Primer combination used for detecting SARS-CoV-2 whole genome, and application method

A technology of whole genome and primer combination, which is applied in the field of primer set for detecting the whole genome of SARS-CoV-2, can solve the problems of not meeting the requirements of in-depth analysis data, relying on electrophoretic separation technology, and low throughput, so as to reduce the cost of detection, High amplification efficiency and high throughput

Pending Publication Date: 2020-09-18
云南科耀生物科技有限公司
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  • Claims
  • Application Information

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Problems solved by technology

But at the same time, as a next-generation sequencing technology, the Sanger method has low throughput, high price, low efficiency, relies on electrophoretic separation technology, and can only detect more than 20% to 30% of quasi-species, which cannot meet the requirements of in-depth analysis data

Method used

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  • Primer combination used for detecting SARS-CoV-2 whole genome, and application method
  • Primer combination used for detecting SARS-CoV-2 whole genome, and application method
  • Primer combination used for detecting SARS-CoV-2 whole genome, and application method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Primer Design and Optimization

[0060] 1. Design of primers

[0061] A. Due to the highly conservative nature of the novel coronavirus, download five G-type, S-type, and V-type sequences from the website of the Global Initiative for Sharing Influenza Data (GISAID) as reference sequences, compare them with the clustalx software, and select the conserved region sequences , the GC content, Tm value, hairpin structure and primer-dimer were analyzed by Primer Select software. Only when all qualified can indicate that the preliminary design of the primer is completed, the full-length sequence gene structure map of SARS-CoV-2 and 5 specific reverse transcription Primer positions and 38 sets of primers to amplify segments such as figure 1 shown;

[0062] B. There is an overlapping region of 100-200bp in every two fragments of the 38 fragments to facilitate splicing and complete splicing. The primer sequences are shown in SEQ ID NO:1-SEQ ID NO:152, see the table b...

Embodiment 2

[0068] Embodiment 2: the extraction of sample RNA

[0069] In this example, the commercialized TIANamp Virus RNA Kit (TIANGEN, Beijing) was used to extract RNA from throat swabs or nasal swabs of 20 patients with new coronary pneumonia. The extraction method refers to the kit instructions.

Embodiment 3

[0070] Embodiment 3: reverse transcription nested PCR reaction

[0071] 1. Reverse transcription

[0072] Carry out RT-PCR with the RNA gained in embodiment 2 as template, and reaction is carried out in two steps, and reaction system and reaction condition are as follows: A, reaction system are as follows:

[0073] The first step reaction system is 20 μL:

[0074]

[0075] The second step reaction system is 40 μL:

[0076]

[0077] B. The reaction conditions are as follows:

[0078] Step 1: 65°C, 5min

[0079] The second step: 47°C, 80min; 70°C, 15min.

[0080] 2. Nested PCR reaction

[0081] The reverse transcription reaction product cDNA obtained in step 1 is diluted one-fold and used as a template for a nested PCR reaction. The reaction system and conditions are as follows, and the nested PCR is carried out in two steps:

[0082] (1) Peripheral PCR:

[0083] The reaction system is 20 μL:

[0084] Reagent components Premix Taq dd H 2 o

upstre...

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Abstract

The invention discloses a primer combination used for detecting SARS-CoV-2 whole genome. The primer combination comprises specific primers used for SARS-CoV-2RNA reverse transcription and a primer group for amplification of the whole genome; by adoption of the primer combination, after target genes are amplified by reverse transcription and nest type PCR, on the basis of a next-generation semiconductor sequencing platform, a next-generation semiconductor sequencing technology used for detecting the SARS-CoV-2 whole genome and mutation sites is constructed; the method has the advantages of being wide in applicability, high in flux and good in accuracy, and can detect multiple samples one time; the convenient method is provided for deep analysis of the SARS-CoV-2 genome; and the method has important meanings for epidemiologic study, clinical early molecular diagnosis and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a primer set, method and application thereof for detecting the whole genome of SARS-CoV-2, and a method for detecting the whole genome and mutation sites of SARS-CoV-2 by using next-generation semiconductor sequencing technology. Background technique [0002] Corona Virus Disease 2019 (COVID-19), referred to as "New Coronary Pneumonia", is a novel coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) that causes respiratory symptoms, fever, An infectious disease with signs such as coughing, shortness of breath, and difficulty breathing; in more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. [0003] The new coronavirus has been identified as a betacoronavirus, which is wrapped in a round or oval virus with a diameter of 60-140nm, widely distributed in humans and other mammals, and its genome is simil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6869C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6869C12Q2600/156C12Q2531/113C12Q2549/119C12Q2525/191C12Q2535/122
Inventor 冯悦贾圆圆杨宪瑶
Owner 云南科耀生物科技有限公司
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