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Primer group and method for rapidly detecting fusobacterium nucleatum of excrement and application of primer group and method

A technology of Fusobacterium nucleatum and detection method, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of false positives and false negatives, and achieve high sensitivity, good specificity, and easy and fast operation. Effect

Inactive Publication Date: 2019-04-26
JIANGXI PRECISION GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the whole gene probe is prone to cross-reaction with other homologous bacteria (such as Escherichia coli, Klebsiella pneumoniae, etc.) in the detection of Fusobacterium nucleatum, and it is easy to produce false negative or false positive results in previous applications

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 is tested bacterial strain source

[0036]The Fusobacterium nucleatum needed for the research comes from the stool samples of the subjects, and the subjects have not taken antibiotics and similar drugs within three months before the collection of the stool samples, refer to Changanyi TM Instructions for the stool collection box, collect the stool samples of the subjects and store them in the laboratory at 2-8°C for no more than 24 hours.

Embodiment 2

[0037] Embodiment 2 primer sequence selection and identification

[0038] According to the bacterial name, the bacterial 16S rRNA sequence was downloaded from the NCBI website (https: / / www.ncbi.nlm.nih.gov / nuccore / ), and then the bacterial-specific primers were designed using the primer design software Primer Primer 5.

[0039] Table 1. Primer information

[0040] Primer

serial number

Sequence 5'-3'

F-P1F

SEQ ID NO.1

TCAGTGTCTTCGGGGAAACC

F-P1R

SEQ ID NO.2

CCCCACCTTCCTCCTACTCA

F-P2F

SEQ ID NO.3

CAGTGTCTTCGGGGAAACCT

F-P2R

SEQ ID NO.4

ACCCATTGTAGCACGTGTGT

F-P3F

SEQ ID NO.5

TCCACGCCGTAAACGATGAT

F-P3R

SEQ ID NO.6

GGTTTCCCCGAAGACACTGA

P-P4F

SEQ ID NO.7

CTGGGGAGTACGTACGCAAG

P-P4R

SEQ ID NO.8

AGGTTTCCCCCGAAGACACTG

P-P5F

SEQ ID NO.9

TCGGGGAAACCTAAAGACAGG

P-P5R

SEQ ID NO.10

TCCACTTCACAGCTTTGCGA

P-P6F

SEQ ID NO.11...

Embodiment 3

[0043] Example 3 V3-V4 primer design and specificity verification

[0044] The microbial 16S rRNA sequence contains 9 variable regions and 10 highly conserved regions. In order to ensure the specificity of the primers, the primers were designed in the conserved regions. Compared with other variable regions, the V3-V4 regions can cover more than 98% of the Bacteria, and the length of the amplified product in the V3-V4 region meets the length requirements of qPCR for the expected product of the primer; on this condition, a pair of primers that meet the requirements are obtained by referring to a large number of literatures, and then the specificity of the primers is verified.

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Abstract

The invention discloses primer group information of fusobacterium nucleatum, a quantitative PCR (Polymerase Chain Reaction) detection method and application of primer group and method. According to a16S rRNA (ribosomal Ribonucleic Acid) nucleotide sequence and a BLAST (Basic Local Alignment Search Tool) function of bacteria in an NCBI (National Center of Biotechnology Information) database, a fusobacterium nucleatum related primer and V3 to V4 primers are designed according to primer design software Primer Primer 5, and the primers are identified. Genome information of total bacteria is extracted from an excrement sample; the qPCR (quantitative Polymerase Chain Reaction) is carried out through utilizing the primers and the relative content of the fusobacterium nucleatum is calculated andobtained. The detection method is used as a principle and an intestinal micro-ecological detection kit is prepared by utilizing the fusobacterium nucleatum primer. A kit is used for carrying out actual detection and a result shows that the content of the fusobacterium nucleatum in the excrement samples of an intestinal micro-ecological patient and a patient with intestinal cancer is remarkably different from that of normal people. A specific primer of the fusobacterium nucleatum, a rapid and accurate detection method and research and development of an intestinal micro-ecological kit provide methods for knowing the distribution of an intestinal micro-ecology and predicating and primarily screening diseases.

Description

technical field [0001] The invention belongs to the technical field of biomedical detection, and is dedicated to inventing a method for quickly identifying Fusobacterium nucleatum in feces, and relates to a primer set of Fusobacterium nucleatum in intestinal tract, a molecular detection method and its application. Background technique [0002] Fusobacterium nucleatum belongs to Gram-negative obligate anaerobic bacteria, which is not only related to oral diseases, but also involved in sepsis, pelvic inflammatory disease, abscesses in brain, liver, lung, spleen and other organs, and colorectal cancer biological process. Fusobacterium nucleatum has a variety of agglutinating proteins that can agglutinate red blood cells and adhere to the surface of epithelial cells and hydroxyapatite. It can not only co-polymerize with early colonizing bacteria such as actinomycetes, but also co-polymerize with late colonizing bacteria such as Porphyromonas gingivalis, Streptococcus sanguis, H...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/11C12R1/01
CPCC12Q1/6851C12Q1/689
Inventor 朱永亮穆延召杨敏靖星宇吴楠王一伟
Owner JIANGXI PRECISION GENE CO LTD
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