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Gene chip for detecting beta-proteobacteria communities in marine environment

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

Inactive Publication Date: 2016-10-26
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Design of probes for gene chips for the detection of β-proteobacterial communities

[0025] The community information of β-proteobacteria in seawater of East China Sea was obtained by high-throughput sequencing technology and clone library technology. According to the 16S rRNA gene sequence information of the β-proteobacteria community, probes were designed for 31 major β-proteobacteriaceae (Alcaligenaceae, Burkholderiaceae, Comamonadaceae, Hydrogenophilaceae, Methylophilaceae, Neisseriaceae, Nitrosomonadaceae, Oxalobacteraceae, Rhodocyclaceae) in the seawater of the East China Sea, and Verify in BLAST to obtain the probe sequence used to prepare the gene chip.

[0026] From the perspective of detection specificity, the applicant screened the above-mentioned sequences that may be used as probes, and finally determined the following probe sequences:

[0027] Wherein the nucleic acid probe for detecting Alcaligenaceae microorganisms has a nucleotide sequence o...

Embodiment 2

[0040] Embodiment 2: the specific detection of the β-proteobacteria colony detection gene chip of the present invention

[0041] The representative clone UnculturalComamonadaceae bacterium clone 2-8 of the seawater dominant family Comamonadaceae in β-proteobacteria was selected to detect the specificity of the gene chip for β-proteobacteria community detection.

[0042] ① DNA extraction: Plasmid DNA was extracted with a plasmid extraction kit.

[0043] ② 16S rRNA gene amplification: The 16S rRNA gene was linearly amplified using the cloning vector pEASY-T1 universal primers M13F (GTA AAA CGACGG CCA GT) and M13R (GTC CTT TGT CGA TAC TG). The amplification program was 94° C. for 5 min, 25 cycles (94° C. for 30 s, 55° C. for 30 s, 72° C. for 1 min and 30 s), and 72° C. for 10 min.

[0044] ③ Fluorescent labeling: Use fluorescently labeled random primers (Cy3-NNN NNN NNN) and Klenow enzyme to label the 16SrRNA gene amplification product. 1.5h at 37°C, 10min at 70°C.

[0045] ④ ...

Embodiment 3

[0047] Example 3: Detection of β-proteobacteria species in seawater samples using chips

[0048] The seawater sample 22A was hybridized with the marine β-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.

[0049] Table 1 Detection results of β-proteobacteria in seawater sample 22A

[0050]

[0051] The gene chip detection results showed that the β-proteobacteria contained in seawater sample 22A included Burkholderiaceae, Comamonadaceae, Methylophilaceae, Nitrosomonadaceae, Oxalobacteraceae; the high-throughput sequencing results showed that seawater sample 22A contained β-proteobacteria including Comamonadaceae, Methylophilaceae, Neisseriaceae, Nitrosomonadaceae, Rhodocyclaceae. The detection result of the gene chip of the present invention is generally consistent with the result of high-throughput sequencing, indicat...

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Abstract

The invention provides a gene chip for detecting beta-proteobacteria communities in a marine environment. The gene chip comprises a chip carrier and a probe which is fixed on the chip carrier and used for detecting Alcaligenaceae bacteria, Burkholderiaceae bacteria, Comamonadaceae bacteria, Hydrogenophilaceae bacteria, Methylophilaceae bacteria, Neisseriaceae bacteria, Nitrosomonadaceae bacteria, Oxalobacteraceae bacteria and Rhodocyclaceae bacteria in seawater. The probe used for the chip can realize rapid and high-throughput detection of beta-proteobacteria communities in seawater, and can detect up to 9 beta-proteobacteria families' information. Therefore, the invention provides powerful technical support for monitoring bacterial communities in the marine environment and has laid a solid foundation for establishing and improving an offshore and marine environmental quality comprehensive assessment system.

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] β-proteobacteria and α, γ, δ, ε-proteobacteria belong to the same phylum Proteobacteria. Among them, α-proteobacteria and γ-proteobacteria are the most common in the marine environment, and β-proteobacteria are relatively rare, but their functions should not be underestimated. When Lee et al. used Fish / MAR technology to study phosphorus-accumulating microorganisms, they determined that phosphorus-accumulating bacteria mainly belonged to β-proteobacteria; Kong et al. believed that β-proteobacteria subclass microorganisms were directly related to phosphorus removal efficiency; Other studies have shown that the microorganisms of the β-Proteobacteria subclass have the ability to degrade and are often found in polluted water bodies. For example, Rhod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C40B40/06C12R1/05C12R1/36C12R1/01
CPCC12Q1/689
Inventor 赵维王敬敬黄志勇
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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