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Virus RNA releasing agent, kit and application of virus RNA releasing agent

A release agent and kit technology, applied in the field of RNA extraction, can solve the problems of decreased sensitivity of nucleic acid detection applications, low efficiency of viral nucleic acid release, and improper SDS ratio, etc., achieving low price, saving reaction time, and short time-consuming effects

Pending Publication Date: 2021-06-25
江苏吉诺思美精准医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, although the above-mentioned nucleic acid extraction technologies or kits have been widely used, they have highlighted their shortcomings in dealing with the novel coronavirus epidemic: long nucleic acid extraction time, complicated process, and prone to cross-contamination, etc.
[0005] Even so, the efficiency of the existing nucleic acid release agents for viral nucleic acid release is low, resulting in a decrease in the sensitivity of nucleic acid detection applications
The actual experimental results prove that the SDS ratio in the existing nucleic acid release agent formulation is slightly inappropriate, resulting in serious inhibition of subsequent PCR experiments

Method used

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  • Virus RNA releasing agent, kit and application of virus RNA releasing agent
  • Virus RNA releasing agent, kit and application of virus RNA releasing agent
  • Virus RNA releasing agent, kit and application of virus RNA releasing agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Nucleic acid release agent configuration process of the present invention

[0024] 1. Reagent preparation: Prepare Dodine, Potassium Chloride, Triton, and Sodium Hydroxide.

[0025] Wherein, Duoguo is purchased from sigma company, product name: Dodin, article number: 45466, dry powder; Triton-X100 is purchased from sigma company, product name: Triton-X100, article number: T8787, liquid (100%); Sodium hydroxide was purchased from sigma company, product name: sodium hydroxidesolution, item number: STBJ3989, liquid (content 50% in H 2 O); Potassium chloride is a dry powder or granule, from domestic commercial brands commonly used, chemically pure. proclin300 is Solarbio brand, 100% liquid, used directly after purchase.

[0026] 2. Reagent configuration process:

[0027] a, mother liquor configuration: according to the state or concentration of the original material, Duoguodin was prepared into a mother liquor of 2mmol / L, and potassium chloride was prepared ...

Embodiment 2

[0030] Embodiment 2 Nucleic acid releasing agent effect application of the present invention

[0031] 1. Prepare the pseudovirus for extraction, and inactivate the pseudovirus at 56°C for 30 minutes. After the inactivation is completed, the pseudovirus is diluted to 200copies / ul, ready for RNA release.

[0032] 2. Prepare the consumables used in the experiment, add 10 ul of diluted pseudovirus in a 1.5ml centrifuge tube, and then add the release agent of the present invention obtained in Example 1 in a ratio of 1:1 or 1:2. React at room temperature for 5 to 10 minutes. Complete the release of RNA, and do 3 parallel controls.

[0033] 3. Take 5ul of the released product, add 15ul of amplification reagent, and perform PCR reaction on Hongshi SLAN-96P fluorescence quantitative PCR instrument. ).

[0034] 4. After PCR amplification, obtain the amplification curve figure 1 ;

Embodiment 3

[0035] Example 3 Sanxiang Biological Nucleic Acid Release Agent Application Test

[0036] 1. According to the same conditions as in Example 2, prepare the pseudovirus for extraction, and inactivate the pseudovirus at 56° C. for 30 minutes. After the inactivation is completed, the pseudovirus is diluted to 200copies / ul, ready for RNA release.

[0037] 2. Prepare the consumables used in the experiment, add 10ul of diluted pseudovirus to a 1.5ml centrifuge tube, and then add the nucleic acid release reagent of Hunan Shengxiang Biological Co., Ltd. in a ratio of 1:1 or 1:2. React at room temperature for 5 to 10 minutes. Complete the release of RNA, and do 3 parallel controls.

[0038] 3. Take 5 ul of the released product, add 15 ul of amplification reagent, and carry out PCR reaction on Hongshi SLAN-96P fluorescent quantitative PCR instrument together with Example 2. The reaction program is 55 ° C for 10 min, 95 ° C for 1 min, (95 ° C 10S, 60°C 1min45cycles).

[0039] 4. After...

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Abstract

The invention discloses a virus RNA releasing agent, a kit and application of a virus RNA releasing agent. The releasing agent comprises 0.01-0.05 mmol / L of dodine, 0.05%-0.5% (v / v) of triton, 5-15 mmol / L of potassium chloride and 0.1%-0.5% (v / v) of sodium hydroxide. Compared with the prior art, reagents used in the formula of the releasing agent are low in price, the preparation method is simple, and the contained reagents do not have chemical danger. The reagent can be used for effectively cracking viruses and safely and quickly releasing nucleic acid, and is simple to operate and short in consumed time. The released product has no inhibition effect on downstream experiments, and does not influence the amplification efficiency of subsequent experiments such as PCR.

Description

technical field [0001] The invention belongs to the technical field of RNA extraction, and in particular relates to a virus RNA release agent, a kit and an application thereof. Background technique [0002] Nucleic acid is the general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is a biological macromolecular compound polymerized by many nucleotide monomers and is one of the most basic substances of life. Nucleic acid is a class of biopolymers, an essential component of all known life forms, the most important substance of all biomolecules, and widely exists in all animal and plant cells and microorganisms. Nucleic acids are composed of nucleotides, and nucleotide monomers are composed of five-carbon sugars, phosphate groups, and nitrogenous bases. If the five-carbon sugar is ribose, the polymer formed is RNA; if the five-carbon sugar is deoxyribose, the polymer formed is DNA. [0003] Nucleic acid extraction is a necessary basic technology for moder...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2527/125C12Q2531/113Y02A50/30
Inventor 贾春蕾曹振龙刘少卿宋晓东
Owner 江苏吉诺思美精准医学科技有限公司