution becomes clear, use a pipette to absorb the supernatant and discard it; Note: Do not remove the magnetic beads, The same below;

[0049](2) Wash once with 1ml PBS solution, put the centrifuge tube on the magnetic separator for 1min, absorb the supernatant and discard it;

[0050] (3) Add 200ul of freshly prepared glutaraldehyde solution (15%), mix well, and react at room temperature in the dark for 1 hour, during which the magnetic beads are kept in a state of mixing;

[0051] (4) Add 1 mg of N protein antibody, mix well, react at room temperature for more than 2 hours, place the centrifuge tube on a magnetic separator for 1 minute, absorb the supernatant and discard it;

[0052] (5) Add 500ul of PBS buffer solution containing 1%BSA, block the reac...

Embodiment 3

[0057] Preparation of antibody immunomagnetic beads: same as Example 2.

[0058] Viral Nucleic Acid Extraction

[0059] (1) Mix the prepared immunomagnetic beads and silanol magnetic beads evenly in a ratio of 2:3;

[0060] (2) Take 100 μl of mixed magnetic beads and add them to the centrifuge tube containing the samples, which can be oral swabs, throat swabs and other samples. After mixing evenly, incubate at room temperature for 10 minutes, place on the magnetic device for 2 minutes, and wait for the solution to be clarified. Clear and discard;

[0061] (3) Add 200ul 80mMTris-HCl, 4M guanidine isothiocyanate, 0.5% Triton X-100, 5mMDTT, 15mMEDTA, pH 5.0 to the mixed magnetic beads, mix well and let stand for 8min;

[0062] (4) Place the mixed magnetic bead tube adsorbing nucleic acid on the magnetic stand for 2 minutes, absorb the supernatant and discard it;

[0063] (5) Add 600 μl of washing solution I (5.5M guanidine hydrochloride, 56% absolute ethanol, 80 μg / mL proteina...

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Abstract

The invention discloses a one-step rapid and efficient viral nucleic acid extraction method. The one-step rapid and efficient viral nucleic acid extraction method comprises the following steps: (1) allowing binding of a virus antibody or receptor to hydroxyl group, carboxyl group, amino group or epoxy group on surface of magnetic microsphere (MNP) so as to form a stable magnetic microsphere-antibody complex (MNP-Ab); (2) uniformly mixing the MNP-Ab with nucleic acid extraction magnetic beads according to a certain proportion so as to obtain a magnetic bead mixture; (3) adding the magnetic beadmixture into a sample so as to allow virus in the sample to be tested with the MNP-Ab so as to obtain enriched virus; (4) causing lysis on the enriched virus by using a lysis solution so as to have nucleic acids released, and allowing combination of the nucleic acids with the nucleic acid extraction magnetic beads in the magnetic bead mixture; and (5) eluting the nucleic acids by using an eluant.The one-step rapid and efficient viral nucleic acid extraction method truly realizes the ''one-step one-tube method'' that immuno-magnetic beads and nucleic acid extraction magnetic beads are contained in the same container, so that neither of the magnetic beads are required to be removed in the process of operation, so that the one-step rapid and efficient viral nucleic acid extraction method iseasy to operate; and moreover, the immuno-magnetic beads are high in specificity and adsorption rate, high in virus recovery rate, and strong in repeatability.

Description

technical field [0001] The invention belongs to the field of viral nucleic acid extraction and detection, and in particular relates to a method for rapidly and efficiently extracting viral nucleic acid in one step. Background technique [0002] Nucleic acid extraction and purification is the basis for downstream nucleic acid detection, biological research or new product development, and the quality and integrity of the obtained nucleic acid directly affects the detection results. Classic nucleic acid extraction and purification methods include phenol-chloroform extraction, alkali cleavage, cetyltrimethylammonium bromide (CTAB) extraction, etc. However, these methods are cumbersome to operate, time-consuming, and the effect of extracting trace amounts of nucleic acid is not good. [0003] As a new nucleic acid extraction method that has emerged in recent years, the magnetic bead method is based on the specific adsorption of magnetic nanomaterials on nucleic acids in a high-s...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2521/537
Inventor 单玉飞曹飞婷方利
Owner CHANGZHOU SMART LIFESCI CO LTD
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