Method for measuring content of the naphthalene degradation gene Nah
A technology of oil pollution and determination method, which is applied in the field of environmental oil pollution monitoring, can solve the problems of not being able to represent the gene type and not reflect the true degradation level of microorganisms, the diversity and distribution of soil microorganisms, etc., to achieve the convenience of hybridization and cloning, and the analysis of microbial degradation performance Comprehensive, high-value effects
Inactive Publication Date: 2014-06-25
NANKAI UNIV
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Problems solved by technology
[0003] The green, economical and efficient way of biodegrading petroleum has aroused the research and advocacy of many domestic scholars, but the traditional method of microbial screening and cultivation can only separate microbial species accounting for 1% to 10% of the total number of petroleum degrading bacteria, which cannot reflect the true degradation level of microorganisms at all. and reflect the diversity and distribution of microorganisms in the soil
In addition, there have been many studies and reports that the aromatic hydrocarbon degradation gene BTEX can encode an important terminal oxidase that metabolizes aromatic hydrocarbons, but their primer design is often limited by the type of degrading bacteria, and the synthetic metabolic enzymes cannot represent most of the degrading bacteria. genotype
Method used
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Embodiment 1
[0025] 1) Collect surface soil at 14 points in total in the oil production area, petroleum processing and refining area, and living area of an oil field. For each sample point, within a diameter of 20 m, select 5 soil samples from the 0-20 cm plow layer according to the "S" shape and mix them. All samples were quickly transferred to a -20°C refrigerator for storage.
[0026] 2) Primer design Follow the primer design principles, compare and design Nahf / Nahr with higher homology in the sequences of strains capable of degrading petroleum hydrocarbons in the soil, as shown in Table 1.
[0027]
[0028] 3) The extracted and purified DNA was subjected to a PCR instrument (Techne TC5000) for Nah degradation gene-specific amplification, and the product was subjected to 2.0% agarose gel electrophoresis to detect whether it was the target fragment.
[0029] 4) According to the operation steps of the AxyPrep DNA Gel Recovery Kit, the target gene was recovered by gel cutting, and the...
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The invention provides a method for measuring content of the naphthalene degradation gene Nah. mutagenic, carcinogenic and teratogenetic toxic aromatic hydrocarbon naphthalene contained in the petroleum-contaminated soil serves as a target degradation component, and the Nah in the naphthalene degradation genes is quantified through real-time fluorescent quantitative polymerase chain reaction (PCR) by adopting a culture-independent molecular biological technique, namely real-time fluorescent quantitative PCR (polymerase chain reaction). The method mainly comprises the following steps: extracting DNA in soil, designing degenerate primers for specific amplification of the naphthalene degradation gene, determining the PCR conditions, making a fluorescent quantitation PCR standard curve, and measuring a sample. The method is a novel method capable of detecting biodegradation activity of aromatic hydrocarbons, can accurately reflect degradation and metabolism pathways of microbes and regional petroleum pollution conditions, and has significant application values in aspects such as oil field pollution monitoring and treatment.
Description
technical field [0001] The invention belongs to the field of environmental oil pollution monitoring, in particular to a method for measuring the content of naphthalene degradation gene Nah in oil-polluted soil. Background technique [0002] Petroleum is an important source of energy to promote social development, but about 1×10 9 tPetroleum and its products enter groundwater, surface water and soil during petroleum exploitation, refining, storage, transportation, use and various leakage accidents, of which my country accounts for more than 600,000 tons, directly causing 5 million hectares of soil in my country to be polluted by petroleum. Aromatic hydrocarbons in petroleum components contain carcinogenic, teratogenic, and mutagenic toxicity, which has brought a series of serious problems such as reduced plant production, hidden food safety hazards, and ecological environment damage to the local area. [0003] The green, economical and efficient way of biodegrading petroleum ...
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IPC IPC(8): C12Q1/68
CPCC12Q1/6851
Inventor 刘庆龙唐景春朱文英张海荣孙克静刘英
Owner NANKAI UNIV