Capture probe set for detecting food allergens as well as preparation method and application of capture probe set

A technology for capturing probes and allergens, applied in the field of capturing probe sets, can solve the problems of detecting allergenic components and allergenic pollutants, and achieve the effect of ensuring richness, high application value, and ensuring capture efficiency

Pending Publication Date: 2021-12-07
ZHEJIANG GONGSHANG UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are not capable of high-throughput detection of allergenic ingredients and allergenic pollutants in highly processed complex foods

Method used

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  • Capture probe set for detecting food allergens as well as preparation method and application of capture probe set
  • Capture probe set for detecting food allergens as well as preparation method and application of capture probe set
  • Capture probe set for detecting food allergens as well as preparation method and application of capture probe set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Conventional allergen detection methods (ELISA, RT-PCR, and LC-MS) cannot achieve high-throughput detection of known allergens and screening of unknown allergens in complex food systems.

[0021] This example proposes a method for preparing a capture probe set for detecting food allergens. Due to the ultra-high sequencing capability of next-generation sequencing, multiple different genomic regions can be analyzed simultaneously to obtain genomic mutations, insertions, Deletion and structural variation, so that all plants, animals and fungi in complex food systems can be identified. This example is based on next-generation sequencing of SNP (single nucleotide polymorphism). The target sequence targeted sequencing technology is a strategy for capturing, enriching and sequencing target genes. The target gene can be captured and enriched through the DNA hybridization reaction between the probe and the target gene, which can effectively reduce the cost of sequencing and impro...

Embodiment 2

[0029] This example proposes a capture probe set for high-throughput sequencing to detect food allergens, including the capture probe set prepared by the preparation method described in Example 1. More specifically:

[0030] The capture probe set has 7482 single-stranded probes, the length of the single-stranded probes is 100-130 bp, and the CG content of the single-stranded probes is 30-70%.

[0031] The nucleotide sequences of the single-stranded probes are shown in SEQ ID NO: 1 to SEQ ID NO: 7482.

[0032] A set of capture probes proposed in this example is shown in Table 1 below.

[0033] Table 1: Allergen gene trap probe sets.

[0034]

[0035]

Embodiment 3

[0037] refer to figure 1 First, design probe sets based on allergen gene fragments obtained from the International Union of Immunization Union (IUIS) and the World Health Organization (WHO) website (WHO / IUIS, www.allergen.org); secondly, construct a sample library ; Third, use the probe set to capture and enrich the allergen gene in the sample library, and perform sequencing to detect the allergen.

[0038] This embodiment proposes an application of a capture probe set for detecting food allergens, including high-throughput sequencing for detecting food allergens, which specifically includes the following steps,

[0039] Synthesis of biotin-labeled probes was commissioned by Sangon Bioengineering (Shanghai) Co., Ltd.;

[0040] Sequencing library construction: Ultrasonic fragmentation of sample DNA, the fragmented DNA is purified by magnetic beads, followed by end repair, connection of sample adapters, purification of ligation products, library amplification, and library purif...

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Abstract

The invention discloses a capture probe set for detecting food allergens as well as a preparation method and application of the capture probe set. The preparation method comprises the following steps of comparing gene segments of at least two allergens in pairs according to sequence similarity; classifying genes with the lengths of more than 120bp and the similarity of not less than 90% among allergen genes into one type; carrying out classification treatment to finally obtain at least one type of target genes, and carrying out multi-sequence comparison on the target genes by utilizing clustalw2 software; obtaining a consensus sequence of each type of target genes as a candidate probe by using a BioPerl module of a Perl language; and for each candidate probe, sequentially intercepting a sequence with the length of 100-130bp, and taking the probe which has the CG content of 30-70% and does not contain simple repetitive sequences as a single-stranded probe. The invention has the beneficial effects that high-throughput detection of sensitization components and sensitization pollutants in a complex food system can be realized, and the application value is very high.

Description

technical field [0001] The invention relates to the technical fields of gene detection technology and allergen detection, in particular to a capture probe set for high-throughput sequencing detection of food allergens, its preparation method and application. Background technique [0002] In recent years, food allergy has become an important public health problem that governments and people in various countries have paid more and more attention to. According to surveys, up to 10% of the world's population is allergic to food. Food allergens are usually proteins with a molecular weight of 5kDa to 70kDa. So far, hundreds of proteins have been identified and named as allergens by the International Union of Immunology (IUIS) and the World Health Organization (WHO). Food allergy usually leads to severe acute systemic allergic reactions in patients, including asthma, urticaria, gastrointestinal spasm, hypotension, shock, and even life-threatening, seriously affecting the health and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806G16B25/20C12N15/11G16B30/10G16B50/30
CPCC12Q1/6806G16B25/20G16B30/10G16B50/30C12Q2565/519C12Q2523/301C12Q2523/308C12Q2525/191C12Q1/6811C12Q1/6895C12Q2537/143C12Q2537/165
Inventor 傅玲琳王彦波周瑾茹彭海
Owner ZHEJIANG GONGSHANG UNIVERSITY
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