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Molecular target for screening flavobacterium and quantitative detection method for flavobacterium

A technology for screening Flavobacterium and molecular targets, applied in the field of microbial inspection, can solve the problems of reporting and difficulties in molecular detection targets of Flavobacterium

Active Publication Date: 2021-09-10
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, PCR detection methods at home and abroad only report molecular detection targets for detection within the genus Flavobacterium. etc., there is no molecular detection target report for Flavobacterium
Moreover, many pathogenic bacteria within the genus often participate in the contamination process of Flavobacterium, and it is still difficult to detect a certain Flavobacterium alone to prevent its contamination.
With the continuous discovery of new species of Flavobacterium, the precise identification of molecular targets based on existing Flavobacterium species is facing great challenges

Method used

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  • Molecular target for screening flavobacterium and quantitative detection method for flavobacterium
  • Molecular target for screening flavobacterium and quantitative detection method for flavobacterium
  • Molecular target for screening flavobacterium and quantitative detection method for flavobacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Screening to obtain the specific molecular target of Flavobacterium

[0030] According to the genome data of 271 strains of Flavobacterium and 40 strains of non-Flavobacteria in the NCBI website, pan-genome analysis was performed; Specific gene fragments of common Flavobacteria such as Flavobacterium sinensis, Flavobacterium meningitidis, Flavobacterium brevis, Flavobacterium halophilum, Flavobacterium psychrophilus), the nucleotide sequence of the gene fragment is as SEQ ID NO: 1.

[0031] The specific molecular targets of the Flavobacterium genus obtained by screening were all from Flavobacterium columnar 94-081, and the corresponding gene loci of the target gene fragment sequences are shown in Table 1 below.

[0032] Table 1 Genomic loci of specific molecular targets of Flavobacterium

[0033]

Embodiment 2

[0034] Embodiment 2 identifies the PCR method of Flavobacterium

[0035] 1) Primer design

[0036] Specific PCR amplification primers (including forward primers and reverse primers) were designed according to the sequence SEQ ID NO: 1 in Example 1, and the primer set sequence is shown in Table 2.

[0037] Table 2 Specific PCR detection primer set

[0038]

[0039] 2) the method for identifying Flavobacterium, the steps are as follows:

[0040] S1 DNA template preparation: the strains to be tested were enriched and cultured in LB liquid medium, and the bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit as the template to be tested;

[0041] S2 PCR amplification: use the primer set 1 to perform PCR amplification of the sample DNA to be tested

[0042] ①PCR detection system:

[0043]

[0044] ②PCR amplification program:

[0045]

[0046] S3: Take the PCR amplification product for gel electrophoresis;

[0047] S4: Observe whe...

Embodiment 3

[0048] The specificity evaluation of embodiment 3 Flavobacterium PCR detection method

[0049] Take 5 strains of Flavobacterium and 44 strains of non-target Flavobacterium, and perform PCR detection according to the method in Example 2. Wherein, the S1 DNA template is prepared by extracting the genomic DNA of each bacterium; the S2 PCR amplification uses primer set 1. A blank control is set, and the template of the blank control is an aqueous solution without genome.

[0050] The strains and test results of the bacteria used are shown in Table 3 below. In the table, "+" in the test result column indicates positive, and "-" indicates negative. The results of electrophoresis of PCR products were as follows: figure 1 Shown; numbers 1 to 5 in the figure are 5 species of Flavobacteria; numbers 6 to 49 are non-target Flavobacteria; M is 2000Maker.

[0051] Table 3 Flavobacterium identification specificity evaluation test result of the present invention

[0052]

[0053]

...

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Abstract

The invention provides a molecular target for screening flavobacterium. The molecular target is a nucleotide fragment, wherein a sequence of the nucleotide fragment is as shown in SEQ ID NO:1. The invention also provides a pair of primers for detecting the molecular target and a corresponding PCR detection method. According to the detection method, more flavobacterium can be detected, the coverage rate is higher, and the practicability is enhanced; and the detection method disclosed by the invention has the advantages of simplicity in operation, easiness in result judgment, short detection time, strong specificity, low cost and good stability for flavobacterium detection.

Description

technical field [0001] The invention belongs to the technical field of microbial inspection, relates to a method for identifying Flavobacterium, in particular to a molecular target for screening Flavobacterium and a quantitative detection method thereof. Background technique [0002] Flavobacterium belongs to the Flavobacteriaceae family and is a class of Gram-negative bacilli with elongated, non-motive, non-capsulated, and non-spore-forming bacteria. Common Flavobacteria include: Flavobacterium columnare, Flavobacterium johnsoniae, Flavobacterium hydatis, Flavobacterium aquatile, Flavobacterium sasangense, succinic acid Flavobacterium succinicans, Flavobacterium sinopsychrotolerans, Flavobacterium meningitidis, Flavobacterium breve, Flavobacterium halophilus, Flavobacterium psychrophilum ). The genus Flavobacterium widely exists in water and soil, and is associated with Pseudomonas, Micrococcus, Alcaligenes, Lactobacillus, Bacillus, Clostridium, Enterococcus, Escherichia ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/6851C12Q1/06C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/686C12Q1/6851C12Q2565/125C12Q2531/113C12Q2545/114C12Q2563/107Y02A50/30
Inventor 吴清平周宝青叶青华刘振杰李凡尚玉婷相欣然王楚芳张菊梅丁郁陈谋通薛亮王涓吴诗曾海燕蔡淑珍万强
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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