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Rapid detection method for bacteroides based on high-throughput sequencing and application

A technology of Bacteroides and genes, applied in the field of molecular biology, to achieve accurate results

Active Publication Date: 2020-08-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is currently no effective technique for rapid species-level identification of all Bacteroidetes in complex samples.

Method used

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  • Rapid detection method for bacteroides based on high-throughput sequencing and application
  • Rapid detection method for bacteroides based on high-throughput sequencing and application
  • Rapid detection method for bacteroides based on high-throughput sequencing and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of Bacteroides-specific primers based on the high-resolution core gene of Bacteroides

[0036] 1. Through the database NCBI (National Center for Biotechnology Information) and EMBL

[0037] (European Molecular Biology Laboratory) research, 42 different Bacteroides species have completed genome sequencing, some have only one genome sequence, and some have nearly a hundred. We downloaded a total of 100 rpsD gene sequences of different species of Bacteroides (at least one sequence for each Bacteroides, and for those with more sequences, select 5 sequences with higher sequencing levels), and constructed a phylogenetic tree with MEGA software as follows: figure 1 As shown, we can see from the figure that this gene can well distinguish different species of Bacteroides.

[0038] 2. Construct the rpsD gene database, download the known rpsD gene sequences of Bacteroidetes from databases such as NCBI and EMBL, and use the downloaded sequences to construct...

Embodiment 2

[0049] Example 2: Validation of Bacteroides-specific primers at the genus level

[0050] Bifidobacterium longum, Lactobacillus breris, Lactobacillus breris, Lactobacillus fermentum, Pediococcus acidilactici, Enterococcus faecalis, Escherichia coli ( Escherichia coli), Akkermansia muciniphila, Bacteroides caccae, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides salyersiae, Bacteroides uniformis, Bacteroides ovatus, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron , Bacteroides kribbi, Bacteroides koreensis, respectively extract the genome, microbial genome extraction refers to the instructions in the bacterial genome DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Beijing, China), with sequences such as SEQ ID NO.1 and SEQ ID The primers shown in NO.2 are used for PCR amplification using the extracted genome as a template, and the amplification conditions ...

Embodiment 3

[0056] Example 3: Construction of a rapid detection method for Bacteroidetes and its verification at the species level

[0057]In order to verify the accuracy of primers SEQ ID NO.1 and SEQ ID NO.2 in the analysis of Bacteroides, we will common Bacteroides (Bacteroides vulgatus), Bacteroides salyersiae, Bacteroides dorei, Bacteroides faecis, Bacteroides uniformis (Bacteroides uniformis) , Bacteroidescaccae, Bacteroides; xylanisolvens, Bacteroides nordii, Bacteroidesstercoris, Bacteroides fragilis, a total of 10 species of Bacteroides (as shown in Table 2) genome according to the concentration of 0.001-50ng / μL Mixed, for the detection of different species of Bacteroides in simulated complex samples. Based on the newly designed Bacteroides identification primers combined with a next-generation sequencer, then perform analog sample sequencing according to the method steps described in Example 2, and finally compare the rpsD gene sequence information obtained by sequencing with th...

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Abstract

The invention discloses a rapid detection method for bacteroides based on high-throughput sequencing and application, and belongs to the technical field of molecular biology. A section of core gene fragment rpsD gene with high resolution is obtained through a molecular biology method to serve as a screening marker, high-throughput identification of the bacteroides can be achieved, the compositionof the bacteroides in a complex sample can be comprehensively identified without separation and purification, and the resolution is higher than that of a 16S rRNA gene fragment. A target strip expanded through a specific primer based on the rpsD gene is about 500bp, and compared with a traditional identification method, the identification cost of the bacteroides is saved by 50% or above. The designed primer is combined with high-throughput sequencing, the distribution of the bacteroides in a feces sample can be accurately identified, all reported bacteroides can be identified, and the identification accuracy rate of the bacteroides reaches 100%.

Description

technical field [0001] The invention relates to a rapid detection method and application of Bacteroides based on high-throughput sequencing, belonging to the technical field of molecular biology. Background technique [0002] Bacteroides are the most abundant Gram-negative bacteria in the human gut flora. As one of the typical next-generation probiotics, it encodes many genes that complement the function of the human genome. These include enhancing our ability to digest dietary fiber polysaccharides, alleviating disease, enhancing human immunity, and even enhancing the suppression of cancer, suggesting a complex relationship between Bacteroides and its host. Notably, some of these functions are related to the ecological distribution and abundance of different species of Bacteroidetes in the human gut. Therefore, it is more and more important to develop a method for rapid detection of Bacteroides species, even at the species level, to assess its diversity and composition in...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6869C12Q1/04C12R1/01
CPCC12Q1/6869C12Q1/689C12Q2535/122C12Q2531/113
Inventor 翟齐啸陈卫王晨陆文伟于雷雷田丰伟赵建新张灏
Owner JIANGNAN UNIV
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