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Method for rapidly separating anaerobic denitrifying bacteria and particular primer thereby

The technology of anaerobic denitrification and separation method is applied in the field of rapid separation of anaerobic denitrification bacteria and the specific primers used, which can solve the problems of large workload, long separation period, and difficulty in obtaining, and achieves low workload. , the effect of shortening the cycle and avoiding interference

Inactive Publication Date: 2009-09-02
HARBIN NORMAL UNIVERSITY
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Problems solved by technology

[0004] The purpose of the present invention is to provide a rapid separation method of anaerobic denitrifying bacteria and the specific method used to solve the problems of long separation period, heavy workload and difficulty in obtaining target functional strains and pure strains of existing anaerobic denitrifying bacteria. sex primer

Method used

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  • Method for rapidly separating anaerobic denitrifying bacteria and particular primer thereby
  • Method for rapidly separating anaerobic denitrifying bacteria and particular primer thereby
  • Method for rapidly separating anaerobic denitrifying bacteria and particular primer thereby

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specific Embodiment approach 1

[0018] Specific embodiment one: the rapid separation method of anaerobic denitrifying bacteria in this embodiment is realized according to the following steps: one, adopt DNA extraction kit to extract the DNA of microorganism in the sample, then adopt specific primer to carry out PCR amplification to DNA, select The samples containing anaerobic denitrifying bacteria are then inoculated in the liquid culture medium of anaerobic bacteria, and Hungate anaerobic operation technology is used to isolate and cultivate anaerobic denitrifying bacteria, and then the bacteria obtained from the isolation and culture are tested. Oxygen denitrification bacteria; 2. Inoculate the anaerobic denitrification bacteria obtained by the preliminary screening in the solid medium of anaerobic bacteria, and make a rolling tube to purify and cultivate the anaerobic denitrification bacteria until colonies grow; 3. Pick a single colony in the working box and inoculate it in an anaerobic liquid medium for ...

specific Embodiment approach 2

[0035] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the composition of the anaerobic bacteria solid medium in step 2 is put into the electromagnetic cooker after being configured, after boiling, add L-cysteine ​​0.5g and quality The resazurin solution (indicator) with a concentration of 0.2% has oxygen at this time, and the color of the culture medium is pink. Heat and boil continuously, cover the pot, and feed in nitrogen gas with a mass purity of 99.99% to drive away oxygen for 30-40 minutes. , until the culture medium turns from pink to colorless, then aliquot the anaerobic tubes, and sterilize at 121°C for 20 minutes. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0036] Specific embodiment three: the difference between this embodiment and specific embodiment two is that in step three, the enrichment culture is carried out for 8-12 days. Other steps and parameters are the same as in the second embodiment.

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Abstract

The invention relates to a method for rapidly separating bacteria and a particular primer thereby, in particular to a method for rapidly separating anaerobic denitrifying bacteria and a particular primer thereby, solving the problems that the prior anaerobic denitrifying bacteria have long separation period and great workload and are difficult to obtain bacterial strains and pure bacterial strains with the destination functions. The method comprises the following steps: 1, extracting microorganism DNA from a sample for PCR augmentation and separation culture and prescreening the anaerobic denitrifying bacteria; 2, preparing particular culture media to be purified until a colony grows up; 3, carrying out the enrichment culture of the colony for the PCR augmentation and re-screening the anaerobic denitrifying bacteria; 4, repeating the separation culture, the purification culture and the enrichment culture, identifying and carrying out the PCR augmentation for removing repetitive bacterial strains and then, repeating the separation culture and the purification culture to obtain the pure bacterial strains. An upstream primer is SEQ ID of NO.1, and a downstream primer is SEQ ID NO.2.The invention shortens the separation period by 40 percent to 50 percent, has little workload and is easy to obtain destination bacterial strains and pure bacterial strains.

Description

technical field [0001] The invention relates to a rapid separation method of bacteria and used primers. Background technique [0002] Anaerobic denitrifying bacteria are mainly used for the treatment of nitrate sewage. At the same time, in recent years, there have been many studies on the use of anaerobic denitrifying bacteria to control sulfate reduction, and good results have been achieved. [0003] At present, the separation cycle of anaerobic denitrifying bacteria is long, the workload in the separation process is heavy, the anaerobic conditions are difficult to control, and it is difficult to obtain pure strains; The degradation effect of nitrate and nitrous acid is not good. The separation of anaerobic denitrification bacteria is difficult, which makes many basic theoretical research impossible. With the deteriorating environment, especially the aggravation of nitrate pollution in groundwater, the separation of anaerobic denitrification bacteria and related research h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12Q1/68C12N15/11C12R1/01
Inventor 胡建民王月媛魏利
Owner HARBIN NORMAL UNIVERSITY
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