CN112063615A

CN112063615A - Genome DNA extracting solution, genome DNA extracting method and application thereof

Info

Publication number: CN112063615A
Application number: CN202010921174.7A
Authority: CN
Inventors: 高社干; 谷变利; 崔天星; 李智涛; 兰子君
Original assignee: First Affiliated Hospital of Henan University of Science and Technology
Current assignee: First Affiliated Hospital of Henan University of Science and Technology
Priority date: 2020-09-04
Filing date: 2020-09-04
Publication date: 2020-12-11

Abstract

The invention discloses a genome DNA extracting solution, a genome DNA extracting method and application thereof. Wherein the genome DNA extracting solution comprises 5-10 mM Tris, 1-5 mM Ethylene Diamine Tetraacetic Acid (EDTA), 1-2.5 mM magnesium sulfate and sterile water, and the pH value of the Tris is 8.0-9.0. The genome DNA extracting solution provided by the invention is simple to prepare, does not need tube replacement pipetting in operation, does not need purification, can effectively avoid genome loss, is quick to operate and cheap in raw materials, and can be directly used for subsequent common PCR or fluorescent PCR, gene detection, molecular cloning, sequence analysis, molecular hybridization and other downstream experiments after being incubated with a sample.

Description

Genome DNA extracting solution, genome DNA extracting method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a genomic DNA extracting solution, a genomic DNA extracting method and application thereof.

Background

Porphyromonas gingivalis (p. gingivalis) is an important pathogenic bacterium of human periodontitis, and it has been shown that its level in the oral cavity can predict disease progression or activity. Preliminary studies of our subject group indicate that p. Henan is a high-incidence province of esophageal cancer in China, and in order to find out the epidemiological characteristics of esophageal squamous carcinoma high-risk population, precancerous lesion and P.gingivalis infection in esophageal cancer patients, epidemiological investigation activities of large sample amount are imperative. Therefore, a method for simply and accurately detecting the p.gingivalis infection state of the oral cavity is necessary.

The methods for detecting p.gingivalis infection mainly include culture methods and genetic detection methods. The culture method has high specificity, however, the method lacks sensitivity, has low culture success rate and needs 5 to 7 days. And therefore not suitable for large-scale molecular epidemiological screening. The gene detection method represented by PCR is developed and matured at present, and the real-time fluorescence quantitative PCR method is widely used for quantitatively detecting P.gingivalis DNA in fresh source samples such as saliva, dental plaque, tongue back and the like.

The fluorescence quantitative PCR method and other gene detection of different types generally involve the extraction and purification process of genome, and the quality and integrity of extracted nucleic acid directly determine the subsequent experimental results. Since it takes a long time and is cumbersome to extract and purify a genome from a sample, the steps of extracting and purifying a genome have been a limiting factor in the speed and range of application of the entire gene test.

The extraction of the genome from the sample mainly comprises two steps: lysing the cells and extracting nucleic acids. Lysis of cells to release nucleic acids is commonly performed by methods including: physical methods (boiling, freeze thawing, microwave, ultrasound, grinding, etc.), chemical methods (high salt, surfactant, phenol, strong base, etc.), and enzymatic digestion methods (lysozyme, proteinase K, etc.). The existing extraction methods all have various defects:

1) phenol-chloroform extraction, isopropanol precipitation and formamide lysis are the most classical methods for extracting DNA, and protease K and Sodium Dodecyl Sulfate (SDS) are used for digesting and lysing cells. In the first two methods, phenol-chloroform or the like contained in the lysate is used for removing proteins, and ethanol or isopropanol is used for precipitating genomic DNA. The formamide cracking method is to destroy the combination of the polyprotein and the chromosome DNA by using high-concentration formamide, and obtain a DNA sample after dialysis. The classical methods can obtain DNA with higher purity without considering the sample amount, can meet various experimental requirements, but have the disadvantages of complicated operation, long time consumption and certain toxicity of used reagents.

2) The alkaline lysis method is to use the damage effect of strong cationic detergent SDS on cell walls under the alkaline condition provided by NaOH (about pH 12.0) to lyse cells, so that intracellular proteins are denatured and genomic DNA is released. The cell wall fragments, denatured proteins and the like generated by the method form a complex, and the complex is effectively precipitated and separated out under the condition of high potassium salt in lysate, so that the genome is retained in the supernatant, and the protein complex is removed and the genome is purified by combining with the steps of absolute ethyl alcohol or ethyl alcohol precipitation and washing. However, the detergent, high salt solution and other components used in this method are liable to remain in the genome and affect the subsequent PCR results.

3) The boiling lysis method is to boil and lyse cells under the action of lysis buffer containing proteinase K, etc., to destroy the combination of protein and chromosome, release DNA, and centrifugally precipitate to obtain purified genome DNA for PCR amplification. The steps of heating, centrifuging and the like of the method need specific equipment, and the protein is solidified due to high-temperature boiling, so that part of nucleic acid is easily wrapped by denatured protein and lost along with centrifugal precipitation, and the initial template genome amount in the supernatant is directly reduced; in addition, even after boiling and high speed centrifugation, the supernatant may still contain a small amount of amplification inhibitors such as proteins, heparin and trace hemoglobin, which may affect the PCR reaction.

4) The magnetic bead method for extracting nucleic acid is characterized in that under the action of cell lysis solution, free nucleic acid molecules after cell lysis are adsorbed by specific magnetic particles, and impurities such as protein and the like are not adsorbed and remain in the solution. Under the action of a magnetic field, separating the magnetic particles from the liquid, recovering the magnetic particles and eluting to obtain the purified DNA. Because the series of reagents do not contain toxic organic solvents such as chloroform, phenol and the like, the extraction steps are simpler, the recovery rate of nucleic acid samples is high, and the obtained nucleic acid has good purity and is widely applied. However, most of the specific magnetic beads and the matched magnetic bead buffer solution are monopolized abroad and are expensive, so that the possibility of the specific magnetic beads and the matched magnetic bead buffer solution in the automatic and high-throughput detection in China is limited.

5) Clinical samples are precious in sources, and the sample size is generally small, such as swabs, puncture washes and the like, through the nucleic acid extraction and purification method, multi-step operation easily causes sample genome loss, and the result accuracy is affected, which limits the detection and research of high-throughput samples.

Therefore, there is an urgent need to develop a reagent capable of rapidly releasing genome, which can retain the initial genome amount as much as possible and reduce genome loss or pollution caused before amplification, and has simple components, no influence on subsequent reaction, and suitability for the gene detection requirement of large-scale samples.

Disclosure of Invention

In order to solve the problems that the extraction and purification of genome DNA in the prior art are long in time consumption and labor-consuming, the cost of reagents and time is high, the consumed materials and reagents for extraction are expensive, and nucleic acid is easily lost in the experimental process.

The invention aims to provide a genome DNA extracting solution, which comprises 5-10 mM Tris, 1-5 mM Ethylene Diamine Tetraacetic Acid (EDTA), 1-2.5 mM magnesium sulfate and sterile water, wherein the pH value of the Tris is 8.0-9.0.

Further, the concentration of EDTA was 1 mM.

The second objective of the invention is to provide a method for extracting genomic DNA, which comprises the following steps:

collecting a sample;

mixing the genomic DNA extract solution according to any one of claims 1 to 3 with a sample at an arbitrary volume ratio to obtain a mixed solution;

uniformly mixing the mixed solution, and standing to obtain a sample treatment solution;

and (3) carrying out denaturation treatment on the sample treatment liquid at 90-100 ℃ to obtain genome DNA.

Further, the volume ratio of the genomic DNA extracting solution to the sample is 5: 1-1: 1.

Further, the mixed liquid is uniformly mixed by adopting any one of vortex oscillation or liquid-transfer gun head blowing.

Further, after the sample is collected, the sample is centrifuged to obtain a precipitate of the sample.

Further, the sample is from one or a mixture of both of a human or a bacterium.

Further, the sample is from a human, and comprises any one or a mixture of any two or more of mouthwash, gingival crevicular fluid, oral or nasopharyngeal swab, and tissue puncture eluate.

Further, the sample is from a bacterium, including a bacterial culture.

The second purpose of the invention is to provide a method for detecting Porphyromonas gingivalis, which adopts the method for extracting the genome DNA of the Porphyromonas gingivalis. .

Compared with the prior art, the invention has the beneficial effects that: (1) the genome DNA extracting solution provided by the invention is simple to prepare, does not need tube replacement and liquid transfer in operation, does not need purification, is quick to operate, has cheap raw materials, and can be directly used for subsequent common PCR or fluorescence PCR, gene detection, molecular cloning, sequence analysis, molecular hybridization and other downstream experiments after being incubated with a sample. (2) When the method for extracting the DNA based on the genome DNA extracting solution provided by the invention is used for gene detection, the detection time can be shortened, the reagent cost can be reduced, and the method is suitable for large-scale high-throughput samples. (3) The genome DNA extracting solution and the application thereof provided by the invention solve the problems that the existing genome extraction and purification technology is time-consuming and labor-consuming, the reagent cost is higher, the genome is easy to lose or pollute, the gene detection speed and efficiency are influenced, and the like.

Drawings

FIG. 1 is a diagram showing the result of fluorescence PCR detection of Porphyromonas gingivalis in a human mouth wash or a mouth swab sample using the genomic DNA extract provided by the present invention;

FIG. 2a is a fluorescence PCR detection amplification curve diagram of the genomic DNA extract for Porphyromonas gingivalis in a bacterial culture sample;

FIG. 2b is a standard curve diagram of the fluorescence PCR detection of Porphyromonas gingivalis in a bacterial culture sample using the genomic DNA extract provided by the present invention;

FIG. 3 is a diagram showing the result of fluorescence PCR detection of Porphyromonas gingivalis in samples of genomic DNA extract used in gingival crevicular fluid, nasopharyngeal swab and tissue biopsy eluate provided by the present invention;

FIG. 4 is a diagram showing the result of fluorescence PCR detection of human PGT gene in a mouth wash or a mouth swab sample using the genomic DNA extract of the present invention;

FIG. 5 is a diagram showing the results of fluorescence PCR detection of Porphyromonas gingivalis by treating an oral swab sample with the genomic DNA extraction solution provided by the present invention and three methods, namely, lysis boiling method and kit extraction method;

FIG. 6 is a comparison of the method for extracting DNA from the genomic DNA extract provided by the present invention with the lysis boiling method, for the general PCR detection of Porphyromonas gingivalis in human buccal swab samples; wherein N represents a negative control and P represents a positive control.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.

The first embodiment is as follows:

the embodiment of the invention provides a genome DNA extracting solution, which comprises 5-10 mM Tris, 1-5 mM Ethylene Diamine Tetraacetic Acid (EDTA), magnesium sulfate with the concentration of 1-2.5 mM and sterile water, wherein the pH value of the Tris is 8.0-9.0.

Tris and EDTA in the genome DNA extracting solution provided by the embodiment of the invention have the function of protecting the genome and have no influence on the subsequent amplification experiment, and magnesium sulfate plays a role in promoting the activity of DNA polymerase in the subsequent amplification reaction. In addition, the pH value of Tris is 8.0-9.0, a slightly alkaline environment is provided, and protein structures such as cells, viruses and bacteria can be quickly cracked through high-temperature denaturation treatment to release genome DNA. The high temperature denaturation treatment is usually a pre-denaturation treatment in the first step of the PCR reaction. In addition, the genomic DNA extracting solution provided by the embodiment of the invention is simple to prepare, does not need tube replacement and pipetting and purification in the operation, and can be directly used for subsequent common PCR or fluorescence PCR, gene detection, molecular cloning, sequence analysis, molecular hybridization and other downstream experiments after being incubated with a sample.

Example two:

sample treatment solutions of bacterial cultures, human mouth rinses, human gingival crevicular fluid, human oral/nasopharyngeal swabs, and human tissue penetrating eluates were prepared.

1. Preparation of a sample treatment solution for mouthwash: the mouthwash sample (approximately 20mL) was centrifuged directly at 4000rpm for 20min and the precipitate collected; adding DNA extracting solution with the volume 2 times that of the precipitate, fully and uniformly mixing by vortex oscillation (or uniformly mixing by blowing with a pipette tip) to resuspend the precipitate, and standing at room temperature to obtain a sample treatment solution. Wherein the DNA extracting solution consists of 7mM Tris, 1mM ethylene diamine tetraacetic acid, magnesium sulfate with the concentration of 2mM and sterile water, and the pH value of the Tris is 8.

2. Preparation of oral/nasopharyngeal swab sample treatment: mixing the oral cavity/nasopharynx swab sample (after sampling, placing the swab in preservation solution), and shaking in vortex to make the preservation solution (iCleanhcy, Shenzhen Huachenyang science and technology Limited) fully dissolve and elute the cell components in the swab; transferring all the preservation solution into a new 1.5mL sterile centrifuge tube, centrifuging at 12000rpm for 10min, pouring out the supernatant, performing instantaneous centrifugation, sucking and discarding the residual supernatant by using a gun head, and avoiding sucking away the precipitate as much as possible; adding DNA extracting solution with the volume 2 times that of the precipitate, fully and uniformly mixing by vortex oscillation (or uniformly mixing by blowing with a pipette tip) to resuspend the precipitate, and standing at room temperature to obtain a sample treatment solution. Wherein the DNA extracting solution consists of 10mM Tris, 1mM ethylene diamine tetraacetic acid, magnesium sulfate with the concentration of 1mM and sterile water, and the pH value of the Tris is 9.

3. Preparing a sample treatment solution of a porphyromonas gingivalis bacterial solution: taking appropriate amount of Porphyromonas gingivalis solution which is subcultured overnight after recovery, measuring OD600, and taking corresponding 10⁹copy/mL of the bacterial solution (OD 1.0 corresponds to 10)⁹copy/mL calculation) to a 1.5mL sterile centrifuge tube, centrifuging at 12000rpm for 10min, and discarding the supernatant; adding DNA extracting solution with the volume 5 times that of the thalli, and fully and uniformly mixing by vortex oscillation to resuspend bacterial precipitates; adding physiological saline into the obtained sample treatment solution according to a ratio of 10 times, and performing gradient dilution until the dilution reaches 10²Concentration of copies/mL; standing at room temperature to obtain a sample treatment solution with a concentration gradient of the bacterial culture. Wherein, the DNA extracting solution consists of 10mM Tris, 2mM ethylene diamine tetraacetic acid, magnesium sulfate with the concentration of 1mM and sterile water, and the pH value of the Tris is 9.

4. Preparation of a sample treatment solution for gingival crevicular fluid: and (3) extracting gingival crevicular fluid of the individual suffering from periodontitis according to a conventional method, adding DNA extracting solution in equal proportion, shaking in a vortex manner, fully and uniformly mixing, and standing at room temperature to obtain a sample treatment fluid. Wherein the DNA extracting solution consists of 8mM Tris, 1mM ethylene diamine tetraacetic acid, magnesium sulfate with the concentration of 1mM and sterile water, and the pH value of the Tris is 8.

5. Preparing a sample treatment solution of the tissue puncture eluent: injecting the tissue puncture eluent into a 1.5mL sterile centrifuge tube, centrifuging at 12000rpm for 10min, and removing the supernatant; adding the DNA extracting solution with the same volume, fully and uniformly mixing by vortex oscillation, and standing at room temperature to obtain a sample treating fluid. Wherein, the DNA extracting solution consists of 8mM Tris, 1mM ethylene diamine tetraacetic acid, magnesium sulfate with the concentration of 1.5mM and sterile water, and the pH value of the Tris is 9.

Example three:

fluorescence PCR detection of Porphyromonas gingivalis:

mu.L of the sample treatment solutions obtained in steps 1 to 5 of example II were each subjected to fluorescent PCR reaction system preparation, subjected to transient centrifugation, and subjected to BioRad CFX96^TMAnd performing real-time fluorescent quantitative PCR amplification analysis on the real-time PCR system. AceQ PCR Probe Master Mix (Vazyme, Q112-03) was purchased from Biotech, Inc. of Nanjing Novowed; all primer probe sequences were obtained from Suzhou Jinweizhi Biotech, IncAnd (4) synthesizing.

The PCR reaction (20. mu.L total) was as follows:

composition (I)	Volume (μ L)
		Upstream primer	0.5
Downstream primer	0.5
		TaqMan fluorescent probe (10. mu.M)	0.2
AceQ PCR Probe Master Mix(2×)	10
		DEPC water	6.8

Wherein:

an upstream primer: 5'-ACCTTACCCGGGATTGAAATG-3'

A downstream primer: 5'-CAACCATGCAGCACCTACATAGAA-3'

And (3) probe: 5 '-FAM-ATGACTGATGGTGAAAACCGTCTTCCCTTC-TARMA-3'

The PCR reaction conditions are as follows:

95℃，10min；

95 ℃, 10s, 60 ℃, 60s (signal acquisition); 40 cycles.

And (3) analyzing an experimental result: after PCR amplification was complete, CFX Maestro was used^TMsoftware componentThe results of the analyses and amplifications are shown in FIGS. 1 to 3. From the amplification curves shown in FIGS. 1-3, the baseline is stable, the exponential region is obvious, the amplification curve is typical S-shaped, and the negative and positive differentiation is clear. Wherein, FIG. 2b shows that after the gradient dilution of the P.gingivalis bacterial liquid, the linear relation among samples with different concentrations is good, and R is²99.9%, amplification efficiency 86.8%. Therefore, the genomic DNA extract provided by the embodiment of the invention can be used for extracting bacterial genomes.

Example four:

fluorescence PCR detection of human PGT genes in mouthwash, buccal swab samples:

mu.L of the sample treatment solutions obtained in step 1 and 2 of example II were used to prepare a fluorescent PCR reaction system, centrifuged instantaneously, and subjected to BioRad CFX96^TMAnd performing real-time fluorescent quantitative PCR amplification analysis on the real-time PCR system. Primer probe sequences were synthesized by jin Wei Zhi Biotech, Suzhou.

The PCR reaction system was the same as that in example three.

Wherein:

an upstream primer: 5'-ATCCCCAAAGCACCTGGTTT-3'

A downstream primer: 5'-AGAGGCCAAGATAGTCCTGGTAA-3'

And (3) probe: 5 '-FAM-CCATCCATGTCCTCATCTC-TARMA-3'

The PCR reaction conditions are as follows:

95℃，10min；

95 ℃, 10s, 60 ℃, 60s (signal acquisition); 40 cycles.

And (3) analyzing an experimental result: after PCR amplification was complete, CFX Maestro was used^TMsoftware analysis amplification results, please refer to fig. 4, the human PGT gene amplification baseline is stable, the exponential region is obvious, and the amplification curve is typical sigmoid. Therefore, the genomic DNA extract provided by the embodiment of the invention can be used for extracting human genomes.

The genomic DNA extracting solution provided by the invention is simple to prepare, cheap in raw materials and quick to operate, can be used for quickly releasing the genomic DNA of samples such as bacterial cultures, gingival crevicular fluid, mouthwash, oral swabs, nasopharyngeal swabs and tissue puncture eluent, does not need purification, and improves the detection efficiency. When the method for extracting the DNA based on the genome DNA extracting solution provided by the invention is used for gene detection, the detection time can be shortened, the reagent cost can be reduced, and the method is suitable for large-scale high-throughput samples.

Example five:

comparative tests for extraction of genomic DNA from buccal swab samples (fluorescent PCR detection of porphyromonas gingivalis) using three methods:

this example compares the method for extracting DNA using the genomic DNA extract provided in the present invention with the lysis boiling method and the kit extraction method. The method comprises the following specific steps:

1. and (3) sample subpackaging: 3 oral swab samples are respectively taken, each oral swab sample is uniformly mixed with 1 part of cell preservation solution (iCleanhcy, Shenzhen Huachenyang science and technology Co., Ltd.), and each mixed solution is subpackaged into 3 centrifuge tubes. The subpackaging steps of each oral swab sample are as follows: after 3 1.5mL centrifuge tubes were prepared and 1mL of the storage solution containing exfoliated buccal cells was mixed well, 300. mu.L of each of the tubes was put into each of the 1.5mL centrifuge tubes and named. Wherein, the first sample is divided into 3 centrifuge tubes which are respectively marked as Q-01, L-01 and K-01; the second sample is divided into 3 centrifuge tubes, which are respectively marked as Q-02, L-02 and K-02; the third sample was dispensed into 3 centrifuge tubes, labeled Q-03, L-03, and K-03, respectively.

2. And (3) centrifuging the sample: the 9 samples after dispensing were centrifuged at 13000rpm for 10min and the supernatant was discarded (removed as clean as possible).

3. Different methods were used for DNA extraction:

3.1 preparing sample treatment solution by using the genomic DNA extracting solution provided by the invention

Taking samples with numbers Q-01, Q-02 and Q-03, adding the same volume of the genomic DNA extracting solution provided by the invention into a centrifugal tube respectively, uniformly mixing by vortex oscillation, resuspending the precipitate, performing instantaneous centrifugation, and standing at room temperature to obtain a first group of sample treatment solution. Wherein the DNA extracting solution consists of 10mM Tris, 1mM ethylene diamine tetraacetic acid, magnesium sulfate with the concentration of 1mM and sterile water, and the pH value of the Tris is 9.

3.2 DNA extraction by lysis boiling

Taking samples with numbers L-01, L-02 and L-03, adding 30 mu L of cell lysate (#18LS11001, Shenzhen bioenergy) into the centrifuge tube respectively, and performing flicking or vortex oscillation to resuspend precipitates; placing in a metal thermostat, and heating at 100 deg.C for 10 min; centrifuging at 12000 r/min for 10min at normal temperature, taking out and standing, and using the supernatant for subsequent PCR.

3.3 extraction of DNA by kit extraction

Samples with numbers K-01, K-02 and K-03 were subjected to nucleic acid extraction and purification by centrifugal column method according to the MicroElute Genomic DNA Kit (# D3096-02, OMEGA) instructions. The method comprises the following steps:

A. dissolving;

B. releasing nucleic acid;

C. adsorbing by a centrifugal column;

D. washing and purifying;

E. eluting; the DNA was eluted with 30. mu.L of Elution Buffer preheated at 70 ℃;

F. storing; directly carrying out subsequent PCR amplification.

4. Fluorescence PCR detection of Porphyromonas gingivalis was performed on genomic DNA obtained by different methods:

2 μ L of the genomic DNA of the sample obtained in steps 3.1-3.3 of this example was used to prepare a fluorescent PCR reaction system, mixed by vortexing, and applied to BioRad CFX96^TMAnd performing real-time fluorescent quantitative PCR amplification analysis on the real-time PCR system.

The PCR reaction system was the same as that of example three.

Wherein:

the primer probe is the same as the primer probe in the third embodiment;

the PCR reaction conditions are as follows:

95℃，10min；

95 ℃, 10s, 60 ℃, 60s (signal acquisition); 40 cycles.

And (3) analyzing an experimental result: after PCR amplification was complete, CFX Maestro was used^TMsoftware analysis of amplification results, see FIG. 5, it can be found by analysis of the results of fluorescence PCR detection of DNA extracted by the three methods,the Ct values of the first samples Q-01, Q-02 and Q-03 are smaller than those of the second samples L-01, L-02 and L-03 and smaller than those of the third samples K-01, K-02 and K-03, respectively. Table 1 shows Ct values of the DNA products obtained by the three extraction methods in the fluorescent PCR detection, and please refer to table 1 for Ct value of each sample. The Ct value is a parameter for measuring the amount of the initial template DNA in the fluorescent quantitative PCR reaction, and under the same amplification condition, the smaller the Ct value of the sample is, the more the initial template DNA amount contained in the sample in the amplification reaction is. The method for extracting DNA by adopting the DNA extracting solution provided by the invention does not need any inter-tube transfer step and has no purification process, the subsequent PCR amplification reaction is directly carried out after the DNA extracting solution is added into a sample, and the Ct value is compared, so that the DNA extracting solution provided by the invention can keep more amount of the sample genome DNA in the extraction process compared with a cracking boiling method and a kit extraction method. Therefore, the DNA extraction solution provided by the invention can effectively release nucleic acid in a sample and reduce the loss of nucleic acid DNA.

TABLE 1

Example six:

comparative validation of genomic DNA extracted from buccal swab samples using two methods (common PCR assay for porphyromonas gingivalis):

this example compares the method for extracting DNA using the genomic DNA extract solution provided by the present invention with the lysis boiling method. The method comprises the following specific steps:

1. and (3) sample subpackaging: 6 oral swab samples were taken, each oral swab sample was mixed with 1 part of cell preservation solution (iclannehcy, shenzhen huachenyang science and technology ltd), and each mixed solution was dispensed into 2 centrifuge tubes. The subpackaging steps of each oral swab sample are as follows: 2 1.5mL centrifuge tubes were prepared, 1mL of a preservative solution (iCleanhcy, Shenzhen Huachenyang science and technology Co., Ltd.) containing exfoliated buccal cells was mixed well, and then the mixture was dispensed into each 1.5mL centrifuge tube, and the names were given separately. Wherein, the first sample is divided into 2 centrifuge tubes, which are respectively marked as Q-01 and L-01; the second sample is divided into 2 centrifuge tubes, which are respectively marked as Q-02 and L-02; the third sample is divided into 2 centrifuge tubes, which are marked as Q-03 and L-03 respectively; the fourth sample is divided into 2 centrifuge tubes, and the samples are marked as Q-04 and L-04 respectively; the fifth sample is divided into 2 centrifuge tubes, which are marked as Q-05 and L-05 respectively; the sixth sample was dispensed into 2 centrifuge tubes, labeled Q-06 and L-06, respectively.

2. And (3) centrifuging the sample: the 12 samples after dispensing were centrifuged at 13000rpm for 10min, and the supernatant was discarded (removed as clean as possible).

3. Different methods were used for DNA extraction:

3.1 extracting DNA with the genomic DNA extract solution provided by the invention

The DNA was extracted from the samples numbered Q-01, Q-02, Q-03, Q-04, Q-05 and Q-06 in the same manner as in example five 3.1.

3.2 DNA extraction by lysis boiling

The samples of numbers L-01, L-02, L-03, L-04, L-05 and L-06 were sampled and extracted for DNA in the same manner as in example five 3.2.

4. The general PCR detection of Porphyromonas gingivalis was performed on genomic DNA obtained by different methods:

1 μ L of the genomic DNA of the samples obtained in steps 3.1 and 3.2 of the experiment of this example was taken to prepare a common PCR reaction system, mixed by vortex shaking and mixed in BioRad CFX96^TMAnd performing real-time fluorescent quantitative PCR amplification analysis on the real-time PCR system. 2 XTaq Plus Master Mix (Vazyme, P-212-01) was purchased from Biotech GmbH of Nanjing Novowed; common PCR primers were synthesized by Jinzhi Biotechnology, Inc., Suzhou.

A common PCR reaction system (25. mu.L total) was as follows:

composition (I)	Volume (μ L)
		Upstream primer (10. mu.M)	0.5
Downstream primer (10. mu.M)	0.5
		Taq Plus Master Mix(2×)	12.5
DEPC water	10.5

Wherein:

an upstream primer: 5'-AGGCAGCTTGCCATACTGCG-3', respectively;

a downstream primer: 5'-ACTGTTAGCAACTACCGATGT-3' are provided.

The PCR reaction conditions are as follows:

at 95 ℃ for 10 min; 36 cycles of 95 ℃, 20s, 60 ℃, 20s, 72 ℃, 20 s; 72 ℃ for 10 min.

The PCR product was electrophoresed through 1.5% agarose gel at 90V for 30 min. In the gel imaging system, an electropherogram was taken and the results are shown in FIG. 6.

And (4) analyzing results: through common PCR detection of sample genomes extracted by the two methods, the brightness of Q-03 and Q-04 bands of a positive sample in the picture 6 is basically equivalent to that of L-03 and L-04 bands, and the brightness of Q-05 and Q-06 bands is slightly stronger than that of L-05 and L-06 bands. Therefore, the DNA extraction solution provided by the invention can effectively release nucleic acid in a sample and reduce the loss of nucleic acid DNA.

The genome DNA extracting solution and the application thereof provided by the invention solve the problems of time consumption, labor waste, high reagent cost, easy genome loss or pollution, influence on gene detection speed and efficiency and the like of the existing genome extraction and purification technology. The invention reduces operation steps to a great extent, basically reserves the initial genome quantity in a sample, increases the detection sensitivity and reduces the time and reagent cost; the subsequent gene detection can be directly carried out by the method, and the stability and the sensitivity of the detection result can not be influenced, so that the method is more beneficial to the detection and analysis of trace samples and large-scale sample genome.

The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims

1. A genome DNA extracting solution is characterized by comprising 5-10 mM Tris, 1-5 mM Ethylene Diamine Tetraacetic Acid (EDTA), 1-2.5 mM magnesium sulfate and sterile water, wherein the pH value of the Tris is 8.0-9.0.

2. The genomic DNA extract solution according to claim 1, wherein the concentration of EDTA is 1 mM.

3. A method for extracting genomic DNA, comprising the steps of:

collecting a sample;

4. The method for extracting genomic DNA according to claim 3, wherein the volume ratio of the genomic DNA extract to the sample is 5:1 to 1: 1.

5. The method for extracting genomic DNA according to claim 3, wherein the mixed solution is mixed uniformly by any one of vortex shaking and pipette tip blowing.

6. The method for extracting genomic DNA according to any one of claims 3 to 5, wherein the sample is collected and centrifuged to obtain a precipitate of the sample.

7. The method for extracting genomic DNA according to any one of claims 3 to 5, wherein the sample is derived from one or a mixture of both of human and bacteria.

8. The method for extracting genomic DNA according to claim 7, wherein the sample is derived from a human, and comprises any one or a mixture of any two or more of mouthwash, gingival crevicular fluid, oral or nasopharyngeal swab, and tissue pricking eluate.

9. The method for extracting genomic DNA according to claim 7, wherein the sample is derived from bacteria, including bacterial cultures.

10. A method for detecting Porphyromonas gingivalis, characterized in that the genomic DNA of Porphyromonas gingivalis is extracted by the genomic DNA extraction method according to any one of claims 3 to 9.