Composition for extracting 2019 novel coronavirus nucleic acid, kit, application and method thereof

A coronavirus and composition technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of small amount of samples, low purity of nucleic acid, inability to adapt to virus detection, etc.

Active Publication Date: 2020-10-23
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the amount of sample used is small and the purity of nucleic acid is low, which makes it unable to adapt to the detection of infected persons with low viral load

Method used

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  • Composition for extracting 2019 novel coronavirus nucleic acid, kit, application and method thereof
  • Composition for extracting 2019 novel coronavirus nucleic acid, kit, application and method thereof
  • Composition for extracting 2019 novel coronavirus nucleic acid, kit, application and method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1, the method for extracting 2019 novel coronavirus nucleic acid by the composition of the present invention

[0067] 1. Preparations

[0068] According to the number of samples to be tested and negative and positive samples, mix proteinase K (20 μL / person) and 2019-nCoV magnetic bead solution (30 μL / person) in proportion to form proteinase K-magnetic bead mixture, shake and mix, centrifugal.

[0069] 2. Nucleic acid extraction process

[0070] 2.1 Take a number of 2mL centrifuge tubes according to the number of samples to be tested, and add 750 μL of sample to each tube;

[0071] 2.2 Add 750 μL of 2019-nCoV extraction solution 1 (2mol / L guanidine hydrochloride, 1mol / L guanidine thiocyanate, 1mol / L urea, 0.2mmol / L surfactin, 150mmol / L chlorine Potassium chloride, 0.5mol / L lithium chloride, triethanolamine dodecyl sulfate with a mass / volume ratio of 0.5%, ethylphenyl polyethylene glycol (NP-40) with a volume / volume ratio of 0.5%, Triton X-100 with a mass / v...

Embodiment 2

[0079] Embodiment 2, composition of the present invention extracts throat swab simulation sample detection

[0080] According to the extraction method described in Example 1, the diluted and standardized novel coronavirus culture (10 copies / mL) and pseudovirus (10 copies / mL) were extracted from negative throat swab samples, and the extracted nucleic acid was tested by fluorescent PCR. Nucleic acid test results such as Figure 1-2 As shown (dark color is ORF1ab gene detection, light color is N gene detection, repeated 24 times), it can be seen from the figure that the composition of the present invention can well extract 10 copies / mL of the new coronavirus sample and pseudovirus , and can detect the corresponding virus.

Embodiment 3

[0081] Example 3, the composition of the present invention extracts the nucleic acid purity and detection of EDTA anticoagulated whole blood simulated samples

[0082] According to the extraction method described in Example 1, the nucleic acid of the EDTA anticoagulated whole blood mock sample (20 copies / mL for the new coronavirus) was extracted, and its concentration and purity are shown in Table 1 below.

[0083]

[0084] As can be seen from the table, the purity extracted using the composition of the present invention all meets the requirements in the art.

[0085] Use the extracted nucleic acid for detection, and the detection results are as follows: Figure 3-4 shown. image 3 is the internal standard amplification curve, Figure 4 It is the amplification curve of the new coronavirus. It can be seen from the figure that the corresponding target can be detected by using the composition of the present invention to extract nucleic acid with low virus concentration.

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Abstract

The invention belongs to the field of molecular biological detection, and particularly belongs to the field of virus nucleic acid extraction. The invention discloses a composition for extracting 2019novel coronavirus nucleic acid. The composition is prepared from an extracting solution 1, an extracting solution 2 and an extracting solution 3, wherein the extracting solution 1 is prepared from guanidine hydrochloride, guanidine thiocyanate, urea, surfactin, potassium chloride, lithium chloride, triethanolamine lauryl sulfate, NP-40, triton X-100 and isopropanol; the extracting solution 2 comprises Tris-HCl, lithium chloride, sodium acetate, triethanolamine lauryl sulfate, NP-40, SDS and ethanol; and the extracting solution 3 comprises Tris-HCl, sodium chloride, and ethanol. By using the composition provided by the invention, the sensitivity of nucleic acid extraction and purification of 2019 novel coronavirus can be specifically improved, and the detection limit can reach 10 copies / mL.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, specifically, to the field of viral nucleic acid extraction, and more specifically, to the field of 2019 novel coronavirus nucleic acid extraction. Background technique [0002] Nucleic acid is one of the most basic substances of all kinds of life. It not only plays a role in storing and transmitting genetic information, but also plays a decisive role in a series of major life phenomena such as growth, inheritance, and variation. Since Swiss physicist Friedrich Miescher first isolated nucleic acid from white blood cells in 1869, researchers from all over the world have carried out unremitting research and exploration on nucleic acid extraction and purification methods. Traditional methods for nucleic acid extraction include phenol extraction, alkaline lysis, CTAB extraction, boiling, etc. Since Vogelstein and Gillespie first used glass fiber (main component SiO2) to recover DNA fragmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/70C12Q1/6851C12R1/93
CPCC12N15/1013C12Q1/6806C12Q1/701C12Q1/6851C12Q2527/125C12Q2563/143C12Q2563/149C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 吴康龙凤英缪为民戴立忠
Owner SANSURE BIOTECH INC
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