Human respiratory virus targeted enrichment capture probe set and application thereof

A technology for targeted enrichment and capture of probes for detection, identification and assessment of respiratory viruses

Inactive Publication Date: 2021-02-09
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no relevant report on NGS diagnostic-specific enrichment probe sets for respiratory viruses

Method used

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  • Human respiratory virus targeted enrichment capture probe set and application thereof
  • Human respiratory virus targeted enrichment capture probe set and application thereof
  • Human respiratory virus targeted enrichment capture probe set and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Design of general target enrichment capture probe set for human respiratory virus

[0053] 1.1 Acquisition of Human Respiratory Virus Gene Sequence

[0054] The human respiratory virus gene sequences used herein were derived from sequence databases published on the Internet. Among them, enterovirus and rhinovirus standard strains and polymorphic sequences come from the picornavirus professional database (http: / / www.picornaviridae.com), and the genetic data of coronaviruses come from CoVDB (https: / / hsls.pitt.edu / obrc / index.php?page=URL1208462413), the genetic data of influenza virus come from GISAID (https: / / www.gisaid.org / ) and ViPR (https: / / www.viprbrc.org / brc / home.spg? decorator=vipr), and the gene sequences of other human respiratory viruses are from GenBank, EMBL and DDBJ three major nucleic acid sequence databases. See Table 1 for specific human respiratory virus species information.

[0055] Table 1. The target species of the universal target enrichm...

Embodiment 2

[0062] Example 2 Preparation of NGS library of human respiratory tract samples and hybridization of probe sets

[0063] 2.1 Nucleic acid preparation of human respiratory samples

[0064] Take 200 μl of bronchoalveolar lavage fluid sample, put it in 2ml EasyMAG automatic nucleic acid extraction system sample lysate, use EasyMAG automatic nucleic acid extraction instrument to extract total nucleic acid, and operate according to the equipment instructions. The elution volume was 60 μl, and the extracted nucleic acid was aliquoted and stored at -80°C.

[0065] 2.2 Reverse transcription of human respiratory sample nucleic acid

[0066] (1) Nucleic acid reverse transcription

[0067] Nucleic acid reverse transcription system using SuperScript TM III reverse transcriptase (Thermo, USA). Take 6 μl of the total nucleic acid prepared in the above step 2.1 for reverse transcription, the system and reaction conditions are as follows:

[0068] DEPC water 5μl dNTP (10...

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Abstract

The invention relates to the field of human respiratory virus diagnosis, and provides a method for generating a targeted enrichment capture probe set for human respiratory viruses. The method comprises the following steps of acquiring a gene sequence of a virus infecting a human respiratory tract; designing a probe through a sliding window method, and performing exhaustion of all possible nucleicacid sequences; filtering the obtained nucleic acid sequences; filtering out sequences highly homologous with the human genome in a nucleic acid sequence library; and clustering the probe sequences. The invention also relates to application of the probe set obtained through the method to preparation of a next-generation sequencing library of human respiratory viruses, so that whole genome sequenceinformation is obtained, the genome variation is identified, potential epidemic propagation characteristics of viruses are evaluated, and early warning and prevention and control of respiratory diseases are guided.

Description

technical field [0001] The invention relates to a human respiratory virus targeted enrichment capture probe set and its use for detecting, identifying and evaluating respiratory viruses. Background technique [0002] Human respiratory virus is an important pathogen that causes human infection, and it is also the main pathogen of new and sudden infectious diseases. Viruses are prone to mutations, which may lead to changes in virulence, drug resistance, or transmission capabilities, and thus cause major public health problems due to rapid spread among the population. Rapid and accurate detection and identification of pathogens, especially obtaining their genome sequence characteristics, is the key to discovering whether there are mutations and new viruses, and then evaluating the potential epidemic characteristics of the virus, which is of great significance for the early warning and prevention of infectious diseases. [0003] The common respiratory viruses that cause human r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6811
CPCC12Q1/6811
Inventor 任丽丽王健伟曹德盼肖艳朱俊琳
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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