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Isothermal amplification kit for detecting SARS-COV-2 and primer probe set

A SARS-COV-2, isothermal amplification technology, applied in the field of isothermal amplification detection kits, can solve the problem of no disclosure of isothermal amplification detection kits, etc., and achieve the effects of convenient interpretation, high sensitivity and high sensitivity

Pending Publication Date: 2020-04-28
上海世和医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the constant temperature amplification detection kit for the newly discovered virus, it is particularly critical to select the appropriate inactivation lysate and the selection of primers and probes. At present, there is no constant temperature amplification detection kit for the SARS-COV-2 virus.

Method used

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  • Isothermal amplification kit for detecting SARS-COV-2 and primer probe set
  • Isothermal amplification kit for detecting SARS-COV-2 and primer probe set
  • Isothermal amplification kit for detecting SARS-COV-2 and primer probe set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1. Composition and configuration of the kit

[0044] In the present invention, the RAA enzyme, buffer A and buffer B were purchased from Hangzhou Zhongce Biology, and the disposable nucleic acid detection device was purchased from Hangzhou Ustar Biotechnology Company.

[0045] RAA enzymes include:

[0046] Reverse transcriptase: MLV is used for RNA chain reverse transcription;

[0047] Strand displacement DNA polymerase: Bsu (Bacillus subtilis Pol I), DNA strand extension;

[0048] Recombinase: T4 uvsX: help primers and templates combine;

[0049] Single-stranded DNA binding protein: T4 gp32 ensures that double-stranded DNA is opened into a single-stranded state;

[0050] NFO Enzyme: An exonuclease that cleaves the probe so that the probe can be extended.

[0051] gp32: 15~30um; 109~200ng / ul uvsX; 3~6um; uVSY (recombinase loading factor) 2~4um; BSu polymerase, 1000Units; 200uM dNTPs; MLV200Untis; NFO 50U.

[0052] Buffer A includes 50mM Tris pH 8.4, 80mM...

Embodiment 2

[0062] Example 2. Screening of inactivated lysate

[0063] Formula 1: 0.1M~0.2M NaOH, 0.1~0.5% TWEEN 80;

[0064] Formula 2: 0.01-0.1M NaOH, 0.1-0.5% TWEEN 20, 0.1%-1% Triton X-100;

[0065] Formulation 3: 0.01-0.1M NaOH, 0.1-0.5% TWEEN 20.

[0066] Use 100ul of lysates from each of the three formulations to process the same oral samples, and select the ACTB gene as an internal reference.

[0067]

[0068]

[0069] The result of the reaction is as follows:

[0070] Recipe 1 Recipe 2 Recipe 3 CT value 27.87 24.52 29.68

[0071] Among the three groups of experiments, formula 2 had the best effect, and formula 2 was selected for treatment.

[0072] Comparison between lysis system and commercial extraction kit:

[0073] Program:

[0074] Two groups of samples were collected, and two samples were collected in parallel for each sample. The two oral swabs collected together were processed with self-prepared lysate and an imported kit respective...

Embodiment 3

[0091] Embodiment 3. Kit detection

[0092] 1. Virus inactivation: Prepare virus inactivation tubes according to the number of test samples required, place the virus inactivation tubes in a centrifuge, and centrifuge at 4000rpm for 3-5 seconds (if there is no centrifuge, you can gently shake the inactivation tubes with your arm), Put the swab into the inactivation solution and squeeze it repeatedly 8-10 times to make the swab fully mix and contact with the inactivation solution. Then press the inactivated solution in the swab to collect at the bottom of the tube. Discard the swab (it is recommended to put the swab in the original sleeve and discard it), cover the inactivation tube, and let it stand for at least 2 minutes to completely inactivate the virus. If the follow-up operation cannot be completed in time, it needs to be stored at -20°C, and if it needs to be stored for a long time, it should be placed at -80°C.

[0093] 2. Constant temperature amplification:

[0094]2...

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Abstract

The invention discloses an isothermal amplification kit for detecting SARS-COV-2 and a primer probe set. The kit comprises (1) inactivated lysate; and (2) an isothermal amplification system: a reaction buffer solution Buffer A, magnesium acetate Buffer B, negative control, nuclease-free water, a primer probe set and RAA enzyme. The lysate is used for rapid inactivation and release of viral nucleicacid, an isothermal amplification technology is utilized for rapidly enriching and amplifying a target area, whether a specific amplified product exists is rapidly determined through combination of aprobe with modification groups and a product with biotin, by a lateral chromatography technology and by use of colloidal gold developing. The kit is simple to operate, professional extraction reagents and detection instruments are not required limitation of detection laboratories and professionals is freed, the kit can perform rapid detection in an instrument-free state outdoors, the whole process only needs 30 min, and reading is very convenient.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a constant temperature amplification detection kit for qualitative detection of SARS-COV-2 virus. Background technique [0002] The new coronavirus (SARS-CoV-2) is a new type of virus that has not been found in humans before. It belongs to the SARS-like coronavirus species, but it is a different strain from SARS-CoV. Corona Virus Disease 2019 (COVID-19 for short), referred to as New Coronary Pneumonia, is an infectious disease caused by a new coronavirus (SARS-CoV-2) transmitted through the respiratory tract. [0003] The detection of new coronavirus needs to be carried out in a professional PCR laboratory by professionals using expensive extraction and detection instruments, which takes at least 3 hours, and the interpretation of the results is difficult. Unable to diagnose in the first time, leading to cross-infection and delay in treatment. Moreover, the positive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2521/507C12Q2521/107C12Q2537/1376C12Q2522/101C12Q2565/625C12Q2545/113
Inventor 方圆宋泽世蔡青青郏肯特田西西
Owner 上海世和医学检验实验室有限公司
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