Method for detecting community structure and abundance of ammonia oxidizing bacteria in wastewater system

Abstract

The invention discloses a method for detecting the community structure and abundance of ammonia oxidizing bacteria in a wastewater system. The method comprises the following specific steps: (A) extracting all genome DNA (Deoxyribonucleic Acid) of microorganisms in active sludge of the wastewater system; (B) performing PCR (polymerase chain reaction) amplification by taking all genome DNA as a template and taking amoAlF with a sequence number of SEQ ID No.1 and amoA2R with a sequence number of SEQ ID No.2 as primers; and (C) sequencing an amplification product by adopting a Roche 454 pyrosequencing process, dividing an operational taxonomic unit for a sequencing result by applying a Mothur program, performing online BLASTn comparison on representative sequences with the similarity of 99% in the operational taxonomic unit and sequences disclosed in a database GenBank, and dividing a system evolutionary tree according to a comparison result to obtain the community structure distribution and abundance of the ammonia oxidizing bacteria. The method is simple to perform, low in cost, quick in detection and high in sequence analysis accuracy, can be used for qualitatively analyzing different community structures of the ammonia oxidizing bacteria and quantitatively analyzing the abundance of different species of the ammonia oxidizing bacteria, can be used for simultaneously analyzing a plurality of samples, and is relatively high in practicality and easy to popularize and apply.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting and analyzing the community structure and abundance of ammonia-oxidizing bacteria in a waste water system. Background technique [0002] With the improvement of living standards and the rapid development of industry, the discharge of wastewater is gradually increasing, and the nitrogen content in wastewater is increasing year by year. How to effectively remove nitrogen in wastewater is a key measure to reduce nitrogen pollution in wastewater and water eutrophication At this stage, the research on denitrification microorganisms in the process of denitrification of wastewater has gradually become a hot spot. The biological denitrification system of wastewater mainly includes nitrification and denitrification processes. Ammonia oxidizing bacteria and nitrite oxidizing bacteria that oxidize nitrite to nitrate. Ammonia oxidizing bacteria are key microorganisms in th...

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Problems solved by technology

[0003]In the wastewater biological system, the microbial flora is an aggregate composed of various microorganisms with a certain spatial distribution. These microorganisms are interdependent and compete with each other, and have complex Population relationship, when the strains are isolated and cultivated, the information on the structure and spatial distribution of the microbial flora under natural conditions will not be obtained. Secondly, because the gene expression and metabolic activity of microorganisms are greatly affected by external environmental factors, the separation of strains Cultivation will change the physiological and biochemical characteristics of microorganisms, thereby reducing the guiding significance for actual engineering practice

In addition, pure culture takes a long time, and some strains (such as Nitrospira in nitrifying bacteria) cannot be isolated through pure culture technology at present, that is, they cannot be isolated and cultured, so molecular biology analysis methods become most of them. Research methods of functional flora, such as denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), real-time fluorescent quantitative PCR (real time Q-PCR), etc. common means

[0004] At present, the DGGE analysis method is the main method for the qualitative analysis of ammonia oxidizing bacteria, such as "DGGE analysis of ammonia oxidizing bacteria 16S rDNA in the artificial rapid infiltration system of sewage" ( Ma Mingchao et al., Journal of Geology of Universities, Vol. 13, No. 4, 2007) based on the number and changes of the bands in the DGGE map, qualitatively analyzed the community structure of ammonia-oxidizing bacteria in the sewage treatment system, but the ammonia-oxidizing bacteria In the qualitative analysis, the DGGE technique used cannot determine the specific species in the ammonia oxidizing bacteria community, and there are large deviations in the analysis process, making it difficult to accurately analyze the composition of different community structures of ammonia oxidizing bacteria

[0005]In addition, the quantitative analysis for ammonia oxidizing bacteria is mainly FISH technology and fluorescent real-time quantitative PCR technology. Base-pairing hybridization between acid probes and nucleic acids, and then quantitative analysis of sample DNA through fluorescence system detection, such as "OLAND Biological Nitrogen Removal System Operation and Molecular Biological Detection of Nitrifying Bacteria" (Zhang Dan et al., Applied and Environmental Microbiology Journal of the Chinese Academy of Sciences, Volume 9, Issue 5, 2003) used FISH technology to quantitatively analyze the changes in the community structure of ammonia-oxidizing bacteria. However, FISH technology has high requirements for probes, complicated operation, and the efficiency of short-chain cDNA probes is significantly reduced. Due to the shortcomings of 100% hybridization efficiency, it is difficult to quantitatively analyze the composition of all ammonia oxidizing bacteria communities

[0006] Fluorescent real-time quantitative PCR technology is to add fluorescent groups to the PCR reaction system, detect the accumulated amount of fluorescence after thermal cycling, and then compare it with the standard curve for quantitative analysis Sample DNA, the patent No. is CN 201110305190.4, and the title of invention "Ammonia Oxidizing Bacteria Real-Time Fluorescent Quantitative PCR Detection Method in Activated Sludge" is used to quantitatively detect the community structure of ammonia oxidizing bacteria with real-time fluorescent quantitative PCR technology; although fluorescent real-time quantitative PCR technology It is applied to the analysis of ammonia oxidizing bacteria, but this technique is difficult to operate and requires the preparation of standard DNA. In the quantitative analysis of DNA, the standard curve is not uniform, the results are not comparable, and the experimental cost is high. It is difficult to determine the community structure of ammonia oxidizing bacteria. Reliable, fast quantitative assays

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