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Method for rapidly analyzing anaerobic ammonia oxidizing bacteria flora

An anammox bacteria, rapid analysis technology, applied in microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc. Quickly analyze problems such as easy digitization, cost reduction, and high repeatability

Inactive Publication Date: 2013-01-09
HARBIN INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to solve the problem that the existing method for analyzing anammox bacteria has a large workload and high cost, and cannot realize the rapid analysis of anammox bacteria, and provides a method for quickly analyzing anammox bacteria

Method used

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  • Method for rapidly analyzing anaerobic ammonia oxidizing bacteria flora
  • Method for rapidly analyzing anaerobic ammonia oxidizing bacteria flora
  • Method for rapidly analyzing anaerobic ammonia oxidizing bacteria flora

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specific Embodiment approach 1

[0024] Embodiment 1: The method for rapid analysis of anammox bacteria in this embodiment is carried out according to the following steps:

[0025] 1. Use the kit to extract the DNA of the sample to be analyzed;

[0026] 2. Specific PCR amplification: take the DNA of the sample as the template, and use Pla46F and 630R as the primers to carry out the first PCR amplification to obtain the amplification product A. The amplification product A is purified by the DNA purification kit to obtain purified product, and then use the purified product as a template and AMX368F and AMX820R as primers to carry out a second PCR amplification to obtain amplification product B;

[0027] 3. Restriction endonuclease digestion: The amplified product B was digested with 1U of mung bean nuclease for 30 min, and the digested amplified product B was purified with a PCR product purification kit, and then MspI and RsaI were used to purify the purified amplified product B is subjected to double-enzymati...

specific Embodiment approach 2

[0033] Specific embodiment 2: This embodiment further describes the first PCR amplification in step 2 of the method for rapid analysis of anammox bacteria described in specific embodiment 1, and the first PCR amplification in step 2 The reaction system was a 50 μL reaction system, consisting of the following components: 10-50 ng of sample DNA, 1 μL primer Pla46F, 1 μL primer 630R, 5 μL 10×Buffer, 4 μL dNTPs, 1 μL Taq enzyme and the balance of ddH 2 O; PCR amplification conditions were as follows: denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 1 min, a total of 30 cycles, then extension at 72°C for 10 min, and incubation at 4°C. Others are the same as the first embodiment.

specific Embodiment approach 3

[0034] Embodiment 3: This embodiment further describes the second PCR amplification in step 2 of the method for rapid analysis of anammox bacteria in Embodiment 1, and the second PCR amplification in step 2 The reaction system is a 50 μL reaction system, which consists of the following components: 10-50 ng of purified product, 1 μL of primer AMX368F, 1 μL of primer AMX820R, 5 μL of 10×Buffer, 4 μL of dNTPs, 1 μL of Taq enzyme and the balance of ddH2 O; PCR amplification conditions were: denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, a total of 30 cycles, and then extended at 72 °C for 10 min, and incubated at 4 °C. Others are the same as the first embodiment.

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Abstract

The invention provides a method for rapidly analyzing anaerobic ammonia oxidizing bacteria flora, relates to the method for analyzing anaerobic ammonia oxidizing bacteria flora and solves the problem that the existing method for analyzing anaerobic ammonia oxidizing bacteria flora is big in workload and high in cost and cannot achieve rapid analysis of anaerobic ammonia oxidizing bacteria flora. The method comprises the steps of (1) extracting deoxyribonucleic acid (DNA) of a to-be-analyzed sample; (2) conducting specific polymerase chain reaction (PCR) amplification; (3) conducting restriction enzyme digestion; (4) purifying digestion products; (5) conducting capillary electrophoresis; and (6) analyzing capillary electrophoresis results, and then obtaining analysis results of anaerobic ammonia oxidizing bacteria. The method does not need clone and sequencing, completes rapid analysis of anaerobic ammonia oxidizing bacteria flora in the sample by analyzing tail end segment length after the digestion directly, reduces workload and lowers cost. The method is used for rapid analysis of anaerobic ammonia oxidizing bacteria flora.

Description

technical field [0001] The present invention relates to a method for analyzing anaerobic ammonia oxidation bacteria group. Background technique [0002] Anaerobic ammonium oxidizing bacteria (ANAMMOX) is a member of the newly discovered Planctomycetes, which belongs to strict anaerobic autotrophic bacteria. 4 + ) is the electron donor, nitrous nitrogen (NO 2 - ) is the electron acceptor, the ammonia nitrogen (NH 4 + ) and nitrous nitrogen (NO 2 - ) directly into nitrogen (N 2 ). The discovery of anammox bacteria has a huge impetus for the study of the earth's nitrogen cycle, enrichment of microbial theories and the development of new biological denitrification processes. At present, it has been proved that anammox bacteria are widely distributed in oceans, lakes, river sediments, groundwater and many sewage treatment devices, and play a pivotal role in the earth's nitrogen cycle. It is reported that its nitrogen production accounts for the ocean nitrogen production....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
Inventor 李相昆储昭瑞张杰
Owner HARBIN INST OF TECH
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