Amplification primer group for detecting SARS-CoV-2 by mNGS and application of amplification primer group

An amplification primer, sars-cov-2 technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of high cost, low load sample false positive results, mutation and other problems , to achieve the effect of improving coverage

Active Publication Date: 2020-08-07
广州微远医疗器械有限公司 +3
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Due to the unbiased nature of metagenomic technology, it is easily affected by factors such as host and pathogen load, and false positive results may also occur in the detection of low-load samples
The sequencing of the virus genome requires virus isolation and cultivation, which is time-consuming and laborious, and mutations may occur during the cultivation process; or deep sequencing of high-load sample nucleic acids is expensive

Method used

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  • Amplification primer group for detecting SARS-CoV-2 by mNGS and application of amplification primer group
  • Amplification primer group for detecting SARS-CoV-2 by mNGS and application of amplification primer group
  • Amplification primer group for detecting SARS-CoV-2 by mNGS and application of amplification primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] An amplification primer and design scheme for detecting novel coronavirus (SARS-CoV-2).

[0036] The present invention targets the detection of the novel coronavirus SARS-CoV-2, combines the advantages of specific primer-targeted reverse transcription and pathogenic metagenomics for unbiased sequencing of all molecules in the sample, and maximizes the performance of pathogenic nucleic acid detection. Among them, the specific primer design method is as follows:

[0037] 1. Genome sequence acquisition.

[0038] Download the 2019 novel coronavirus reference genome with accession number GCF_009858895.2 and the human reference genome with accession number GCF_000001405.39 from the NCBI website.

[0039] 2. Generating a short sequence of the 2019 novel coronavirus genome.

[0040] Sliding and intercepting short sequences on the 2019-nCoV genome sequence with a window length of 15 and a step of 1 until the entire genome is traversed, generating 392 short-sequence sets of the...

Embodiment 2

[0048] Experimental verification of the amplification primer set for detection of SARS-CoV-2 by mNGS (different amounts).

[0049] 1. Sample source.

[0050] The nucleic acid of the sample was verified as positive by the CFDA-approved in vitro diagnostic reagent (fluorescent quantitative PCR method), and the copy number of SARS-CoV-2 was about 2×10 by standard curve quantification. 6 copies / mL.

[0051] The specific calibration method is: transcribe the 509 bp partial sequence of ORF1ab of the new coronavirus in vitro, calculate the copy number of the target fragment in the purified product by the molecular weight of the sequence and the total amount of the purified product transcribed, and use a reagent with a medical device registration certificate after doubling dilution A standard curve of copy number and cycle number was established, and then the copy number in the new coronavirus positive clinical samples was calibrated.

[0052] 2. Experimental sample preparation.

[0...

Embodiment 3

[0078] Experimental verification of the amplification primer set for detection of SARS-CoV-2 by mNGS (different primer combinations).

[0079] 1. Sample source and sample preparation are the same as in Example 2.

[0080] Two, under the condition of embodiment 2 primer sets, increase or reduce the number of primer types by half, all consider uniform genome coverage when increasing or reducing, experimental design is as follows:

[0081] Table 4. Confirmation design of SARS-CoV-2 specific primer combinations

[0082]

[0083] Note: "16 specific primers" in the above table refer to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 in Table 1 of Example 1 , the sequence of 29, 31;

[0084] "32 specific primers" in the above table refers to the sequence of SEQ ID NO:1-32 in Table 1 of Example 1;

[0085] "48 specific primers" in the above table refers to the sequence of SEQ ID NO: 1-48 in Table 1 of Example 1.

[0086] Three, carry out transcription to set up lib...

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Abstract

The invention relates to an amplification primer group for detecting SARS-CoV-2 through mNGS and an application of the amplification primer group, and belongs to the technical field of gene detection.The amplification primer group comprises a random primer and an SARS-CoV-2 specific primer, the random primer is oligonucleotide of 5 bp to 7 bp, and the SARS-CoV-2 specific primer is oligonucleotideof 10 bp to 20 bp designed for an SARS-CoV-2 sequence. According to the amplification primer group, specific amplification is carried out on the novel coronavirus in a library building mode before library building, high-coverage targeted enrichment library building is carried out, and the coverage degree of pathogen detection, identification of pathogen genome mutation, the sensitivity of pathogen detection and the diagnosis capacity of novel coronavirus infection are improved.

Description

technical field [0001] The present invention relates to the technical field of gene detection, in particular to a mNGS amplification primer set for detection of SARS-CoV-2 and its application. Background technique [0002] Metagenome (mNGS) technology has gradually become a widely used technology in many aspects of discovery and translational research. It has important applications in the pathogenic diagnosis and analysis of urgent and critical infectious diseases. It can conduct unbiased analysis of all nucleic acids in samples. Sequencing, including nucleic acids of human and microbial origin. [0003] Novel coronavirus, named 2019-nCoV or SARS-CoV-2, can be transmitted through droplets and contact, and is the pathogen of easily transmitted diseases. The disease infected with SARS-CoV-2 is called COVID-19 pneumonia, and the symptoms are fever, dry cough, fatigue, etc., which are similar to ordinary pneumonia, making it difficult for clinicians to distinguish. Treatment a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/701C12Q2531/113C12Q2535/122C12Q2527/127C12Q2521/101C12Q2521/107Y02A50/30
Inventor 许腾谢淑媚陈文景曾伟奇周晓思李永军王小锐苏杭
Owner 广州微远医疗器械有限公司
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