阅读说明：本技术 煮沸法提基因组dna在浸矿菌中的应用 (Application of boiling method for extracting genome DNA in mineral leaching bacteria ) 是由 开雷 孙文娟 于 2021-08-13 设计创作，主要内容包括：本发明提供一种煮沸法提基因组DNA在浸矿菌中的应用,涉及浸矿菌基因提取领域。该煮沸法提基因组DNA在浸矿菌中的应用,包括以下步骤：步骤一、对浸矿菌进行培养；步骤二、对浸矿菌进行收集；步骤三、清洗；步骤四、进行煮沸处理；步骤六、进行测定。通过合理的提取步骤,使步骤简化,节约提取的时间,提取迅速,且成本消耗低,通过煮沸水浴裂解,可以有效使内部的DNA提取率较高,使得其可以确保低丰度的微生物被检测到,得到的DNA较为灵敏、保真,整个提取步骤在一个试管中完成,可以有效避免在转移的过程中被污染,提高了内部的纯度,同时减少了操作上的失误。(The invention provides application of genome DNA extracted by a boiling method in mineral leaching bacteria, and relates to the field of gene extraction of the mineral leaching bacteria. The application of the boiling method for extracting the genome DNA in the mineral leaching bacteria comprises the following steps: step one, culturing mineral leaching bacteria; step two, collecting the leaching bacteria; step three, cleaning; step four, boiling treatment is carried out; and step six, measuring. Through reasonable extraction step, make the step simplify, practice thrift the time of extracting, extract rapidly, and with low costs consumption, through boiling the water bath schizolysis, can effectively make inside DNA extraction rate higher for it can ensure that the microorganism of low abundance is detected, and the DNA that obtains is comparatively sensitive, fidelity, and whole extraction step is accomplished in a test tube, can effectively avoid being contaminated at the in-process of transfer, has improved inside purity, has reduced the error in the operation simultaneously.)

1. The application of the genomic DNA extracted by a boiling method in mineral leaching bacteria is characterized in that: the method comprises the following steps:

step one, culturing mineral leaching bacteria: adding corresponding culture substances into a 9K culture medium, adjusting the pH value by using dilute sulfuric acid, sterilizing at 121 ℃ for 20 minutes, putting ore leaching bacteria into the culture medium, and culturing at a proper temperature;

step two, collecting the leaching bacteria: collecting bacteria liquid when the ore leaching bacteria is expanded to logarithmic phase, centrifuging for 3 min at 12000r/min, collecting bacteria, washing, suspending each bacteria liquid with sterile water, counting with a blood counting chamber, selecting two parts, each part having bacteria content of 10 ⁸A plurality of;

step three, cleaning: adding the obtained mineral leaching bacteria into a centrifugal tube of 1, 5mL, centrifuging, and then sucking supernatant;

step four, boiling treatment: adding 60 mu LTE buffer solution into the cells in the third step, suspending the cells, and bathing in boiling water at 100 ℃ for 2 minutes;

step five, extracting DNA: centrifuging the cell sap after water bath at 13000r/min for 1 min, extracting supernatant, and storing at-20 ℃ for later use;

step six, determination is carried out: the internal concentration, PCR amplification and sensitivity were measured.

2. The use of genomic DNA extracted by boiling in an ore leaching bacteria according to claim 1, wherein: the leaching bacteria in the step 1 comprise acidithiobacillus ferrooxidans, acidithiobacillus thiooxidans, leptospirillum ferriphilus, acidithiobacillus caldus, acidithiobacillus thiooxidans and acidithiobacillus thermophilus.

3. The use of genomic DNA extracted by boiling in an ore leaching bacteria according to claim 1, wherein: the 9K culture medium comprises: (NH4)₂SO₄3g/L，KCl0.1g/L，K₂HPO₄0.5g，MgSO₄·7H₂O0.5g/L，Ca(NO₃)₂0.01g/L。

4. The use of genomic DNA extracted by boiling in an ore leaching bacteria according to claim 2, wherein: the culture conditions of the acidophilic thiobacillus ferrooxidans and the acidophilic thiobacillus thiooxidans are that the temperature is 30 ℃ and the PH is 2.0, the culture conditions of the leptospirillum ferriphilus are that the temperature is 40 ℃ and the PH is 1.6, the culture conditions of the acidophilic thiobacillus thiooxidans are that the temperature is 40 ℃ and the PH is 2.0, the culture conditions of the acidophilic thiobacillus thiooxidans are that the temperature is 40 ℃ and the PH is 1.5, and the culture conditions of the acidophilic thiobacillus thermophilus are that the temperature is 40 ℃ and the PH is 1.0.

5. The use of genomic DNA extracted by boiling in an ore leaching bacteria according to claim 1, wherein: the washing sequence in the second step is as follows: the membrane was washed twice with sterile water at pH2.0 and then twice with sterile water at pH 7.0.

6. The use of genomic DNA extracted by boiling in an ore leaching bacteria according to claim 1, wherein: the rotating speed of the centrifugation in the third step is 12000r/min, and the centrifugation time is 3 minutes.

7. The use of genomic DNA extracted by boiling in an ore leaching bacteria according to claim 1, wherein: in the sixth step, a micro-spectrophotometer is adopted for concentration, EB dyeing and gel imaging are adopted for PCR amplification, and a corresponding reaction curve is prepared by adopting amplification reaction for sensitivity.

8. The use of genomic DNA extracted by boiling in an ore leaching bacteria according to claim 1, wherein: and the instrument for carrying out water bath in the fourth step is a super constant temperature water bath.

Technical Field

The invention relates to the technical field of gene extraction of ore leaching bacteria, in particular to application of boiling method extracted genome DNA in ore leaching bacteria.

Background

Most of mineral leaching microorganisms involved in microbial metallurgy are chemoautotrophic bacteria growing in extreme environments, including bacteria and archaea, and have the characteristics of slow growth speed and generally low thallus concentration compared with microorganisms in other environments. In the study of the molecular biological characteristics of the low-abundance microorganisms, a large amount of samples need to be collected to meet the requirement of DNA extraction, and the DNA extraction is difficult due to the interference of pH, ion concentration, slag and the like in a leaching system in the extraction process.

There are many methods for DNA extraction, but none of them is a standard established in the scientific community, and each method has its advantages and disadvantages. At present, the DNA extraction of environmental samples can be divided into a direct extraction method, an indirect extraction method and improvement thereof, the direct extraction method can obtain DNA with high recovery rate, but the purity is low, and the subsequent PCR amplification can be influenced and community analysis is distorted; indirect extraction gives a higher purity of DNA and a diversity of fidelity DNA, but this method is only applicable to bacteria [75 ]. The methods for extracting bacterial DNA commonly adopted in laboratories include an alkaline lysis method, an SDS lysis method, a Chelex-100 method, a CTAB method, a liquid nitrogen grinding method and the like, but the conventional methods for extracting bacterial DNA are only used for one type of gram-positive bacteria or gram-negative bacteria and lack of universality.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides the application of the genomic DNA extracted by the boiling method in the mineral leaching bacteria, and solves the problems of low purity and lack of universality in the existing extraction of the genomic DNA from the mineral leaching bacteria.

(II) technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme: the application of the genomic DNA extracted by the boiling method in the mineral leaching bacteria comprises the following steps:

Step one, culturing mineral leaching bacteria: adding corresponding culture substances into a 9K culture medium, adjusting the pH value by using dilute sulfuric acid, sterilizing at 121 ℃ for 20 minutes, putting ore leaching bacteria into the culture medium, and culturing at a proper temperature;

step two, collecting the leaching bacteria: collecting bacteria liquid when the ore leaching bacteria is expanded to logarithmic phase, centrifuging for 3 min at 12000r/min, collecting bacteria, washing, suspending each bacteria liquid with sterile water, counting with a blood counting chamber, selecting two parts, each part having bacteria content of 10⁸A plurality of;

step three, cleaning: adding the obtained mineral leaching bacteria into a centrifugal tube of 1, 5mL, centrifuging, and then sucking supernatant;

step four, boiling treatment: adding 60 mu LTE buffer solution into the cells in the third step, suspending the cells, and bathing in boiling water at 100 ℃ for 2 minutes;

step five, extracting DNA: centrifuging the cell sap after water bath at 13000r/min for 1 min, extracting supernatant, and storing at-20 ℃ for later use;

step six, determination is carried out: the internal concentration, PCR amplification and sensitivity were measured.

Preferably, the leaching bacteria in step 1 include acidithiobacillus ferrooxidans, acidithiobacillus thiooxidans, leptospirillum ferriphilus, acidithiobacillus caldus, acidithiobacillus thiooxidans, and acidithiobacillus thermophilus.

Preferably, the 9K medium comprises: (NH4)₂SO₄3g/L，KCl0.1g/L，K₂HPO₄0.5g， MgSO₄·7H₂O0.5g/L，Ca(NO₃)₂0.01g/L。

Preferably, the culture conditions of the acidophilic thiobacillus ferrooxidans and the acidophilic thiobacillus thiooxidans are temperature 30 ℃ and pH2.0, the culture conditions of the leptospirillum ferriphilus are temperature 40 ℃ and pH1.6, the culture conditions of the acidophilic thiobacillus thiooxidans are temperature 40 ℃ and pH2.0, the culture conditions of the thermophilic thiobacillus thiooxidans are temperature 40 ℃ and pH1.5, and the culture conditions of the thermophilic acidophilic iron bacterium are temperature 40 ℃ and pH 1.0.

Preferably, the washing sequence in the second step is as follows: the membrane was washed twice with sterile water at pH2.0 and then twice with sterile water at pH 7.0.

Preferably, the rotation speed of the centrifugation in the third step is 12000r/min, and the centrifugation time is 3 minutes.

Preferably, the concentration in the sixth step adopts a micro-spectrophotometer, EB staining and gel imaging are adopted for PCR amplification, and a corresponding reaction curve is prepared by adopting amplification reaction in sensitivity.

Preferably, the apparatus for performing water bath in the fourth step is a super constant temperature water bath.

(III) advantageous effects

The invention provides application of genome DNA extracted by a boiling method in mineral leaching bacteria. The method has the following beneficial effects:

1. the invention simplifies the steps, saves the extraction time, and has rapid extraction and low cost consumption through reasonable extraction steps.

2. According to the invention, the whole extraction step is completed in one test tube, so that the pollution in the transfer process can be effectively avoided, the internal purity is improved, and the operation errors are reduced.

3. According to the invention, by boiling water bath cracking, the extraction rate of the internal DNA can be effectively higher, so that low-abundance microorganisms can be detected, and the obtained DNA is sensitive and fidelity.

4. The extraction method can be used for carrying out PCR (polymerase chain reaction) and subsequent ecological community analysis at a molecular level on the mineral leaching bacteria with low thallus density in an extreme environment, and can solve the problems that the DNA of the mineral leaching bacteria is difficult to extract due to the coating of extracellular polymers, is broken by grinding or is polluted by organic reagents and the like.

Drawings

FIG. 1 is a gel electrophoresis analysis diagram of PCR products using genomic DNA extracted by different methods of the present invention as a template;

FIG. 2 is a Real-time PCR standard curve of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first embodiment is as follows:

the embodiment of the invention provides application of genome DNA extracted by a boiling method in mineral leaching bacteria, which comprises the following steps:

step one, culturing mineral leaching bacteria: adding corresponding culture substances into a 9K culture medium, adjusting the pH value by using dilute sulfuric acid, sterilizing at 121 ℃ for 20 minutes, putting ore leaching bacteria into the culture medium, and culturing at a proper temperature;

step two, collecting the leaching bacteria: when the ore leaching bacteria rise to a logarithmic phase, collecting bacteria liquid, centrifuging for 3 minutes under the condition of 12000r/min, collecting bacteria, washing twice by using sterile water with the pH of 2.0, then washing twice by using sterile water with the pH of 7.0, then suspending each bacteria liquid by using sterile water, counting by using a blood counting plate, and selecting two parts, wherein the bacteria content of each part is 108;

step three, cleaning: adding the obtained mineral leaching bacteria into a 1, 5mL centrifuge tube, centrifuging for 3 minutes at the centrifugal rotation speed of 12000r/min, and then sucking the supernatant;

step four, boiling treatment: adding 60 mu LTE buffer solution into the cells in the third step, suspending the cells, and carrying out water bath for 2 minutes in boiling water at 100 ℃ through a super constant-temperature water bath tank;

Step five, extracting DNA: the cell fluid after water bath is centrifuged for 1 minute at 13000r/min, and the supernatant is extracted and stored at-20 ℃ for later use.

The mineral leaching bacteria in the step 1 comprise Acidithiobacillus ferrooxidans (Acidithiobacillus ferrooxidans), Acidithiobacillus thiooxidans (Acidithiobacillus thiooxidans), Leptospirillum ferrophilum (Leptobacillus ferrophilum), Acidithiobacillus caldus (Acidithiobacillus caldus), thiobacillus thermosulfidooxidans (Sulfobacillus thermosulfidooxidans) and siderobium thermophilum (Ferroplasma thermophilum).

The culture conditions of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospira ferruginea, Acidithiobacillus caldus, Acidithiobacillus thiooxidans and Acidithiobacillus thermophilus are as follows:

table shows the culture conditions of various bacteria

Comparative example one: the comparative example differs from the first example in that: in the embodiment, a boiling cracking method is adopted, the comparative example adopts a traditional kit method to extract DNA, and the specific extraction steps are as follows:

(1) taking bacterial suspension containing 108 bacteria into a centrifugal tube of 1.5mL, centrifuging at 12000r/min for 3min, then completely sucking supernatant, adding 150 mu LTE to suspend cells, adding 10 mu L lysozyme, uniformly mixing, and standing at room temperature for 10 min;

(2) Adding 500 mu LDigestionbuffer, mixing, adding 3 mu LProteaseK, mixing, and keeping the temperature at 55 ℃ for 20 min;

(3) centrifuging at 12000r/min at high speed for 5min, transferring the supernatant into a 1.5mL sterile centrifuge tube, adding 300 μ LSolutionB, and mixing;

(4) transferring the sample to 3S column, placing the column in 2.0ml COLLECTIONTUBE, and sampling with 1ml LTip head; centrifuging at 12000r/min for 1min at room temperature by using a desk centrifuge;

(5) taking down the 3S column, and discarding waste liquid in a centrifuge tube; putting the column back into the same centrifuge tube, adding 500 μ L of ashsolution, and centrifuging at 12,000r/min for 1min at room temperature; repeating the steps once;

(6) taking down the 3S column, discarding all waste liquid in the centrifugal tube, putting the column back into the same centrifugal tube, centrifuging at room temperature for 2min at 10000r/min to remove residual Washsolution;

adding 60 mu LTE into the center of the column, putting the column into a new clean 1.5mL or 2.0mL centrifuge tube, and standing for 2min at room temperature or 37-55 ℃; 12000r/min, centrifuging for 1min at room temperature; the liquid in the collection tube was genomic DNA, which was transferred to a 200 μ LEP tube and stored at-20 ℃ for future use.

(I) analyzing the concentration, purity and recovery rate

Taking the 12 DNA samples extracted by the two methods, taking TE as a blank control, and measuring the DNA concentration and OD on a NanoDrop micro-spectrophotometer ₂₆₀/OD₂₈₀Three measurements were made for each concentration and averaged. Calculating the recovery rate: recovery (ng/108) ═ concentration (ng/μ L) × 60 μ L.

The following results were obtained:

TABLE 2 genomic DNA concentration, purity and recovery

(II) PCR amplification

mu.L of each of the upstream and downstream primers, 2 XPCR MasterMix12.5. mu.L, and 1. mu.L of template were supplemented to 25. mu.L with sterile ddH2O, and amplified in a PCR instrument in a microfuge tube according to the following parameters. PCR conditions were as follows: pretreating at 95 ℃ for 3min, at 95 ℃ for 30s, at 56 ℃ for 30s, at 72 ℃ for 30s (32 cycles), extending at 72 ℃ for 10min, carrying out 2% agarose gel electrophoresis on 10 mu L of amplification product, taking 250 bp-I DNAsader as a molecular weight standard, taking a blank control as a negative control, carrying out EB (Epstein-Barr) dyeing, and observing under a gel imaging system.

The 6 pairs of primers used for PCR amplification were specific primers designed for the gyrB genes of a. ferrooxidans, a. thiooxidans and l. ferrophilum, the arsB genes of a. caldus and S. thermosulfidans, and the 16S rRNA of f. thermophilum, respectively. The length and sequence of the various primers are shown in the following table:

table three: primers and sequences

The electrophoresis results are shown in FIG. 1: the strains corresponding to 1 and 7 in the figure are Thiobacillus ferrooxidans (the length of the product is 160 bp); 2. the strain corresponding to 8 is thiobacillus thiooxidans (the length of the product is 175 bp); 3. the strain corresponding to 9 is leptospirillum ferriphilum (the length of the product is 168 bp); 4. the strain corresponding to 10 is Acidithiobacillus caldus (the length of the product is 150 bp); 5, 11 is thermophilic sulfur oxidation sulfureted bacillus (the length of the product is 261 bp); 6, the strain corresponding to 12 is thermoacidophilic siderophyte (the length of the product is 248bp), A is a kit method; and B, boiling.

(III) sensitivity test

The sensitivity measurement of the two methods selects Acidithiobacillus ferrooxidans commonly used in the ore leaching process, and 6 groups of bacteria liquid are taken as samples after the bacteria liquid is diluted by 1:10 times, and the bacteria amount is respectively 1, 102, 103, 104, 105 and 106. The genomic DNA of the 6 groups of samples was extracted by the above two methods, and then subjected to Real-timePCR amplification under the same conditions as the PCR amplification in the previous step. Carrying out PCR amplification by using the whole genome DNA of the pure Acidithiobacillus ferrooxidans as a template, diluting a product by 1:10 times (copy number is 10-109 copy/mL) to be used as a Real-timePCR standard product, and taking the standard curve as the standard curve. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; then denaturation at 95 ℃ for 30s, annealing at specific annealing temperature for 30s, extension at 72 ℃ for 30s, and real-time fluorescence detection for 40 cycles; and (3) melting curve analysis: 95 ℃ for 1min, 55 ℃ for 10s, the temperature rises by 0.5 ℃ after each cycle, the time is 10s, the product specificity is analyzed, and the total time is 80 cycles.

The resulting curve is shown in FIG. 2: as is clear from FIG. 2, the boiling lysis method enables DNA to be extracted from a small number (102) of cells, and the number of copies extracted at different concentrations is increased by approximately the same factor (10 times) as the amount of the original cells, and the change is more uniform. The kit method is to extract DNA from a sample containing 103 cells, but the extracted genomic DNA tends to increase by 2 times with 10 times of increase of the cells, the deviation of the DNA copy number from the initial cell amount is large, and the more the cell amount is, the more the DNA loss is, in the extraction process.

The result shows that the boiling lysis method can extract DNA from bacteria liquid with lower concentration more sensitively than a kit method, has better fidelity on the extraction of genome DNA of mixed bacteria during community analysis, and can more accurately analyze the proportion of various bacteria in the original mixed system.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.