Genome chip for analyzing microbial community structure in acidic environment

A microbial community and genome technology, applied in the field of gene chips, can solve the problems of difficult microbial community structure, large workload of gene cloning and sequence analysis, limitations of fluorescent dye types, etc., and achieve the effect of overcoming the complexity of the process

Inactive Publication Date: 2007-09-12
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, methods such as nucleic acid restriction fragment length polymorphism (RFLP / Restriction Fragment Length Polymorphism) and denaturing gradient gel electrophoresis (DGGE / denatured gradient gel electrophoresis) currently used in microbial community analysis have low sensitivity and low specificity , while the workload of gene cloning and sequence analysis is heavy
Although FISH detection technology has high sensitivity and strong specificity, there are restrictions on the types of fluorescent dyes, and the types of microorganisms detected each time are limited
Therefore, these methods are difficult to simultaneously, quickly, accurately and sensitively analyze the microbial community structure

Method used

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  • Genome chip for analyzing microbial community structure in acidic environment
  • Genome chip for analyzing microbial community structure in acidic environment
  • Genome chip for analyzing microbial community structure in acidic environment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Embodiment 1: Preparation of genome chip of the present invention

[0011] At least 10 strains of Acidithiobacillus ferrooxidans, at least 2 strains of Acidithiobacillus thiooxidans, at least 1 strain of Acidithiobacillus caldus, and at least 1 strain of Acidithiobacillus albertensis , At least 6 strains of Leptospirillum sp., at least 8 strains of Acidiphiliums sp., at least 1 strain of Sulfolobus Metallicus, at least 1 strain of Acidophilus sp. (Acidianus sp.), at least one strain of Metallosphaera sedula and at least two strains of Sulfobacillus were extracted with a DNA extraction kit. Using the extracted whole genome DNA as a probe, it was dissolved in 50% DMSO to a final concentration of 50 pmol / μl, and then the OmniGridAccent Arraying System (Genomic Solutions, USA ) spotting the probe on the siliconized glass slide treated with amination surface. There were 8 probe array replicates on each slide. The spotted chip was dried overnight at room temperature, and f...

Embodiment 2

[0012] Example 2: Chip hybridization specificity assessment

[0013] Acithiobacillus ferrooxidans (Acidithiobacillus ferrooxidans), referred to as A.f bacteria, was selected to detect the hybridization specificity of the chip probe. The DNA of the whole genome of A.f bacteria was labeled with cy3 fluorescent dye and then hybridized with the oligonucleotide chip at 50°C. The results showed that strong fluorescent signals were generated at the probe positions corresponding to A.f bacteria on the chip (see Figure 1 shown), and no non-specific cross-hybridization with non-target genes was found on the chip. It shows that there is a strong specificity between the specific oligonucleotide probe and its corresponding target gene.

Embodiment 3

[0014] Example 3: Sensitivity detection of chip hybridization

[0015] The pure genomic DNA of Acidithiobacillus ferrooxidans 23270 was used to test the sensitivity of the chip of the present invention. The pure genomic DNA was diluted according to a certain concentration gradient (concentrations were 0.11, 1.15, 11.5, 115 ng / ul), labeled with cy3 and then hybridized with the chip. From the chip hybridization analysis results (see accompanying drawing 2), it is found that the detection limit of the chip to the purely cultured microbial genomic DNA is 0.11ng / ul.

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Abstract

The invention discloses a kind of genome chip for analyzing the microbial community structure in an acidic environment. The probe of chip includes the entire genomic DNA of at least ten thiobacillus ferrooxidans, at least two thiobacillus acidophilus sulfur oxides, at least one eosinophils thiobacillus, at least one acidithiobacillus albertensis bacteria, at least six thiobacillus oxidation of ferrous - Lo, at least eight heterotrophic bacteria acidophilus, at least one metal leaf sulfur bacteria, at least one Acidianus tengchongensis, at least a Metallosphaera sedula and at least two of thermophilic bacillus curing.

Description

technical field [0001] The invention relates to a gene chip, in particular to a genome chip for analyzing the microbial community structure in an acidic environment. Background technique [0002] Traditional microbial community research methods are based on isolation and culture, including the determination and analysis of microbial cell morphology, growth temperature, pH value, GC content, microbial total, biomass, respiratory efficiency, enzyme activity and other properties. Although these traditional methods are still the basis of microbial community research, due to the differences in the cultivation requirements of different types of microorganisms, microbial community analysis based on isolation and culture cannot provide sufficiently accurate and rich information. In particular, most of the microorganisms existing in an acidic environment are chemoautotrophic acidophilic bacteria, which are difficult to isolate and culture under artificial conditions, especially on so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 邱冠周刘学端覃文庆刘新星申丽王军
Owner CENT SOUTH UNIV
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