Method for detecting and identifying subspecies of fusobacterium nucleatum

A subspecies, nucleotide sequence technology, applied in the field of biomedicine, can solve the problems of inability to reach the subspecies level, lack of accuracy, and technical limitations of isolation and culture

Pending Publication Date: 2021-06-18
SHANGHAI TENTH PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some studies have developed a method based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), but this method requires specific expensive equipment, and the accuracy (about 80%) is somewhat lacking.
These methods can only be used after successful isolation and culture, but for samples containing a large number of mixed bacteria, such as feces and intestinal tissue, the isolation and culture technology still has great limitations, making the isolation and culture based on such samples require a lot of manpower Material investment
In addition, high-throughput sequencing technologies such as 16S rRNA gene amplicon sequencing and metagenomic sequencing commonly used in such samples cannot yet achieve subspecies identification.
[0004] Therefore, there is still a lack of an accurate and intuitive detection method to distinguish the different subspecies of Fusobacterium nucleatum

Method used

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  • Method for detecting and identifying subspecies of fusobacterium nucleatum
  • Method for detecting and identifying subspecies of fusobacterium nucleatum
  • Method for detecting and identifying subspecies of fusobacterium nucleatum

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Obtaining of subspecies-specific DNA sequences

[0043] In this example, first, the subspecies-specific DNA sequence of Fusobacterium nucleatum was obtained by comparative genomics. The specific method is to download the sequenced Fusobacterium nucleatum strains from the NCBI public database (such as image 3 shown) genomes, including completed maps and drafts; kSNP software was used to construct a genome-wide phylogenetic tree to determine the strains belonging to different subspecies; PGAP software was used to perform pan-genome analysis on all included genomes. From the obtained gene clusters, genes that are conserved in all strains of a certain subspecies of Fusobacterium nucleatum but absent in other subspecies are selected. Compare the corresponding gene sequence with the NCBI nucleotide database to observe whether there are homologous sequences in other subspecies and species other than Fusobacterium nucleatum, and confirm whether it is truly specific...

Embodiment 2

[0049] Example 2 Example of the application of subspecies-specific primers in a single strain sample

[0050] extract as figure 1 Total DNA of known Fusobacterium nucleatum strains of the subspecies listed in . PCR amplification was performed using the subspecies-specific primers in Table 1. In addition, human whole blood DNA was used as negative control for PCR, and 16S rRNA gene amplification primers (27F and 1429R) were used as positive control for bacterial DNA template. Agarose gel electrophoresis results ( figure 1 ) shows that each specific primer has a single specific amplification band at the corresponding position in the corresponding subspecies of Fusobacterium nucleatum strains, and there is no amplification in other subspecies and other Fusobacterium strains. In the detection of 10 Fusobacterium nucleatum strains covering 4 subspecies, the detection results were completely consistent with the strain subspecies classification, and the accuracy was 100%.

Embodiment 3

[0051] Example 3 Application example of subspecies-specific primers in samples containing mixed bacteria

[0052] Take the tumor tissue of 20 colorectal cancer patients as an example. Tissue DNA was extracted and PCR amplified using the subspecies-specific primers in Table 1. In addition, a universal primer for the V6 region of the 16S rRNA gene was used as a DNA template positive control. The results of agarose gel electrophoresis were as follows: figure 2 shown. The results of 6 pairs of primers in nucleatum subspecies were different, but they were relatively consistent, and the results of N3-F / R and N4-F / R were completely consistent. The positive amplification of other primers could not completely cover the strains with positive amplification of N3-F / R and N4-F / R, it is speculated that they may have differences in sensitivity under the background of complex flora; another individual primer (N1 -F / R and N5-F / R) were positively amplified in strains where other primers wer...

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Abstract

The invention relates to the technical field of biological medicines, in particular to a method for detecting and identifying subspecies of fusobacterium nucleatum. The fusobacterium nucleatum is closely associated with various diseases, and recent research shows that the fusobacterium nucleatum can promote generation and development of colorectal tumors. However, an accurate and visual detection method for distinguishing different subspecies of the fusobacterium nucleatum is lacked at present. Different subspecies of the fusobacterium nucleatum are different in biological characteristic, colonization and pathogenicity, so that accurate subspecies identification has far-reaching significance for knowing the pathogenic mechanism of the fusobacterium nucleatum. According to the invention, a group of specific DNA sequences for identifying the fusobacterium nucleatum subspecies are provided, corresponding detection primers are designed, and the application of the detection primers in subspecies level detection and identification of the fusobacterium nucleatum is explained. According to the invention, the composition and the source of the subspecies of the fusobacterium nucleatum in the focus can be disclosed, and the relationship between different subspecies and diseases and related epidemiological characteristics can be explored.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to a subspecies detection and identification method of Fusobacterium nucleatum, in particular to a set of Fusobacterium nucleatum subspecies-specific DNA sequences, and describes its function in Fusobacterium nucleatum Applications in subspecies level detection and identification. Background technique [0002] Fusobacterium nucleatum (Fusobacterium nucleatum) is a common colonizing bacterium in the oral cavity, which is involved in a variety of oral diseases under certain conditions, such as periodontitis and oral tumors. Fusobacterium nucleatum can also spread to other body parts other than the oral cavity, and cause a variety of diseases related to bacterial infection, including coronary heart disease, liver abscess, inflammatory bowel disease, premature birth and other pregnancy complications. In particular, it has recently been found that there is a high abundance o...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686
Inventor 秦环龙蔚青毕德玺朱崟
Owner SHANGHAI TENTH PEOPLES HOSPITAL
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