A method and application for detecting covid-19 based on mngs

A COVID-19, purpose-based technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve problems such as reducing the enrichment effect of mutated virus sequences, false negatives, etc. The effect of recording efficiency, increasing extension length and improving enrichment efficiency

Active Publication Date: 2022-01-25
天津金匙医学科技有限公司
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AI Technical Summary

Problems solved by technology

The mutation speed of the new coronavirus is fast, and the sequence diversity is very rich. New virus subtypes are constantly being discovered and uploaded around the world. So far, NCBI has collected 50,326 new coronavirus gene sequences, which are only designed for a fixed genome sequence. Primers will reduce the enrichment effect of mutant virus sequences, resulting in false negatives

Method used

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  • A method and application for detecting covid-19 based on mngs
  • A method and application for detecting covid-19 based on mngs
  • A method and application for detecting covid-19 based on mngs

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0066] Experimental Example 1 Primer Design

[0067] 1. Generate multiple genomes

[0068] First download more than 100 COVID-19 novel coronavirus genomes from the NCBI website, and use fast Fourier transform (MAFFT) (v.7.388) to match and align all the downloaded genomes; Note, to ensure the mutation detection rate of subsequent primers , where the source sequences of multiple genomes must be at least greater than 100.

[0069] 2. Candidate primer design.

[0070] Use PYFASTA software to segment the matched multiple genomes into 394nt short fragments with 197nt overlap, and intercept 50nt at both ends of each fragment as the primer design region, design multiple 13nt forward or reverse primers to form the region The primer set to be merged, and then all the primers in the primer set to be merged in each region are sorted from high to low frequency, select the primer with the highest frequency and does not contain uncertain bases, and then delete the primer sequence All the...

Embodiment 1

[0120] Example 1 Experimental verification of reverse transcription primer set

[0121] 1. Sample source

[0122] The new coronavirus pseudovirus used in the preparation of the positive reference product was purchased from Shanghai Yisheng, using a retroviral vector loaded with the partial sequence of the COVID-19 virus ORF1 a / b gene, the entire sequence of the E gene and the N gene coding region, with a length of 2000bp . It was verified positive by in vitro diagnostic reagents (fluorescent quantitative PCR method), and the standard curve was quantified as 2*10^8copies / mL. The cells used were purchased from Nanjing Kebai, and the cell pellet was 10^6 / tube.

[0123] 2. Experimental sample preparation

[0124] Since the detection limit of metagenomic detection of RNA viruses in clinical respiratory samples is expected to be 1000 copies / mL, the concentration of COVID-19 for preparing positive samples is 1×10^3 copies / mL. In order to simulate the host content of alveolar lavag...

Embodiment 2

[0136] Example 2: Experimental verification of the reverse transcription primer set (the effect of the primer set on detection enhancement).

[0137] 1. The sample source is the same as in Example 1.

[0138] 2. Experimental sample preparation

[0139] Prepare positive samples P1 and P2 with concentrations of 1×10^3copies / mL and 1×10^4copies / mL of COVID-19 pseudovirus respectively. In order to simulate the host content of alveolar lavage fluid, cultured human cell lines were added to P1 and P2 to make the cell concentration 10^5Cell / mL. And prepare a negative sample N1 without pseudovirus.

[0140] 3. Experimental method

[0141] The experimental scheme selected in Example 1 was adopted, that is, after ribosomal RNA was removed, full-length reverse transcription was performed using specific primers and random primers, and then the double-stranded cDNA was digested and interrupted to build a library, compared with and without COVID-19 The number of reads detected by multipl...

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Abstract

The present invention provides a mNGS-based method for detecting COVID‑19 novel coronavirus and its application. In the method, multiple genome-specific reverse transcription primer sets for COVID‑19 novel coronavirus are designed and prepared to improve the detection of novel coronavirus RNA. Reverse transcription efficiency reduces the problem of aerosol pollution, and also improves the ability to enrich nucleic acid sequences of different mutant viruses.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a method for detecting COVID-19 based on mNGS and its application. Background technique [0002] The symptoms of novel coronavirus pneumonia are similar to those of ordinary pneumonia, so rapid and accurate diagnostic techniques play a vital role in the treatment of patients. At present, the mainstream detection method for disease diagnosis and epidemic screening is real-time fluorescent RT-PCR detection technology. This technology is simple to operate, short detection time, and high sensitivity. It is the first choice for rapid diagnosis and large-scale population screening. However, this technology also has drawbacks. The current commercial kits usually target two specific gene loci of the new coronavirus and can only detect two short sequences. The nucleic acid of the new coronavirus is RNA, which is easier to degrade than DNA. After the virus dies and splits, the in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C40B50/06
CPCC12Q1/701C12Q1/6844C40B50/06C12Q2600/16C12Q2521/107C12Q2537/143Y02A50/30
Inventor 王棪梁永李玉龙李立锋蒋智
Owner 天津金匙医学科技有限公司
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