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Method for determining content of petroleum hydrocarbon anaerobic degradation gene bAMA in petroleum-contaminated anaerobic environment

A method for measuring oil pollution, which is applied in the field of measuring bamA content of petroleum hydrocarbon anaerobic degradation gene in oil-polluted anaerobic environment, to achieve the effect of simple hybridization and cloning, high application value, and comprehensive analysis of microbial degradation performance

Inactive Publication Date: 2020-01-21
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] For the complexity of petroleum components and the deficiencies of the prior art, the object of the present invention is to provide a method for determining the content of petroleum hydrocarbon anaerobic degradation genes in petroleum-polluted anaerobic environments

Method used

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  • Method for determining content of petroleum hydrocarbon anaerobic degradation gene bAMA in petroleum-contaminated anaerobic environment
  • Method for determining content of petroleum hydrocarbon anaerobic degradation gene bAMA in petroleum-contaminated anaerobic environment
  • Method for determining content of petroleum hydrocarbon anaerobic degradation gene bAMA in petroleum-contaminated anaerobic environment

Examples

Experimental program
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Effect test

Embodiment 1

[0072] Concentration X, copy number, lg(CN) and cycle threshold C of 1 recombinant plasmid and 9 recombinant plasmids after dilution in Example 1 (masD) t As shown in Table 1.

[0073] Table 1 Example 1-masD standard curve

[0074]

[0075] The formula of the typical curve of embodiment 1 is:

[0076] C t =-5.4911lg(CN)+42.27

[0077] The correlation coefficient R of the standard curve 2 =0.9997, the amplification efficiency E of fluorescent quantitative PCR was 53.0%.

Embodiment 2

[0078] Concentration X, copy number, lg(CN) and cycle threshold C of 1 recombinant plasmid and 10 diluted recombinant plasmids in Example 2 t As shown in table 2.

[0079] Table 2 embodiment 2-bamA standard curve

[0080]

[0081]

[0082] The formula of the typical curve of embodiment 2 is:

[0083] C t =-4.7832lg(CN)+40.856,

[0084] The correlation coefficient R of the standard curve 2 =0.998, the amplification efficiency E of fluorescent quantitative PCR is 63.6%

[0085] 8) Determine the cycle threshold C of the sample to be tested by fluorescent quantitative PCR t , the cycle threshold C t Substitute into the formula of the standard curve obtained in step 7) to obtain the copy number of the degraded gene of the sample to be tested.

[0086] In a specific embodiment of the present invention, the method for obtaining the sample to be tested is as follows: add 0.01 g of anaerobic activated sludge obtained from the secondary sedimentation tank of Tianjin Jinnan Di...

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Abstract

The present invention discloses a method for determining content of petroleum hydrocarbon anaerobic degradation gene bamA in a petroleum-contaminated anaerobic environment. The method takes alkanes and benzenes in the petroleum-contaminated anaerobic environment as target degradation components, and adopts non-culture molecular biology technology real-time fluorescence quantitative PCR to quantifytwo degradation genes. The method mainly comprises DNA extraction, design of degenerate primers specifically amplifying the two degradation genes, determination of PCR conditions, manufacture of fluorescence quantitative PCR standard curve, and sample determination. The method is a new method capable of detecting activity of microorganism degradation of petroleum hydrocarbons under anaerobic conditions, at the same time can accurately reflect microorganism degradation metabolic pathway and regional petroleum pollution status, and has important application value in environmental pollution monitoring and treatment.

Description

technical field [0001] The invention belongs to the technical field of environmental oil pollution monitoring, and in particular relates to a method for measuring the content of bamA, an anaerobic degradation gene of petroleum hydrocarbons in an anaerobic environment polluted by oil. Background technique [0002] In recent decades, with the rapid development of the petroleum industry, the pollution of petroleum hydrocarbons in soil and water has become a common environmental problem. Aerobic bioremediation is considered to be an effective method for dealing with petroleum hydrocarbon pollution. However, in many contaminated sites such as sediment and groundwater environments, it is very difficult and costly to stimulate microbial growth by providing oxygen. A large number of studies in recent years have shown that the degradation of petroleum hydrocarbons can also be carried out under anoxic or anaerobic conditions. This novel point of view is suitable for in-situ bioremedi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/64
CPCC12Q1/6851C12Q2531/113C12Q2563/107
Inventor 宋本如唐景春甄梅楠刘小妹
Owner NANKAI UNIV