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Gene chip for detecting gamma-proteobacteria communities in marine environment

A technology of gene chip and marine environment, applied in the field of gene chip for detecting gamma-proteobacteria community in marine environment

Inactive Publication Date: 2016-04-13
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a gene chip for detecting the γ-proteobacterial community in the marine environment, which can detect the γ-proteobacterial community in the marine environment in parallel, quickly and with high throughput, so that the gene chip technology can be better applied It is used in marine environmental monitoring to make up for the shortcomings of existing detection technologies

Method used

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  • Gene chip for detecting gamma-proteobacteria communities in marine environment
  • Gene chip for detecting gamma-proteobacteria communities in marine environment
  • Gene chip for detecting gamma-proteobacteria communities in marine environment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Design of probes for gene chips for gamma-proteobacteria community detection

[0048] High-throughput sequencing technology and clone library technology were used to obtain the community information of γ-proteobacteria in seawater of East China Sea. According to the 16S rRNA gene sequence information of the γ-proteobacteria community, the 31 main γ-proteobacteriaceae (Aeromonadaceae, Alcanivoracaceae, Alteromonadaceae, Chromatiaceae, Colwelliaceae, Coxiellaceae, Ectothiorhodospiracea, Enterobacteriaceae, Thiotrichaceae, Xanthomonadaceae, Hahellaceae, Hahellaceae, Halomonadaceae、Idiomarinaceae、Legionellaceae、Litoricolaceae、Oceanospirillaceae、Zooshikella、Vibrionaceae、Moraxellaceae、Thiotrichales_incertae_sedis、Pasteurellaceae、Piscirickettsiaceae、Pseudoalteromonadaceae、Pseudomonadaceae、Psychromonadaceae、Salinisphaeraceae、Shewanellaceae、Sinobacteraceae、Ferrimonadaceae、Francisellaceae、Methylococcaceae)进行探针设计,并在BLAST中进行 To verify, obtain the probe sequence used to pr...

Embodiment 2

[0085] Embodiment 2: Specific detection of the gamma-proteobacteria colony detection gene chip of the present invention

[0086] The representative clone Francisellaphilomiragia1-5 of Francisellaceae, a seawater dominant family Francisellaceae in γ-proteobacteria, was selected to detect the specificity of the gene chip for γ-proteobacteria community detection.

[0087] ① DNA extraction: Plasmid DNA was extracted with a plasmid extraction kit.

[0088] ②Amplification of 16SrRNA gene: The 16SrRNA gene was linearly amplified using the cloning vector pEASY-T1 universal primers M13F (GTAAAACGACGGCCAGT) and M13R (GTCCTTTGTCGATACTG). The amplification program was 94°C for 5min, 25 cycles (94°C for 30s, 55°C for 30s, 72°C for 1min30s), and 72°C for 10min.

[0089] ③Fluorescent labeling: Use fluorescently labeled random primers (Cy3-NNNNNNNNNN) and Klenow enzyme to label the 16SrRNA gene amplification products. 1.5h at 37°C, 10min at 70°C.

[0090] ④ Hybridization: 15 μL of fluoresc...

Embodiment 3

[0092] Embodiment 3: Utilize the chip to detect the gamma-proteobacteria species in the seawater sample

[0093] The seawater sample 22A was hybridized with the marine γ-proteobacteria community detection gene chip of the present invention, the specific method is as in Example 2. And compared with the results of high-throughput sequencing. The results are shown in Table 1.

[0094] Table 1 Detection results of γ-proteobacteria in seawater sample 22A

[0095]

[0096]

[0097] Note: + means detected, - means not detected.

[0098] 基因芯片检测结果表明海水样品22A中含有zooshikella、Xanthomonadaceae、Vibrionaceae、Thiotrichales_incertae_sedis、Thiotrichaceae、Sinobacteraceae、Shewanellaceae、Psychromonadaceae、Pseudomonadaceae、Pseudoalteromonadaceae、Piscirickettsiaceae、Pasteurellaceae、Oceanospirillaceae、Moraxellaceae、Methylococcaceae、Litoricolaceae、Legionellaceae、Idiomarinaceae、Halomonadaceae、Hahellaceae、 Francisellaceae, Enterobacteriaceae, Ectothiorhodospiraceae, Coxiellaceae, Colwelliaceae, Ch...

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Abstract

The invention provides a gene chip for detecting gamma-proteobacteria communities in a marine environment. The gene chip can detect the gamma-proteobacteria communities in the marine environment in a parallel, rapid and high-flux manner, so that the gene chip technology is better applied to marine environment monitoring. A probe used by the chip can detect the gamma-proteobacteria communities in seawater in the rapid and high-flux manner and can detect information of gamma-proteobacteria of 31 families to the maximum, so that powerful technical support is provided for monitoring bacterial communities in the marine environment, and a firm foundation is laid for establishing and perfecting a comprehensive evaluation system of marine environment quality of offshore areas.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a gene chip for detecting gamma-proteobacterial community in marine environment. Background technique [0002] γ-Proteobacteria is the main group of microorganisms in the marine environment. Some studies have shown that during the outbreak of some natural ecological disasters (enteromorpha), γ-Proteobacteria is the most abundant bacterial group in the corresponding coastal areas. Taking Qingdao as an example , the diversity of microbial species in the coastal environment of Qingdao during the outbreak of Enteromorpha was very high, and the gamma-proteobacteria accounted for the vast majority of the measured species, and many of them did not appear before the outbreak of Enteromorpha. This shows that γ-proteobacteria have potential environmental indicators. In addition, according to the research on the impact of bacterial community and environmental factor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C40B40/06
CPCC12Q1/689C12Q2600/166Y02A50/30
Inventor 赵维王敬敬黄志勇
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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