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Method for carrying out molecular detection on fusobacterium or carrying out classified authentication on culture level of fusobacterium

A molecular detection technology for Fusobacterium, applied in the field of biomedicine, which can solve the problems of non-specific amplification, lack of a method for classification and identification of strains for molecular detection of Fusobacterium, and inability to exclude interference from other strains.

Active Publication Date: 2020-06-09
SHANGHAI TENTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although some studies have established a qPCR quantitative method for Fusobacterium nucleatum, and based on this, it has been confirmed that Fusobacterium nucleatum is closely related to colorectal cancer, but the primers used are not specific for Fusobacterium nucleatum, and still have non-specificity for other species of Fusobacterium nucleatum. Amplification, interference from other strains cannot be ruled out
Therefore, there is still a lack of effective molecular detection and species-level classification and identification methods for Fusobacteria in microecological samples.

Method used

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  • Method for carrying out molecular detection on fusobacterium or carrying out classified authentication on culture level of fusobacterium
  • Method for carrying out molecular detection on fusobacterium or carrying out classified authentication on culture level of fusobacterium
  • Method for carrying out molecular detection on fusobacterium or carrying out classified authentication on culture level of fusobacterium

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Embodiment 1

[0045] This embodiment provides the application of the above-mentioned method for molecular detection of Fusobacterium or classification and identification of its species level in the sequenced genome.

[0046] The whole-genome sequencing data of Fusobacterium strains were downloaded from the NCBI genome database, and the strains with wrong species names in the database were corrected according to the ANIb value between the whole genomes. Among them, Fusobacterium nucleatum was classified at the subspecies level; Fusobacterium naviforme and genomes that did not meet the RefSeq criteria were not included in the analysis. Furthermore, the complete sequence of rpoB gene and the fragment corresponding to rpoB_subregion_3 were extracted from each genome, and the phylogenetic tree was established with MEGA software after multiple sequence alignment by MUSCLE software.

[0047] like figure 1 As shown, it shows that the fragment corresponding to rpoB_subregion_3 can distinguish Fusob...

Embodiment 2

[0049] This embodiment detects the amplification specificity of the primers shown in SEQ ID NO.4 to SEQ ID NO.5 in a single isolate of Fusobacterium and clinical samples.

[0050] Since the primer sequence provided by the present invention has degenerate bases, it needs to effectively amplify different Fusobacterium species in the same genus, so different Fusobacterium strains are used to test its versatility. DNA extracted from stool samples of colorectal cancer patients was also tested for positive amplification.

[0051] like figure 2 As shown, the primers provided by the present invention all obtain specific bright bands with uniform fragment lengths in different single isolates of Fusobacterium, which fully verifies the good universality of the primers. In stool samples, specific bands with uniform fragment lengths can also be amplified, and the negative amplification was negative in the negative control using human blood DNA as a template, indicating that the primers h...

Embodiment 3

[0053] This example provides the application of the above method in the feces of 20 patients with colorectal cancer, so as to verify the reliability of the method.

[0054] The preoperative feces of 20 patients with primary colorectal cancer were collected, amplicon sequencing was performed according to the method of the present invention, and the data were analyzed.

[0055] like image 3 As shown, the presence of Fusobacterium was detected in a total of 12 samples. In these samples, 96% of the reads (sequenced fragments) obtained by amplification and sequencing were Fusobacterium-specific target sequences, and only 4% of the reads is a non-specific sequence. The resulting sequences could be combined into 18 OTUs belonging to 16 Fusobacterium species / subspecies, of which 8 were known and 8 were unknown. Analysis of the Fusobacterial composition in each sample revealed that the Fusobacterial population in the feces of these patients was often dominated by a specific species....

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Abstract

The invention provides a method for carrying out molecular detection on fusobacterium or carrying out classified authentication on a culture level of the fusobacterium. By taking a rpoB gene as a detection and the classified authentication target of the fusobacterium, the method comprises the following steps of: amplifying the gene of a sample; carrying out high-throughput sequencing and sequencecomparison and analysis on an amplicon, and establishing an OTU (Operational Taxonomic Unit) by obtained data according to 100% consistency; and comparing an above OTU sequence with a sequence corresponding to a characteristic sequence segment in a fusobacterium whole genome, after a non-specific OTU sequence without a similarity is rejected, jointly establishing an evolutionary tree, and annotating a culture corresponding to each OTU according to an evolutionary relationship so as to realize the classified authentication of a fusobacterium group in a sample and the abundance quantification ofdifferent cultures. The method disclosed by the invention is combined with a high-throughput sequencing technology to measure an amplicon sequence obtained after a specific primer is amplified, and through a method of evolutionary tree construction, fusobacterium cultures even subspecies in the sample can be authenticated so as to be favorable for disclosing a culture level structural compositionof the fusobacterium in a micro-ecology sample.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for molecular detection of Fusobacterium or classification and identification of its strain level. Background technique [0002] Fusobacterium, a group of strictly anaerobic, Gram-negative bacteria, are commensal to humans and commonly colonize the oral cavity. Some Fusobacterium species are opportunistic pathogens and in some cases can cause oral diseases such as periodontitis. Fusobacteria can also cause infections in other parts of the body. Recent studies have shown that Fusobacterium is also associated with the development of colorectal cancer. 16SrRNA gene sequencing showed that the abundance of Fusobacterium in the feces of colorectal patients was significantly higher than that of normal people; cell and mouse experiments also showed that Fusobacterium nucleatum, the representative strain of Fusobacterium, can promote the occurrence of colorectal cancer develop and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/06
CPCC12Q1/6851C12Q1/689C12Q2537/165C12Q2535/122C12Q2531/113Y02A90/10
Inventor 毕德玺秦环龙蔚青朱崟张扬
Owner SHANGHAI TENTH PEOPLES HOSPITAL
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