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Loop-mediated isothermal amplification detection primer group for detecting 2019-nCoV and application of loop-mediated isothermal amplification detection primer group

A 2019-ncov, loop-mediated isothermal technology, applied in the fields of microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve complex problems, requires relatively several hours or even a day or two, and is not suitable for large-scale pathogen screening and detection. , the limitations of virus isolation and culture technology, etc., to achieve the effect of saving detection time, fast detection speed, and high detection sensitivity

Active Publication Date: 2020-06-12
SUZHOU HEALTH COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation and culture technology has great limitations, often has shortcomings such as low sensitivity, cumbersome and time-consuming operations, and cannot be applied to practical applications such as large-scale disease source screening and detection
At present, molecular biology technology mainly adopts fluorescent quantitative PCR detection method and sequencing method. This method is fast, sensitive, and suitable for processing large-scale samples, and has shown great advantages in virus detection. Interpretation requires special precision equipment, and the operation procedure is relatively complicated, and it takes a few hours or even a day or two, and it also has certain limitations.

Method used

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  • Loop-mediated isothermal amplification detection primer group for detecting 2019-nCoV and application of loop-mediated isothermal amplification detection primer group
  • Loop-mediated isothermal amplification detection primer group for detecting 2019-nCoV and application of loop-mediated isothermal amplification detection primer group
  • Loop-mediated isothermal amplification detection primer group for detecting 2019-nCoV and application of loop-mediated isothermal amplification detection primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Design and synthesis of primers

[0024] According to the gene sequence of the new coronavirus 2019-nCoV released by Genebank, and the gene sequence of coronaviruses with high homology (such as SARS), all sequences were compared and analyzed using the sequence comparison software Clustal X, and 2019 was found. - Conserved region of nCoV, use the online primer design software Primer Explorer V4 to design 5 primers, the primer set is shown in Table 1, the primer sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd., diluted to 10 μM or 20 μM after synthesis Store in a -20°C refrigerator in the dark for later use.

[0025] Table 1

[0026]

Embodiment 2201

[0027] Example 2 Establishment of 2019-nCoV ring-mediated isothermal amplification detection method

[0028] Loop-mediated isothermal amplification of 2019-nCoV was performed using the novel coronavirus (2019-nCoV) S gene (GeneBank ID: NC_045512.2) sequence plasmid (purchased from Nanjing GenScript Biotechnology Co., Ltd.) constructed on the PUC57 vector as a template The establishment of the detection method and the reaction system are shown in Table 2. Bst DNA polymerase, supporting reaction buffer, and fluorescent visual detection reagents were purchased from Rongyan Biotechnology Co., Ltd.

[0029] Table 2

[0030]

[0031] The reaction conditions are: constant temperature reaction at 63° C. for 50 minutes, treatment in boiling water for 2 minutes for enzyme inactivation treatment, and termination of the reaction.

Embodiment 3

[0032] Example 3 Analysis of kit detection sensitivity

[0033] Using the plasmid described in Example 2 as a template, use 0.5x10 5 The initial concentration of copy / μL is diluted 10 times with ultrapure water, and diluted into 4 concentration gradients: 0.5x10 5 copy / μL, 0.5x10 4 copy / μL, 0.5x10 3 copy / μL and 0.5x10 2 copy / μL, use water instead of the template as a negative control, and perform detection according to the following steps:

[0034] (1) Add samples to different PCR tubes according to the reaction system in Example 2, and the template concentration of each PCR tube is 0, 0.5x10 5 copy / μL, 0.5x10 4 copy / μL, 0.5x10 3 copy / μL and 0.5x10 2 copy / μL;

[0035] (2) Gently flick the bottom of the test tube with your fingers to mix the solution thoroughly, put it in a centrifuge for a few seconds, and let the reaction solution sink to the bottom;

[0036] (3) Place the configured reaction test tube in a water bath, and react at a constant temperature of ...

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Abstract

The invention provides a loop-mediated isothermal amplification detection primer group for detecting 2019-nCoV and application of the loop-mediated isothermal amplification detection primer group. Theprimer group comprises a forward outer primer NCP-F3-2 shown as SEQ ID NO. 1, a reverse outer primer NCP-F3-2 shown as SEQ ID NO. 1, a reverse outer primer NCP-B3-2 as shown in SEQ ID NO. 2, a forward inner primer NCP-FIP-2 as shown in NO. 3, a reverse inner primer NCP-BIP-2 as shown in SEQ ID NO. 4, and a loop primer NCP-LB-2 as shown in SEQ ID NO. 5. According to the invention, the detection time for the 2019-nCoV is short, the sensitivity is high, the specificity is high, the detection result can be observed by naked eyes, and the detection efficiency of the 2019-nCoV is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a loop-mediated isothermal amplification detection primer set for detecting 2019-nCoV and its application. Background technique [0002] The novel coronavirus (2019-nCoV) belongs to a large family of viruses known to cause colds as well as more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The new coronavirus is a new strain of coronavirus that has never been found in humans before. The common signs of people infected with coronavirus include respiratory symptoms, fever, cough, shortness of breath and dyspnea. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. At present, there is no specific treatment for the disease caused by the new coronavirus, which poses a great threat to human society. [0003] At present, the di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107C12Q2565/125Y02A50/30C12Q2531/101
Inventor 刘松柏
Owner SUZHOU HEALTH COLLEGE