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Method for high-throughput extraction of trace nucleic acid for large-volume mixed swab sample detection

A sample detection and large-volume technology, which is applied in the fields of life science and biology, can solve problems such as insufficient work efficiency, low detection sensitivity, and reduced sensitivity, and achieve the effect of less impurity content, high sensitivity, and improved sensitivity

Pending Publication Date: 2021-05-14
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even so, although the above mixed detection method has improved the ability of nucleic acid detection, its detection sensitivity is low, and the work efficiency is still not efficient enough, especially for samples containing low copy nucleic acid may produce "false negative" detection results
[0006] In the prior art, the magnetic bead method is mostly used for nucleic acid extraction, but when the current magnetic bead method is used for nucleic acid extraction in samples, the applicable sample volume is usually 200-300ul, which is not suitable for large volume requirements (for example, multiple samples Mixed detection) nucleic acid detection needs
And importantly, multiple samples are mixed, affected by the total volume, the samples are diluted with each other, which inevitably dilutes the target nucleic acid, and reduces the sensitivity when the sampling volume is only 200-300ul

Method used

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  • Method for high-throughput extraction of trace nucleic acid for large-volume mixed swab sample detection
  • Method for high-throughput extraction of trace nucleic acid for large-volume mixed swab sample detection
  • Method for high-throughput extraction of trace nucleic acid for large-volume mixed swab sample detection

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: used material and specific steps of large volume sample extraction method, such as figure 1 .

[0049] 1. Reagent components

[0050] 1. Collection of extraction reagents

[0051] 1a. Lysis Buffer (LB).

[0052] 1b. Adsorption solution Binding Buffer (BB).

[0053] 1c. Elution Buffer (EB).

[0054] 1d. Proteinase K (PK), store at -20°C.

[0055] 1e. Wash solution (WB).

[0056] 2. Bring your own

[0057] 1. Lysis tube: 50ml centrifuge tube.

[0058] 2. Eluent collection tube: 1.5ml microcentrifuge tube.

[0059] 3. 30ml disposable syringe (screw connector).

[0060] 4. 5-10ml disposable syringe (self-prepared).

[0061] 5. Low-speed centrifuge (≥4000g).

[0062] 5. High-speed microcentrifuge (≥12000g).

[0063] 6. 60°C water bath or dry heat module.

[0064] 7. Closed column positive pressure nucleic acid extraction device: the extraction container in the Chinese authorized patent CN201580073867.2. The closed column method positive pressure n...

Embodiment 2

[0074] Embodiment 2: comparison and verification test

[0075] Pre-experimental reagents and devices:

[0076] Xpress SARS-CoV-2, Cepheid, 904 Caribbean Drive, Sunnyvale, CA 94089 USA

[0077] Comma XP TM Viral DNA / RNA Extraction Kit (Column Method), CommaXP TM Serum Plasma Cell-Free DNA Mid-Prep Kit, CommaVac TM Luer interface negative pressure device, Shenzhen Comma Biotechnology Co., Ltd. (abbreviation: Comma Company)

[0078] 12-channel negative pressure column adsorption rack

[0079] Portable oil-free vacuum pump

[0080] Manually Operated Extraction System (MOES), Occam Biolabs, Inc., DE, USA (referred to as OBI company)

[0081] BDS new crown pseudovirus quality control product: S2 standard product (see the table below for specific concentration), Guangzhou Bangdesheng Biotechnology Co., Ltd. (referred to as BDS company)

[0082] Concentration number Mean (X)coples / mL X±2SD(coples / mL) S1 (low value) 2.01E+03 6.24E+02~6.48E+03 S2 (m...

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Abstract

The invention relates to a method for high-throughput extraction of trace nucleic acid for large-volume mixed swab sample detection, which comprises the following steps: carrying out lysis treatment on a to-be-detected sample by using a lysis solution to release nucleic acid contained in the sample, to obtain the lysis solution mainly containing the nucleic acid; extracting the lysis solution containing the nucleic acid through a closed-column-method positive pressure nucleic acid extraction device to obtain an extract; and preparing a test liquid for virus detection by using the extract. According to the method disclosed by the invention, the nasopharyngeal swab liquids of different individuals from multiple sources are extracted by a column method, and the steps of high-speed centrifugation of 2000-4000 g and 10-20 min are added after the swab liquids are merged to remove precipitates, so that the resistance during column passing is greatly reduced; and as a pretreatment step, 1-2 times of cleaning and centrifuging steps are reduced compared with common extraction, and the nucleic acid extraction efficiency is improved.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a method for extracting trace nucleic acid with high throughput for detection of large-volume mixed swab samples. Background technique [0002] At present, there are two main methods for clinical virus detection: nucleic acid detection and antigen antibody immunological detection. Nucleic acid detection needs to extract viral nucleic acid RNA from various body fluid samples, and the volume of body fluid samples usually detected is limited by existing commercial extraction devices, which cannot exceed 200-300 μl, thus limiting the sensitivity of detection. [0003] For example, the novel coronavirus COVID-19 virus is an RNA virus, which mainly invades the body through the upper respiratory tract and digestive tract. Binds, enters host cells, and utilizes host organelles for viral protein synthesis and viral replication. The samples used for the detection ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 李同心钱明伟杨凡
Owner FUZHOU ADICON CLINICAL LAB INC
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