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Kit for detecting new coronavirus and detection method

A kit and virus technology, applied in the field of new coronavirus detection, can solve problems such as nucleic acid detection failure, and achieve the effect of reducing detection difficulty and improving detection efficiency

Pending Publication Date: 2022-04-12
INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, nucleic acid detection methods mainly target the open reading frame OFR1 region and the nucleocapsid protein region of the new coronavirus to design primers and probes. Continuously mutating viruses may cause nucleic acid detection to fail.

Method used

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  • Kit for detecting new coronavirus and detection method
  • Kit for detecting new coronavirus and detection method
  • Kit for detecting new coronavirus and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The composition of the kit used to detect the new coronavirus is shown in Table 2:

[0068] Table 2

[0069]

[0070] The method of use of the kit is:

[0071] 1. Sample processing:

[0072] Take 2 μL of nucleic acid extract from nasal swab or throat swab, mix with 12.5 μL nucleic acid amplification enzyme and 0.5 μL fluorescent dye reagent, mix well and add to the microfluidic chip loading area. At the same time, take 2 μL of positive control and negative control reagents, mix them with nucleic acid amplification reagents and fluorescent dyes, add them to the chip plate loading area after mixing, seal the chip plate with parafilm, and remove air bubbles with a scraper.

[0073] 2. Detection:

[0074] Fix the packaged chip disc to the microfluidic isothermal amplification accounting analyzer, set the reaction program to 65°C and 60 minutes, and set the detection wells, positive control wells and negative control wells according to the order of sample addition. Af...

experiment example 1

[0075] Experimental Example 1: Primer Screening Test

[0076] Design isothermal amplification primers according to the ORF7b region with the lowest mutation rate on the new coronavirus, and the designed primers are shown in Table 3:

[0077] table 3

[0078]

[0079]

[0080] The primers were tested using common Taq enzymes, and the reaction system is shown in Table 4:

[0081] Table 4

[0082] Element Volume (μL) TaKaRa Ex Taq (5U / μl) 0.2 10×Ex Taq Buffer (20mM Mg 2+ plus)

2 dNTP (2.5mM each) 1.6 template 1 Primer 1 (10 μM) 1 Primer 2 (10 μM) 1 h 2 o

13.2

[0083] Reaction program: 98°C for 10s, 60°C for 15s, 72°C for 40s, 40cycle, 72°C for 5min, 4°C∞. Wherein, primer 1 and primer 2 represent a pair of upstream and downstream primers, including each pair of F3 and B3 primers or FIP and BIP primers.

[0084] The obtained experimental results are as figure 1 shown. Depend on figure 1 It can be seen t...

experiment example 2

[0086] Screening of Temperature Conditions in Isothermal Amplification Experiments

[0087] Set 4 gradients: 60°C, 63°C, 65°C, 67°C. According to the primer combination determined in Experimental Example 1 and the LAMP reaction system, the reaction solution was prepared for amplification. The LAMP isothermal amplification reaction system is shown in Table 5, and the reaction time was 60 minutes.

[0088] Table 5: LAMP isothermal amplification reaction system

[0089] Element volume WarmStart Multi-Purpose RT-LAMP 2X Master Mix(with UDG) 12.5μL Fluorescent dye (50×) 0.5μL template 2μL SEQ ID NO:3 (40μM) 0.8μL SEQ ID NO: 4 (40μM) 0.8μL SEQ ID NO: 1 (10μM) 0.4μL SEQ ID NO:2 (10μM) 0.4μL Supplement H 2 O to

[0090] Amplified products were identified by agarose gel electrophoresis, and the amplification results were as follows: figure 2 shown. Depend on figure 2 The results of agarose gel electrophoresis show...

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Abstract

The invention relates to the field of biological detection, in particular to a kit for detecting new coronavirus and a detection method. The kit contains a nucleic acid amplification reagent, and the nucleic acid amplification reagent contains isothermal amplification primers with nucleotide sequences as shown in SEQ ID NO: 1-SEQ ID NO: 4. Or isothermal amplification primers containing nucleotide sequences as shown in SEQ ID NO: 9 to SEQ ID NO: 12. The invention develops a new coronavirus detection reagent capable of realizing rapid real-time field detection, the detection efficiency can be improved, and an effective technical means is provided for prevention and control of the new coronavirus.

Description

technical field [0001] The invention relates to the field of biological detection, in particular, to a kit and a detection method for detecting a new coronavirus. Background technique [0002] The new type of coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) has caused a large spread of the epidemic worldwide. Its strong infectivity and high fatality rate have posed a huge challenge to the global epidemic prevention and control work. In order to effectively control the epidemic It is particularly important to develop accurate and efficient detection methods. Current detection methods include pathogenic detection, molecular biological detection and immunological detection. [0003] Etiological detection refers to the direct detection of the pathogen itself using techniques such as virus isolation and culture methods or electron microscope observation, which can provide direct evidence for the identification of pathogens in the early stages of disease ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
Inventor 师丹阳王东帅金敏李君文杨栋尹静李海北杨忠委陈天姣周书青王华然
Owner INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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