A kind of ngs primer set and kit for detecting respiratory virus pathogens

A technology of primer sets and kits, which is applied in the fields of life sciences and biology, can solve the problems of reduced ability to detect viral pathogens and complex nucleic acid sources, and achieve the effects of improving detection sensitivity, avoiding base preference, and simple and fast operation

Active Publication Date: 2022-05-03
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the shortcomings are also very obvious. For clinical samples, the source of nucleic acid in each sample is complex, and random primers will try to transcribe all possible RNAs, especially the ribosomal RNA in human samples, which has a high content. The ability to detect viral pathogens is reduced

Method used

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  • A kind of ngs primer set and kit for detecting respiratory virus pathogens
  • A kind of ngs primer set and kit for detecting respiratory virus pathogens
  • A kind of ngs primer set and kit for detecting respiratory virus pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: detect the specific reverse transcription primer design of flow A, flow B, syncytial virus and novel coronavirus (SARS-CoV-2)

[0043] First, download the reference genome sequences of influenza A, influenza B, syncytial virus and new coronavirus from the NCBI GeneBank nt database.

[0044]After multiple sequence alignment of these genome sequences, use PYFASTA (http: / / pypi.python.org / pypi / pyfasta / ) to cut each genome into 400-base long fragments, allowing 200 bases between these fragments base overlap. Then search for kmer (13mer) of 13 bases in the window region of 50 bases at the beginning and end of these 400-base long fragments (it can be the sense strand or the antisense strand), and all kmers are based on the appearance The frequency is sorted in descending order, and the 13mer with the highest frequency (the most conservative sequence) is selected as the candidate primer selected in the first round, and then all 500-base long fragments containing ...

Embodiment 2

[0047] Using the sensitivity reference products in the national reference products for detection of influenza A, influenza B, syncytial virus and new coronavirus nucleic acid of the National Institute of Inspection and Quarantine, the effect of using 594 virus-targeting primers and using random primers for metagenomic library sequencing was tested.

[0048] 1) Source of samples: Standard Plate of Respiratory Pathogens of China Institute of Inspection and Quarantine, and New Coronavirus Standard.

[0049] 2) Sample pretreatment method: In order to simulate the alveolar lavage fluid sample, a certain amount of human cells need to be mixed into the pure virus culture to provide the host nucleic acid background. Use 1 × 10 for the fixed value of A flow, B flow, syncytial virus and SARS-CoV-2 sensitivity reference 5 Immortalized human leukocytes / mL were diluted to 100,000 copies / mL and marked as S1; then the cells of this concentration were used to make a 2-fold concentration gradi...

Embodiment 3

[0109] NGS testing was performed on 4 real sputum / throat swab samples with nucleic acids of influenza A, influenza B, syncytial virus, and new coronavirus to verify the effect of the reverse transcription primer set.

[0110] 1) Sample source and nucleic acid extraction: sputum / throat swab samples from 4 patients with the target virus, passed through Qiagen Total RNA extracted with Viral RNA extraction kit.

[0111] 2) The experimental steps of reverse transcription and library construction are the same as in Example 2.

[0112] 3) All libraries were single-ended 75bp sequencing (SE75) using the illumina Nextseq platform, the host sequence was removed by bioinformatics analysis, and the number of reads detected by the new coronavirus and the genome comparison rate after splicing with parameters were analyzed, as follows surface:

[0113]

[0114] figure 2 is the genome coverage map of influenza A in the sample, image 3 is the B genome coverage map in the sample, Fi...

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Abstract

The invention discloses an NGS primer set and a kit for respiratory RNA pathogens (flu A, flu B, syncytial virus, and new crown). The primer set consists of 594 primer sequences. The present invention specifically designs 594 primers for influenza A, influenza B, syncytial virus and novel coronavirus genomes, covering the entire viral genome, improving the detection of viruses and greatly improving the possibility of full-length detection of viral genome sequences without PCR amplification. It can quickly achieve full coverage of the viral genome.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to an NGS primer set and a kit for detecting influenza A, influenza B, syncytial virus and the new crown genome. Background technique [0002] High-throughput sequencing technology (NGS) has the ability to achieve full coverage of pathogen genomes quickly and at low cost. The traditional method of detecting RNA pathogens uses random primers for reverse transcription of RNA, which has the advantage of low cost, and a random primer of 6 to 9 bases can cover various RNA possibilities. But the shortcomings are also very obvious. For clinical samples, the source of nucleic acid in each sample is complex, and random primers will try to transcribe all possible RNAs, especially the ribosomal RNA in human samples, which has a high content. The ability to detect viral pathogens is reduced. [0003] Among respiratory infections, AB syncytial virus and the new coronav...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6869C12N15/11
CPCC12Q1/701C12Q1/6869C12Q2600/16C12Q2521/107C12Q2535/122C12Q2537/143C12Q2525/191Y02A50/30
Inventor 杨启文贾沛瑶朱盈丁文超王珺
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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