An automatic detection method for the abundance of methanotrophs

Abstract

The invention discloses an automatic detection method of abundance of methane-oxidizing bacteria. The automatic detection method comprises the following steps: building an automation workstation of an original state microbial oil gas and hydrate exploration technology, treating samples, finishing pipetting by the adoption of an automatic liquid treatment device at the workstation, extracting and purifying DNA by automatic nucleic acid extraction equipment, carrying out agarose electrophoresis detection, carrying out real-time fluorogenic quantitative PCR detection on abundance of methane-oxidizing bacteria, obtaining the abundance result and the like. Based on an automatic analysis method, nucleic acid extraction and purification of methane-oxidizing bacteria are automated; preparation of a specific gene PCR reaction system is automated, errors caused by human factors introduced by manual operation are minimized, operation steps are carried out in a clean environment, the whole detection process is controlled through computer software, detection procedures can be traced, and system errors can be corrected uniformly.

Description

technical field [0001] The invention relates to the technical field of oil gas and natural gas hydrate exploration and oil gas and natural gas hydrate reservoir characterization, and more specifically relates to an automatic detection method for the abundance of methane-oxidizing bacteria. Background technique [0002] China is a big energy-consuming country. In 2013, my country's dependence on foreign oil was close to 60%. With the acceleration of industrialization and urbanization, my country's demand for oil, gas and hydrate energy has entered a period of continuous growth. Although my country is implementing strategies for energy conservation, emission reduction and new energy development, my country still cannot get rid of its dependence on oil and gas resources for a long period of time. Therefore, my country's energy security has been put on the important agenda. In order to alleviate my country's energy tension, it is imminent to intensify exploration and develop o...

AI Technical Summary

Problems solved by technology

However, because more than 99% of the microorganisms in soil, sediment and water cannot be cultivated, the traditional microbial exploration method based on the culture method is difficult to meet the exploration requirements in terms of detection accuracy; because gaseous hydrocarbon micro-leakage is dynamic and discontinuous , so traditional microbial exploration methods cannot find micro-seepage of gaseous hydrocarbons in blocks where micro-cracks are in a squeezed state; it is difficult to carry out exploration in today's deserts, Gobi and deep ocean environments with a single anomalous index of live bacteria; Two samplings are required for oil and gas reservoir characterization, which not only significantly increases the cost, but also cannot be implemented in many production areas, because most oil and gas reservoirs in my country have not carried out microbial characterization

[0004] The immaturity of microbial oil and gas and hydrate exploration technology is mainly manifested in: (1) In the face of complex samples of obligate or facultative microorganisms with low abundance, indicative significance for oil and gas, traditional methods based on microbial culture methods Microbial oil and gas exploration technology is not only difficult to measure the microbial abundance (because more than 99% of the microorganisms in the soil and sediments cannot be cultivated), but also has poor data reliability; (3) In the face of complex and diverse oil and gas and hydrate exploration areas, especially in deserts, Gobi, saline-alkali lands and ocean deep water areas, traditional microbial oil and gas and hydrate exploration technologies based on microbial culture methods basically fail; The main reason is that the traditional microbial oil, gas and hydrate exploration technology only relies on the abnormality of live bacteria with a single index; (4) The traditional oil, gas and hydrate exploration technology based on the culture method has a very low processing efficiency for samples, and usually a batch of samples is at least It takes 14 days to obtain the results, which has long cycle, low efficiency and high cost; (5) There are many factors affecting the data and large fluctuations, which will introduce a large number of human errors in the analysis and measurement process; the detection process is not traceable, and it is impossible to carry out systematic error analysis Correction

[0005] Based on the original state detection technology of microbial single cells and genes, Wang Jianghai and others took the lead in proposing the concept, principle and technical scheme of original state microbial oil and gas and hydrate exploration technology, and performed specific fluorescent staining, methane- Significant progress has been made in the rapid quantitative detection of dead and viable butane oxidizing bacteria, the establishment of multiple indicators for microbial oil and gas exploration, and the PCR strategy for denaturing gradient gel electrophoresis analysis of microbial communities in oil and gas fields. The live bacteria, dead bacteria and total bacteria abnormalities of fungi are multiple exploration indicators of microbial oil and gas and hydrate exploration technology, but manual methods are still required to detect microbial abundance, which cannot fundamentally solve the defects of microbial oil and gas and hydrate exploration technology

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