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Nucleic-acid visual detection kit for novel coronavirus SARS-CoV-2

A coronavirus, visual detection technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve the problems of high technical requirements, unfavorable clinical application, expensive instruments, etc.

Pending Publication Date: 2020-07-07
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In order to solve the novel coronavirus SARS-CoV-2 fluorescent RT-PCR detection mentioned in the above-mentioned background technology, it takes a long time, requires expensive special instruments, has high technical requirements for operators, and is not conducive to clinical application. The present invention provides A LAMP detection primer set, kit and detection method for loop-mediated isothermal amplification of SARS-CoV-2 with high detection sensitivity

Method used

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  • Nucleic-acid visual detection kit for novel coronavirus SARS-CoV-2
  • Nucleic-acid visual detection kit for novel coronavirus SARS-CoV-2
  • Nucleic-acid visual detection kit for novel coronavirus SARS-CoV-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preparation of Visual Detection Kit for Novel Coronavirus SARS-CoV-2 Nucleic Acid

[0034] (1) RT-LAMP primer design, synthesis and preparation of primer set solution:

[0035] Log in to the novel coronavirus gene sequence database of the National Genome Science Data Center (https: / / bigd.big.ac.cn / ncov). Search and download novel coronavirus, other coronaviruses and other pneumonia-causing viruses (influenza virus, respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza virus, enterovirus, herpes simplex virus, human metapneumovirus, reovirus , measles virus, cytomegalovirus, etc.) genome sequences, using CLUSTAL, DNASTAR, BIOEDIT and other bioinformatics software to compare and analyze the genome sequences. The analysis found that there was no significant variation in the genome sequences of the recently popular new coronavirus SARS-CoV-2 isolates, so the representative strain of the new coronavirus SARS-CoV-2, BetaCoV / Wuhan / WH-01 / 2019, was selec...

Embodiment 2

[0051] Example 2: Optimization of the detection temperature of the new coronavirus SARS-CoV-2 nucleic acid visual detection kit

[0052] To each RT-LAMP reaction system (25ul), add 12.5μL of 2×LAMP reaction solution, 7ul of primer set aqueous solution, 1ul of positive template, and make up the volume to 25ul with sterile DEPC water. The final concentration of each component in each 25ul RT-LAMP reaction system is 20mM Tris-HCl (pH8.8), 10mM KCl, 8mM MgSO 4 , 10mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 0.8M betaine, 1.4mM dNTPs, 8U Bst DNA polymerase, 200U AMV reverse transcriptase, 2.5U RNase inhibitor, 0.25mMHNB, the moles of each primer are: primers F3, B3 each 5pmol, primers FIP, BIP each 40pmol. A negative control and a blank control were set up in the experiment.

[0053] The temperature of RT-LAMP reaction was set at 58, 59, 60, 61, 62, 63, 64, and 65 °C, respectively, and the reaction time was 50 minutes. Then, the reaction was terminated at 85 °C for 5 minutes. Visually...

Embodiment 3

[0057] Example 3: Detection time optimization and sensitivity determination of SARS-CoV-2 nucleic acid visual detection kit

[0058] Positive templates were diluted 10-fold with DEPC water. The amount of positive template in tubes 1# to 9# was 1.6ng, 0.16ng, 0.016ng, 1.6pg, 0.16pg, 0.016pg, 1.6fg, 0.16fg, 0.016fg / ul, respectively. Tubes 10# and 11# are negative control and blank control, respectively.

[0059] The RT-LAMP reaction system is the same as the reaction system in Example 2. That is, to each RT-LAMP reaction system (25ul), add 12.5μL of 2×LAMP reaction solution, 7ul of primer set aqueous solution, 1ul of positive templates of different concentration gradients, and make up the volume to 25ul with sterilized DEPC water. The final concentration of each component in each 25ul RT-LAMP reaction system is 20mM Tris-HCl (pH8.8), 10mM KCl, 8mM MgSO 4, 10mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 0.8M betaine, 1.4mM dNTPs, 8U Bst DNA polymerase, 200U AMV reverse transcriptase, ...

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Abstract

The invention discloses a nucleic-acid visual detection kit for a novel coronavirus SARS-CoV-2. The kit disclosed by the invention comprises a primer group and ingredients such as a RT-LAMP reaction solution, a positive control, a negative control and DEPC water. The primer group for detecting the SARS-CoV-2 comprises 6 primers in all, i.e., outer primers F3 and B3, inner primers FIP and BIP, andloop primers LF and LB and has a sequence shown in SEQ ID NO: 1 to NO: 6. The primer group and the kit can accurately detect an S gene of the SARS-CoV-2 and are free of a cross reaction with other coronaviruses and other viruses causing pneumonia. The SARS-CoV-2 can be detected in 15 minutes at the soonest through isothermal amplification at a temperature of 63 DEG C. The detection sensitivity reaches 1.6fg / microliter. The kit has the advantages of simplicity, convenience, rapidness, sensitiveness and specificity, a result can be directly decided through observing color changes of a reaction tube with naked eyes under natural light without expensive instruments, rapid and accurate detection on the SARS-CoV-2 can be achieved, and thus, the kit is applicable to on-site detection and clinicalcheckout in primary hospitals.

Description

technical field [0001] The invention belongs to the technical field of viral nucleic acid detection, and relates to a nucleic acid isothermal amplification visual detection kit for novel coronavirus SARS-CoV-2. Background technique [0002] Novel coronavirus pneumonia (referred to as new coronary pneumonia, English abbreviation COVID-19) is extremely contagious, and is mainly characterized by fever, dry cough and pneumonia lesions, which can seriously lead to death. [0003] The pathogen of new coronary pneumonia is the new coronavirus SARS-CoV-2. SARS-CoV-2, Severe Acute Respiratory Syndrome Virus (SARS-Cov) and Middle East Respiratory Syndrome Virus (MERS-Cov) belong to the genus Betavirus of the family Coronaviridae, but SARS-CoV-2 is closely related to SARS-Cov and MERS-Cov. The homology of Cov is only 79.5% and 40%, respectively. The above three coronaviruses can cause severe pneumonia in humans, and there are also endemic human coronaviruses that cause mild respirato...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2521/107C12Q2545/113C12Q2527/127C12Q2563/107Y02A50/30C12Q2527/101
Inventor 颜世敢朱丽萍
Owner QILU UNIV OF TECH
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