KR20130090296A

KR20130090296A - Contamination testing method for water purification microbe material

Info

Publication number: KR20130090296A
Application number: KR1020120011523A
Authority: KR
Inventors: 윤석호
Original assignee: 윤석호
Priority date: 2012-02-03
Filing date: 2012-02-03
Publication date: 2013-08-13

Abstract

In the present invention, in order to determine whether aerobic microorganisms that can be contaminated during the cultivation of nitrobacter Winogradsky, a microorganism for water purification, by analyzing the real-time polymerase chain reaction of 16S rRNA gene to measure the total microbial amount and nitrobacter By analyzing the results of real-time polymerase chain reaction of the specific genes of the Winogradsky microorganisms, the total amount of the Nitrobacter Winogradsky microorganisms among the total microbial amounts can be measured to determine whether the contamination is achieved by using the ratio of the two gene products. Thus, according to the present invention, by using the 16S rRNA gene can be confirmed whether the contamination is not limited to a specific microorganism can quantitatively analyze the contamination.

Description

{Contamination testing method for water purification microbe material}

The present invention is to measure the degree of contamination of microbial agents, in particular nitrosomonas, which decomposes nitrogen metabolism, a method for analyzing and confirming the degree of microorganisms that may be contaminated when culturing microorganisms in nitrobacter by analyzing in real time.

Nitrogen occupies more than 70% of the atmosphere and is a major component of proteins and nucleic acids, essential for life. These nitrogenous substances are used as constituents of living organisms and genetic materials through metabolic processes of life. In particular, the circulation of nitrogenous substances in the aquatic environment is very important for the survival of life. In the aquatic environment, these nitrogen metabolites are replaced by stable substances, such as nitrates, in which nitrogenous substances such as ammonia are relatively harmless through the nitrogen cycle, and nitrates are removed by plants such as plants.

Nitrogen organics in fish metabolites in the aquatic environment are primarily broken down into ammonia and nitrite by microorganisms. However, ammonia and nitrite are very harmful to ornamental fish and must be quickly decomposed and removed. The decomposition of harmful ammonia and nitrite occurs in stages (ammonia → nitrite → nitrate). In this process, microorganisms that decompose nitrogen play an important role. nitrosomonas spp.) and nitrobacter spp.

Nitrosomonas is a rod-shaped chemical synthesizer that is capable of flagellar movement and oxidizes ammonia to nitrite. Nitrosomonas is very useful for the purification of contaminated water and plays a very important role in the nitrogen cycle.

Representative species of the genus Nitrosomonas include N. aestuarii, N. communis, N. europaea, N. eutropha, N. halophila, N. marina, N. nitrosa, N. oligotropha and N. ureae.

Nitrobacter, like Nitrosomonas, is a rod-shaped chemical synthesizer that plays an important role in the nitrogen cycle by oxidizing nitrite to nitrate. Representative species include N. alkalicus, N. hamburgensis, N. vulgaris and N. winogradskyi.

In addition, Bacillus spp. Can decompose these harmful nitrogenous substances and are used in a variety of biological purification processes.

In the conventional microbial culture for water purification, in order to prevent contamination of other microorganisms, the medium is sterilized by autoclaving and the microorganisms to be cultured on the sterilized plate medium are separated and purely cultured. However, it is possible to be contaminated by various microorganisms during the cultivation process, so it is necessary to confirm whether there is an accurate contamination.However, the conventional identification method must be cultured using a selective medium, and the exact species or genus is identified by biochemical analysis or DNA sequencing. It was possible through molecular biological methods such as This process requires a large number of experiments and takes two to three days longer than a week to obtain accurate results, which has many disadvantages in terms of efficiency.

In addition, in the case of aerobic bacteria, the growth time between generations is very short, so it is usually possible to check whether there is contamination through the culture process for one day, but in the case of a species having a long growing time between microorganisms or special generations such as anaerobic bacteria In addition, it is difficult to analyze by the culture method, so the improvement of this inefficient method is necessary in the same industry.

In order to solve the above problems of the present invention, there is no need for additional culturing process, and it is possible to confirm the presence or absence of contamination quickly, and by using a real-time polymerase chain reaction method capable of measuring the degree of contamination, The purpose of the present invention is to obtain a method of purely cultivating only the desired microorganisms by determining whether or not and trace back problems of the production process.

As a result of solving the technical problems of the present invention and repeating the research to best meet the object of the present invention, the inventors have configured all the assays to be completed within 4 hours compared to the cultivation method, so that the quantitative analysis can be performed. Real time polymerase chain reaction was applied.

In order to check whether aerobic microorganisms such as Escherichia coli have a relatively short intergenerational growth time in culturing Nitrobacter winoderadsky, the present inventors select a gene specific for Nitrobacter winodegradsky, The probe is designed by selecting a microorganism 16S rRNA gene which is a common gene.

The design of the probe consists of 10 to 40 base pairs to enable real-time polymerase chain reaction, and to label the fluorescent material on one side of the probe in order to measure the amount of amplification products in real time, and to match the matched quencher of the labeled fluorescent material ( A quencher is labeled in the opposite direction to design a probe for real-time polymerase chain reaction.

The designed probe performs real-time polymerase chain reaction to measure the amplified product of each specific gene in real time, and the quantitative value of 16S rRNA gene product that can amplify all microorganisms is specific to nitrobacter winogradsky. Subtract the quantitative value of to determine the presence of other microorganisms except Nitrobacter Winogradsky in the total culture.

As described above, the present invention can quantitatively measure the degree of contamination of other microorganisms in the cultivation of microorganisms for water purification, which is very effective in the production of pure microorganisms.

Figure 1 is a graph showing the amplification amount of Nitrobacter Winogradsky specific amplification products relative to the total amount of microorganisms.

In the present invention, in order to confirm the presence of other microorganisms that may be contaminated when culturing the microorganisms of nitrobacter species, quantitative analysis using real-time polymerase chain reaction was selected.

The first step of the quantitative analysis is to select specific nucleotide sequences of 16S rRNA genes that have very high genetic homology among microbial species among the microorganisms, and to select them as primers for real-time polymerase chain reaction and to use the microorganisms present in the culture medium. The total amount of is measured quantitatively. In this process, it is possible to quantify by using a fluorescent material. In the present invention, EvaGreen can be used, which is used in the same industry, and can also use generic substances such as SYBR Green, LcGreen, etc., which can be commonly used. I never do that.

The second step is to select a primer that can specifically amplify Nitrobacter Winogradsky and proceed in the same manner as the 16S rRNA gene in real time polymerase chain reaction.

In this step, in order to measure gene products specific to Nitrobacter Winogradsky, one or more specific fluorescent materials, such as JOE, VIC, NED, TAMRA, Texas-red, CY5, which have different wavelengths from SYBR Green, may be used. Select and measure simultaneously and compare and quantify each Ct value. At this time, 16S rRNA amplification Ct value is lowered when contamination of other microorganisms occurs, while Ct value does not change in the case of Niobacter Winogradski specific products.

These results indicate that microorganisms other than Nitrobacter winogradsky survived in the culture. In other words, if the ratio of the 16S rRNA gene product and Nitrobacter Winogradsky-specific gene product can be quantified, the contamination during the culturing process can be confirmed, and since the quantitative analysis is possible, the degree of contamination can also be confirmed. In addition, by selecting the conserved nucleotide sequence of the 16S rRNA gene, it is possible to check whether any contaminated microorganisms are contaminated, and thus not only the growth conditions of the contaminated microorganisms but also the contamination of one or more microorganisms can be measured. Has an advantage.

Symbol A represents the 16S rRNA genetic product which represents the total amount of microorganisms.
Code B represents Nitrobacter Winogradsky specific gene product.
The code | symbol C represents the point which shows the time point of the change of an amplification fluorescence value.
The code D represents the amplification repetition number at the time point when the amplification fluorescence value is changed.

Claims

Real-time polymerase chain amplification of the Nitrobacter winobradski-specific genes, and 16S rRNA genes capable of amplifying all microorganisms by amplification of the real-time polymerase chain method, resulting in the nitrobacter winograd in the 16S rRNA gene amplification product Method for measuring the source of contamination in the culture medium using the subtracted ski-specific gene amplification products.

The method of claim 1,
Nitrobacter Winogradsky-specific genes include the nitrite oxidoreductase beta subunit.

The method of claim 1,
Nitrobacter Winogradsky-specific genes include the nitrite oxidoreductase gamma subunit.

The probe used for the real-time polymerase chain reaction is a test probe, characterized in that 10 to 40 base pairs.

Primer used in the real-time polymerase chain reaction is an amplification primer, characterized in that 10 to 40 base pairs.