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Novel coronavirus metagenome sequencing primer, sequencing method and application

A coronavirus and metagenomic technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as low accuracy, unfavorable genomic data acquisition, and long metagenomic sequencing process, and achieve increased efficiency. data, reduce sequencing time, and improve accuracy

Pending Publication Date: 2021-11-26
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
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Problems solved by technology

[0005] In view of this, the purpose of the present invention is to provide a novel coronavirus metagenomic sequencing primer, sequencing method and application, which can solve the problem that the current novel coronavirus metagenomic sequencing accuracy is low, and the metagenomic sequencing process takes a long time, which is not conducive to genome sequencing. Data acquisition, increasing the difficulty of bioinformatics analysis

Method used

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  • Novel coronavirus metagenome sequencing primer, sequencing method and application
  • Novel coronavirus metagenome sequencing primer, sequencing method and application
  • Novel coronavirus metagenome sequencing primer, sequencing method and application

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Embodiment 1

[0032] This embodiment is aimed at the design of 60 pairs of specific PCR amplification primers for the complete genome sequence of the novel coronavirus isolate, and the specific sequence information is shown in Table 1 below:

[0033] Table 1, the sequence information of primers SED ID No.1-SED ID No.120 of the present invention

[0034]

[0035]

[0036]

[0037]

Embodiment 2

[0039] In this example, nine cases of COVID-19 positive throat swab RNA nucleic acid samples were sequenced, as follows:

[0040] 1. PCR amplification of sequencing samples

[0041] 1.1. Reverse transcription of RNA from sequencing samples

[0042]Use the Qubit 2.0 Fluorescence Quantitator to accurately quantify the initial sample, and take 5 μL (total RNA <100ng) to perform reverse transcription of the RNA nucleic acid of the sequencing sample according to the instructions of the QIAseq SARS-CoV-2 Primer Panel. Among them, RP primers were diluted 11 times. Sample mixture reaction system: 5 μl of sample, 1 μl of diluted RP primer, 6 μl of RNase-free water; reaction conditions: 65°C for 5 minutes. 20 μl reverse transcription reaction system: RNA / RP mixture 12 μl, Mμltimodal buffer 4 μl, RNase-free water 1 μl, RI 1 μl, EZReverse Transcriptase 1 μl; reaction conditions: 42°C for 50 minutes, 70°C for 15 minutes.

[0043] 1.2. Specific amplification of cDNA from sequencing sampl...

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Abstract

The invention relates to the technical field of gene detection, and discloses a novel coronavirus metagenome sequencing primer, a sequencing method and application. The sequencing primer comprises 60 pairs of specific PCR amplification primers designed for a complete sequence of a novel coronavirus isolated strain genome, and the sequences of the 60 pairs of specific PCR amplification primers are SED ID No. 1-SED ID No. 120 in sequence. The sequencing method comprises the steps that sequencing sample RNA reverse transcription is conducted; specific amplification of sequencing sample cDNA is conducted, and a PCR amplification product is purified by means of a purified magnetic bead reagent; a sample library is prepared by means of a three-generation library construction kit; and computer sequencing is conducted on library construction samples. Accordingly, the problems that at present, the novel coronavirus metagenome sequencing accuracy is low, and the metagenome sequencing process time is long can be solved.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a novel coronavirus metagenomic sequencing primer, sequencing method and application. Background technique [0002] Novel coronavirus pneumonia is an acute infectious pneumonia, and its pathogen is a new type of coronavirus named "2019-nCoV" by the World Health Organization. The new type of coronavirus belongs to the genus of coronavirus beta. It is a non-segmented single-stranded positive-sense RNA virus with an envelope. It is mainly transmitted through droplets and has a strong infectivity. The initial symptoms after infection are mostly fever, fatigue and dry cough. , and gradually appear severe manifestations such as dyspnea, most of which have a good prognosis, and some severe cases can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. There is still a lack of effective antiviral drugs against the new coronavirus. [0003] At pres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6806C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6806C12Q2531/113C12Q2521/107C12Q2535/122
Inventor 郭彦
Owner ACADEMY OF MILITARY MEDICAL SCI
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