CN110643599B - Extraction method of fecal microorganism genome DNA suitable for PCR amplification - Google Patents
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Abstract
The invention relates to an extraction method of fecal microorganism genome DNA suitable for PCR amplification, which comprises the following steps: step S1: collecting a fecal sample; step S2: adopting PBS buffer solution and ethanol to carry out centrifugal re-suspension on the fecal sample in the step S1 for a plurality of times to obtain a precipitate, and utilizing Tris-HCl to re-suspend and dissolve the precipitate to obtain bacterial liquid containing microorganisms in the fecal sample; step S3: sequentially adding colloidal gold solution and proteinase K into the bacterial liquid in the step S2, uniformly mixing, heating and centrifuging to obtain supernatant, and diluting the supernatant by using Tris-HCl to obtain the microbial genome DNA which can be used as a PCR amplification template. Compared with the prior art, the method is extremely simple and convenient to operate and good in stability, is a practical and reliable fecal microorganism DNA extraction method, and can be widely applied to the research of intestinal microecology.
Description
Technical Field
The invention relates to the related fields of scientific research, clinical diagnosis, drug development and the like of intestinal microorganisms, in particular to an extraction method of fecal microorganism genome DNA suitable for PCR amplification.
Background
The human intestinal canal is covered with a large number of intestinal flora with various types, and a mutually beneficial symbiotic survival relationship is formed between the intestinal flora and the human body, so that the human intestinal canal has important influence on the health condition of the human body. With the continuous and intensive research on the influence of intestinal flora on human health in recent years, more than 30 diseases have been found to be related to the intestinal ecosystem. Thus, the physiological health of each individual is regulated by the intestinal flora in the individual's body, in addition to the effect of the individual's own gene regulation. The intestinal flora in the digestive system has even been referred to as the "human second genome", which is associated not only with intestinal dysfunction and metabolism, but also with tumors and even with diseases of the mental nervous system. It is known that about 1000-1150 bacteria species exist in the intestinal tract of a human body, wherein the dominant species is about 160 species, and the distribution of the microbial species in the intestinal tract is in a relatively balanced state under the normal physiological state of a healthy human body, but the bacterial species are accompanied by obvious dysregulation of the flora during the disease period of various diseases of the human body.
With the increasing perfection of high-throughput sequencing technology in recent years, the method for quantitatively analyzing the abundance of the intestinal microorganisms of a human body by utilizing a method for carrying out PCR amplification and library-building sequencing on the 16S rRNA genes of the microorganisms is widely applied, and the method eliminates the limitation that the microorganisms can be identified only after being separated and cultured, and provides a powerful research tool for further researching the interrelationship between the intestinal microorganisms and the physiological health of the human body. However, for most nucleic acid-related biotechnology, the quality of DNA extraction can have a major impact on the end result. It is known that various compounds represented by phenols existing in human intestinal feces have remarkable inhibition effect on subsequent PCR amplification. Therefore, there are many kits designed specifically for extracting microbial DNA from intestinal feces on the market, and these kits generally provide microbial genomic DNA with higher purity, but the procedures are often complicated, time-consuming, labor-consuming and expensive. Therefore, on the premise of not affecting the PCR amplification efficiency, the method for extracting the fecal microorganism DNA has the advantages of simple steps, time and labor saving and relatively low cost, and has important practical application value.
Patent CN1632581a discloses a method for extracting DNA of a prokaryotic microorganism in a sample and a special reagent thereof. The special reagent for extracting the DNA of the prokaryotic microorganism in the sample is an anti-DNA monoclonal antibody marked by colloidal gold; the anti-DNA monoclonal antibody is obtained by immunizing mice with a conjugate of a prokaryotic microorganism genomic DNA and Salmonella (Minesota strain) as an antigen. A method for extracting DNA from a prokaryotic microorganism in a sample using the reagent, comprising the steps of: 1) Lysing prokaryotic microorganism cells in a sample to obtain a lysate containing prokaryotic microorganism DNA; 2) Adsorbing the prokaryotic microorganism DNA in the lysate obtained in the step 1) by using the colloidal gold labeled anti-DNA monoclonal antibody. The immune colloidal gold adsorbed with DNA obtained by the method can be directly used for PCR amplification. However, the invention needs to prepare the anti-DNA monoclonal antibody marked by colloidal gold, and the preparation process is complex and the price is high. Calf serum is also used in the invention to eliminate the influence of PCR inhibitors, and the price is not low. Compared with the invention, the invention only uses colloid Jin Zheyi reagent except conventional biochemical reagent, avoids the complicated monoclonal antibody preparation process, has low cost and obviously has higher practical application value.
Disclosure of Invention
The invention aims to solve the problems and provide an extraction method of fecal microorganism genome DNA suitable for PCR amplification, which has simple steps, time and labor saving, relatively low cost and no influence on the subsequent PCR amplification reaction.
The aim of the invention is achieved by the following technical scheme:
an extraction method of fecal microorganism genomic DNA suitable for PCR amplification, the extraction method comprising the steps of:
step S1: collecting a fecal sample;
step S2: adopting PBS buffer solution and ethanol to carry out centrifugal re-suspension on the fecal sample in the step S1 for a plurality of times to obtain a precipitate, and utilizing Tris-HCl to re-suspend and dissolve the precipitate to obtain bacterial liquid containing microorganisms in the fecal sample;
step S3: sequentially adding colloidal gold solution and proteinase K into the bacterial liquid in the step S2, uniformly mixing, heating and centrifuging to obtain supernatant, and diluting the supernatant by using Tris-HCl to obtain the microbial genome DNA which can be used as a PCR amplification template.
Preferably, the step S1 specifically includes the following steps:
step S1 (a): performing high-pressure sterilization treatment on the sampling spatula and the sampling centrifuge tube to ensure that no other foreign strains pollute the sample spatula;
step S1 (b): placing the feces into a sterilized centrifuge tube by holding a sterilized sampling spatula, immediately covering the centrifuge tube after filling the centrifuge tube with 1/5-1/2 of the volume, putting the centrifuge tube into dry ice for freezing, and then preserving the centrifuge tube in an environment of an ultralow temperature refrigerator at-80 ℃ to obtain a feces sample. The freezing storage is favorable for the strain not to deteriorate.
Preferably, the step S2 specifically includes the following steps:
step S2 (a): taking a certain amount of fecal sample, adding 3.5-4.5 ℃ precooled PBS buffer solution into the sample, fully oscillating and uniformly mixing after the sample is melted, centrifuging for 12-18min at 3.5-4.5 ℃ with a centrifugal force of 80-120g, sucking supernatant, continuously centrifuging for 8-12min at 3.5-4.5 ℃ with a centrifugal force of 12000-14000g, and discarding the supernatant to obtain a tube bottom sediment;
step S2 (b): adding 3.5-4.5 ℃ precooled PBS buffer solution into the tube bottom sediment in the step S2 (a), re-suspending and centrifuging for 8-12min at the temperature of 3.5-4.5 ℃ by using 12000-14000g centrifugal force, discarding supernatant to obtain tube bottom sediment, adding 3.5-4.5 ℃ precooled ethanol into the tube bottom sediment, re-suspending and centrifuging for 8-12min at the temperature of 3.5-4.5 ℃ by using 12000-14000g centrifugal force, discarding supernatant to obtain tube bottom sediment, and repeating ethanol re-suspending and centrifuging operation on the tube bottom sediment to obtain final bacterial liquid sediment;
step S2 (c): and (3) carrying out resuspension dissolution on the bacterial liquid precipitate obtained in the step S2 (b) by adopting 10mM Tris-HCl to obtain bacterial liquid. Multiple times of resuspension is beneficial to the acquisition of microorganisms, can obtain microorganisms with fewer impurities, and is beneficial to the subsequent DNA extraction and PCR amplification;
the fecal sample in step S2 (a): the PBS buffer in step S2 (a): the PBS buffer in step S2 (b): the ethanol in step S2 (b): the addition ratio of Tris-HCl in the step S2 (c) is 1: (1/5-1/3) mL/mg: (1/40-5/40) mL/mg: (1/40-5/40) mL/mg: (4-6) mu L/mg.
Preferably, the pH of the PBS buffer in step S2 is in the range of 7.1-7.3, and the pH of the Tris-HCl is in the range of 7.9-8.1. The pH value does not damage cells and DNA, and is beneficial to maintaining the integrity of the DNA.
Preferably, the step S3 specifically includes the following steps:
step S3 (a): taking a certain amount of bacterial liquid, adding colloidal gold solution, fully oscillating and uniformly mixing, and heating at 90-100 ℃ for 8-12min to obtain a premixed solution;
step S3 (b): adding proteinase K into the pre-mixed solution in the step S3 (a), firstly, carrying out warm bath for 25-35min at 50-60 ℃, and then, heating for 8-12min at 90-100 ℃ to obtain a mixed solution;
step S3 (c): centrifuging the mixed solution obtained in the step S3 (b) for 2.5-3.5min by using a centrifugal force of 12000-14000g, taking supernatant, and diluting the supernatant by 90-110 times by using 10mM Tris-HCl to obtain the microbial genome DNA which can be used as a PCR amplification template;
the bacterial liquid in the step S3 (a): the colloidal gold solution in step S3 (a): the addition ratio of proteinase K in step S3 (b) is 1: (1/20-1): (1/400-1/600) mg/. Mu.L. At the high temperature, the nano heat transfer effect of the colloidal gold can quickly destroy cells of microorganisms, release of genome DNA is realized, and meanwhile, the influence of other inhibitors on subsequent PCR reactions can be eliminated by diluting supernatant.
Preferably, the colloidal gold solution in step S3 is a colloidal gold solution prepared in a sterile state, and the particle size of gold particles in the colloidal gold solution is in the range of 5-20nm. The nano heat transfer effect in this size range is more excellent.
Preferably, the final concentration of the colloidal gold solution in the step S3 after dilution is 1.5-15. Mu.g/mL.
Preferably, when the microbial genomic DNA extracted by the extraction method is used for PCR amplification reaction, the total volume of a PCR amplification reaction system is 25 mu L, and the PCR amplification reaction system comprises: 2-3U Taq polymerase, dATP, dCTP, dGTP and dTTP substrates in the concentration range of 180-220nM, and deoxyribonucleotide oligonucleotide primer in the concentration range of 180-220 nM.
Preferably, the thermal cycling conditions of the PCR amplification reaction are: preheating at 90-100deg.C for 1.5-2.5min, performing 30-45 cycles, and extending at 70-75deg.C for 4-6min. Further preferably, the cycle comprises: after denaturation at 90-100℃for 25-30s, annealing for 18-22s and extension at 70-75℃for 0.8-1.2min/Kb product length.
According to the invention, the release process of the microbial genome DNA is realized by adding colloidal gold and proteinase K into microbial flora washed by PBS and ethanol and then performing heat treatment, the nano heat transfer effect of the colloidal gold can rapidly break cells at high temperature, the rapid release of the genomic DNA is realized, the influence of various possible inhibitors in feces on the subsequent PCR reaction is eliminated by a dilution mode, and the diversity and community composition structure of microorganisms in intestinal feces can be comprehensively reflected. The colloidal gold solution used can be prepared by white phosphorus reduction, sodium citrate reduction or sodium citrate-tannic acid reduction, or can be prepared by commercial colloidal gold reagent such as colloidal gold solution sold by Sigma company in the United states (product number is G1527). In the actual operation process, the colloidal gold solution is prepared under the aseptic condition, so that the colloidal gold solution does not need to be sterilized again and does not need to be diluted before use.
Compared with the prior art, the invention has the following characteristics:
1) The method has the advantages that the colloidal gold is utilized to increase the microbial wall breaking efficiency, meanwhile, the simple dilution method is utilized to eliminate the influence of various potential inhibitors on the subsequent PCR in the extraction process, the operation is extremely simple and convenient, and the microbial DNA suitable for PCR amplification can be extracted from the excrement within one hour, so that the stability is good;
2) The colloidal gold can be stored for a long time, has stable property, is not easy to inactivate and has long service life;
3) The method has the advantages of low cost, easy preservation, wide application, easy separation of optimized materials from the system and good stability, is a practical and reliable fecal microorganism DNA extraction method, and can be widely applied to the research of intestinal microecology.
Drawings
FIG. 1 is a comparative diagram of the extraction method of genomic DNA of fecal microorganisms suitable for PCR amplification according to the present invention versus the extraction of genomic DNA of the same fecal sample using a commercial kit (Tiangen DNA extraction kit);
FIG. 2 is a comparison of PCR amplification assays using genomic DNA from different microorganisms extracted according to the present invention.
In the figure: 1-lane M1 (23 kb is the longest DNA fragment length in the DNA marker); 2-lane A; 3-lane B; 4-lane M2; 5-lane 1; 6-lane 2; 7-lane 3; 8-lane 4; 9-lane 5; 10-lane 6.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
Example 1
The extraction method of the fecal microorganism genome DNA suitable for PCR amplification comprises the following specific steps:
(1) Collection of fecal samples
Autoclaving a sampling spatula and a sampling centrifuge tube (5 mL);
during sampling, the disposable sterile latex gloves are worn by both hands, the sterilized sampling spatula is used for loading fecal excrement into a sterilized centrifuge tube, the centrifuge tube is immediately covered after being filled to half volume, the sterilized centrifuge tube is put into dry ice for freezing storage after marking, and the sterilized centrifuge tube is stored in an environment of minus 80 ℃ after being retrieved from a laboratory;
(2) Acquisition of microorganisms
Taking 20mg of fecal sample, adding 5mL of PBS buffer solution (pH 7.2) precooled at 4 ℃ into the fecal sample, fully vortex oscillating and uniformly mixing after the sample is melted, and centrifuging for 15min at 4 ℃ under the centrifugal force of 100g, and sucking the supernatant;
centrifuging the supernatant at 13000g at 4deg.C for 10min, discarding supernatant, adding 1.5mL pre-cooled PBS buffer solution (pH 7.2) at 4deg.C into the bottom sediment, and centrifuging at 13000g at 4deg.C for 10min;
removing supernatant, adding 1.5mL of ethanol precooled at 4 ℃ for resuspension, centrifuging at 4 ℃ for 10min by using 13000g of centrifugal force, and removing supernatant;
after repeating the ethanol resuspension step described above, the bacterial pellet was resuspended and dissolved with 100. Mu.L of 10mM Tris-HCl (pH 8.0).
(3) Extraction of fecal microorganism genomic DNA
Adding colloidal gold solution with the diameter of 10nm into 100 mu L of the resuspended bacterial liquid, fully vortex oscillating and uniformly mixing, and heating at 95 ℃ for 10min;
then adding 0.2mg proteinase K, then carrying out a warm bath at 56 ℃ for 30min, and finally heating at 95 ℃ for 10min;
the supernatant was collected by centrifugation at 13000g for 3 min.
Comparing the extracted fecal microorganism genome DNA with DNA extracted from the same fecal sample by agarose gel electrophoresis using a root DNA extraction kit, the results are shown in FIG. 1:
lane M1 is the molecular weight marker (bottom to top, respective, 514 bp,2027bp,2322bp,4361bp,6557bp,9416bp,23130 bp) of lambda phage DNA digested with HindIII restriction enzyme, lane A is fecal microorganism genomic DNA extracted using the Tiangen DNA extraction kit, and lane B is fecal microorganism genomic DNA extracted using the method of the present invention. It is evident that the brightness of the main light band in lane B at about 23kb is far higher than that of the main light band in lane A at about 23kb, i.e.the total amount of fecal microorganism DNA obtained by the method of the present invention is significantly higher than that obtained by the commercial radix Santali DNA extraction kit.
Example 2
The extraction method of the fecal microorganism genome DNA suitable for PCR amplification comprises the following specific steps:
(1) Collection of fecal samples
Autoclaving a sampling spatula and a sampling centrifuge tube (5 mL);
during sampling, the disposable sterile latex gloves are worn by both hands, the sterilized sampling spatula is used for loading fecal excrement into a sterilized centrifuge tube, the centrifuge tube is immediately covered after being filled to half volume, the sterilized centrifuge tube is put into dry ice for freezing storage after marking, and the sterilized centrifuge tube is stored in an environment of minus 80 ℃ after being retrieved from a laboratory;
(2) Acquisition of microorganisms
Taking 20mg of fecal sample, adding 5mL of PBS buffer solution (pH 7.2) precooled at 4 ℃ into the fecal sample, fully vortex oscillating and uniformly mixing after the sample is melted, and centrifuging for 15min at 4 ℃ under the centrifugal force of 100g, and sucking the supernatant;
centrifuging the supernatant at 13000g at 4deg.C for 10min, discarding supernatant, adding 1.5mL pre-cooled PBS buffer solution (pH 7.2) at 4deg.C into the bottom sediment, and centrifuging at 13000g at 4deg.C for 10min;
removing supernatant, adding 1.5mL of ethanol precooled at 4 ℃ for resuspension, centrifuging at 4 ℃ for 10min by using 13000g of centrifugal force, and removing supernatant;
after repeating the ethanol resuspension step described above, the bacterial pellet was resuspended and dissolved with 100. Mu.L of 10mM Tris-HCl (pH 8.0).
(3) Extraction of fecal microorganism genomic DNA
Adding colloidal gold solution with the diameter of 10nm into 100 mu L of the resuspended bacterial liquid, fully vortex oscillating and uniformly mixing, and heating at 95 ℃ for 10min;
then adding 0.2mg proteinase K, then carrying out a warm bath at 56 ℃ for 30min, and finally heating at 95 ℃ for 10min;
the supernatant was collected by centrifugation at 13000g for 3 min.
Diluting the extracted fecal microorganism genome DNA with 10mM Tris-HCl (pH 8.0) 100 times;
1. Mu.L of the above diluted solution was added with 2U of Taq polymerase and 2.5. Mu.L of 10 Xreaction buffer, 2. Mu.L of dNTP (containing dATP, dCTP, dGTP and 2.5mM of dTTP each), 0.5. Mu.L of 10. Mu.M primers (for amplifying Mycobacterium, bacteroides, bifidobacterium, bluet, E.coli and faecalis respectively), and finally the volume was fixed to 25. Mu.L with water;
preheating the PCR reaction system at 95 ℃ for 2 minutes, then performing 35 cycles, and finally extending at 72 ℃ for 5 minutes; wherein each cycle comprises: denaturation at 95℃for 30 seconds, annealing at 60℃for 20 seconds, extension at 72℃for 15 seconds;
the PCR reaction products were analyzed by agarose gel electrophoresis, and the results are shown in FIG. 2:
lane M2 is a molecular weight marker (500 bp,400bp,350bp,300bp,250bp,200bp,150bp,100bp,50bp, respectively from top to bottom). Lane 1 is another bacillus amplification product, lane 2 is a bacteroides amplification product, lane 3 is a bifidobacterium amplification product, lane 4 is a lauteromycete amplification product, lane 5 is an escherichia coli amplification product, and lane 6 is a faecalis amplification product. As can be seen from FIG. 2, all lanes 1-6 have luminescent bands which meet the expected size, namely, the target strain bands can be amplified from the fecal microorganism genomic DNA extracted by the method of the invention by a PCR method, which proves that the fecal microorganism genomic DNA extracted by the method of the invention has reliable and stable quality.
Example 3
The extraction method of the fecal microorganism genome DNA suitable for PCR amplification comprises the following specific steps:
(1) Collection of fecal samples
Autoclaving a sampling spatula and a sampling centrifuge tube (5 mL);
during sampling, the disposable sterile latex gloves are worn by both hands, the sterilized sampling spatula is used for loading fecal excrement into a sterilized centrifuge tube, the centrifuge tube is immediately covered after 1/3 volume of the centrifuge tube is filled, the sterilized centrifuge tube is put into dry ice for freezing storage after marking, and the sterilized centrifuge tube is stored in an environment of minus 80 ℃ after being retrieved from a laboratory;
(2) Acquisition of microorganisms
Taking 40mg of fecal sample, adding 8ml of PBS buffer solution (pH 7.1) precooled at 3.5 ℃ into the fecal sample, fully vortex oscillating and uniformly mixing after the sample is melted, centrifuging for 18min at 3.5 ℃ by using a centrifugal force of 120g, and sucking the supernatant;
centrifuging the supernatant at 3.5deg.C with 12000g for 12min, discarding supernatant, adding 5mL of 3.5deg.C pre-cooled PBS buffer (pH 7.1) into the precipitate at the bottom of the tube, and centrifuging at 3.5deg.C with 12000g for 12min;
discarding supernatant, adding 5mL of ethanol precooled at 3.5deg.C for resuspension, centrifuging at 3.5deg.C for 12min with 12000g centrifugal force, discarding supernatant;
after repeating the ethanol resuspension step described above, the bacterial pellet was resuspended and dissolved with 160. Mu.L of 10mM Tris-HCl (pH 8.1).
(3) Extraction of fecal microorganism genomic DNA
Adding 160 mu L of colloidal gold solution with the diameter of 20nm into 160 mu L of the re-suspended bacterial liquid, fully vortex oscillating and uniformly mixing, and heating at 100 ℃ for 12min;
then adding 0.4mg proteinase K, then heating at 50deg.C for 35min, and finally heating at 90deg.C for 12min;
centrifuging for 3.5min by using a centrifugal force of 12000g, taking supernatant, and diluting the supernatant by 110 times by using 10mM Tris-HCl to obtain the microbial genome DNA which can be used as a PCR amplification template.
The whole extraction method is simple and convenient to operate, microbial DNA suitable for PCR amplification can be extracted from feces in half an hour, 1 mu L of the dilution is taken, 3U of Taq polymerase and 2.5 mu L of 10x reaction buffer solution are added, 2 mu L of dNTP (containing dATP, dCTP, dGTP and dTTP respectively 2.5 mM) are added, 10 mu M of primers are respectively 0.5 mu L, and finally water is used for fixing the volume to 23 mu L; preheating the PCR reaction system at 100 ℃ for 1.5 minutes, then performing 30 cycles, and finally extending at 70 ℃ for 4 minutes; wherein each cycle comprises: denaturation at 100℃for 30 seconds, annealing at 60℃for 22 seconds, and extension at 70℃for 15 seconds. The light of the lane luminous stripes can be found to be high, namely the extraction quantity of the microorganism genome DNA is high, and the amplification effect is obvious.
Example 4
The extraction method of the fecal microorganism genome DNA suitable for PCR amplification comprises the following specific steps:
(1) Collection of fecal samples
Autoclaving a sampling spatula and a sampling centrifuge tube (5 mL);
during sampling, the disposable sterile latex gloves are worn by both hands, the sterilized sampling spatula is used for loading fecal excrement into a sterilized centrifuge tube, the centrifuge tube is immediately covered after 1/5 volume of the centrifuge tube is filled, marked and put into dry ice for freezing storage, and the laboratory is retrieved and then stored in the environment of-80 ℃;
(2) Acquisition of microorganisms
Taking 12mg of fecal sample, adding 4ml of 4.5 ℃ precooled PBS buffer solution (pH 7.3) into the fecal sample, fully vortex oscillating and uniformly mixing after the sample is melted, centrifuging for 12min at 4.5 ℃ under the centrifugal force of 80g, and sucking the supernatant;
centrifuging the supernatant at 14000g at 4.5deg.C for 8min, discarding supernatant, adding 0.3mL of 4.5deg.C pre-cooled PBS buffer (pH 7.3) into the bottom sediment, and centrifuging at 14000g at 4.5deg.C for 8min;
discarding supernatant, adding 0.3mL of ethanol precooled at 4.5 ℃ for resuspension, centrifuging at 4.5 ℃ for 8min by using 14000g of centrifugal force, and discarding supernatant;
after repeating the ethanol resuspension step described above, the bacterial pellet was resuspended and dissolved with 72. Mu.L of 10mM Tris-HCl (pH 7.9).
(3) Extraction of fecal microorganism genomic DNA
Adding 3.6 mu L of colloidal gold solution with the diameter of 5nm into the resuspended 72 mu L of bacterial liquid, fully vortex oscillating and uniformly mixing, and heating at 90 ℃ for 8min;
then adding 0.12mg proteinase K, then heating at 100deg.C for 8min, and then heating at 60deg.C for 25 min;
after centrifugation at 14000g for 2.5min, the supernatant was diluted 90-fold with 10mM Tris-HCl to obtain a microbial genomic DNA which was used as a template for PCR amplification.
The whole extraction method is simple and convenient to operate, target microorganism genome DNA is used for PCR amplification, 1 mu L of the diluent is added with 3U Taq polymerase and 2.5 mu L10 x reaction buffer solution, 2 mu L dNTP (containing dATP, dCTP, dGTP and dTTP 2.5mM respectively) and 10 mu M primers 0.5 mu L respectively, and finally water is used for constant volume to 27 mu L; preheating the PCR reaction system at 90 ℃ for 2.5 minutes, then carrying out 45 cycles, and finally extending at 75 ℃ for 6 minutes; wherein each cycle comprises: denaturation at 90℃for 25 seconds, annealing at 60℃for 18 seconds, and extension at 75℃for 15 seconds. All lanes can be found to have luminous stripes, which indicates that the quality of the microbial genome DNA extracted from the feces is reliable and stable.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (6)
1. A method for extracting genomic DNA of fecal microorganisms suitable for PCR amplification, characterized in that it comprises the following steps:
step S1: collecting a fecal sample;
step S2: adopting PBS buffer solution and ethanol to carry out centrifugal re-suspension on the fecal sample in the step S1 for a plurality of times to obtain a precipitate, and utilizing Tris-HCl to re-suspend and dissolve the precipitate to obtain bacterial liquid containing microorganisms in the fecal sample; the method specifically comprises the following steps:
step S2 (a): taking a certain amount of fecal sample, adding 3.5-4.5 ℃ precooled PBS buffer solution into the sample, fully oscillating and uniformly mixing after the sample is melted, centrifuging for 12-18min at 3.5-4.5 ℃ with a centrifugal force of 80-120g, sucking supernatant, continuously centrifuging for 8-12min at 3.5-4.5 ℃ with a centrifugal force of 12000-14000g, and discarding the supernatant to obtain a tube bottom sediment;
step S2 (b): adding 3.5-4.5 ℃ precooled PBS buffer solution into the tube bottom sediment in the step S2 (a), re-suspending and centrifuging for 8-12min at the temperature of 3.5-4.5 ℃ by using 12000-14000g centrifugal force, discarding supernatant to obtain tube bottom sediment, adding 3.5-4.5 ℃ precooled ethanol into the tube bottom sediment, re-suspending and centrifuging for 8-12min at the temperature of 3.5-4.5 ℃ by using 12000-14000g centrifugal force, discarding supernatant to obtain tube bottom sediment, and repeating ethanol re-suspending and centrifuging operation on the tube bottom sediment to obtain final bacterial liquid sediment;
step S2 (c): carrying out resuspension dissolution on the bacterial liquid precipitate obtained in the step S2 (b) by adopting 10mM Tris-HCl to obtain bacterial liquid;
the fecal sample in step S2 (a): the PBS buffer in step S2 (a): the PBS buffer in step S2 (b): the ethanol in step S2 (b): the addition ratio of Tris-HCl in the step S2 (c) is 1: (1/5-1/3) mL/mg: (1/40-5/40) mL/mg: (1/40-5/40) mL/mg: (4-6) μL/mg;
step S3: sequentially adding colloidal gold solution and proteinase K into the bacterial liquid in the step S2, uniformly mixing, heating and centrifuging to obtain supernatant, and diluting the supernatant by using Tris-HCl to obtain the microbial genome DNA which can be used as a PCR amplification template; the method specifically comprises the following steps:
step S3 (a): taking a certain amount of bacterial liquid, adding colloidal gold solution, fully oscillating and uniformly mixing, and heating at 90-100 ℃ for 8-12min to obtain a premixed solution;
step S3 (b): adding proteinase K into the pre-mixed solution in the step S3 (a), firstly, carrying out warm bath for 25-35min at 50-60 ℃, and then, heating for 8-12min at 90-100 ℃ to obtain a mixed solution;
step S3 (c): centrifuging the mixed solution obtained in the step S3 (b) for 2.5-3.5min by using a centrifugal force of 12000-14000g, taking supernatant, and diluting the supernatant by 90-110 times by using 10mM Tris-HCl to obtain the microbial genome DNA which can be used as a PCR amplification template;
the bacterial liquid in the step S3 (a): the colloidal gold solution in step S3 (a): the proteinase K was added in a ratio of 1. Mu.L in step S3 (b): (1/20-1) μL: (1/400-1/600) mg;
the colloidal gold solution in the step S3 is prepared under a sterile state, and the particle size range of gold particles in the colloidal gold solution is 5-20nm;
the final concentration of the colloidal gold solution in the step S3 after dilution is 1.5-15 mug/mL.
2. The method for extracting genomic DNA from fecal microorganisms suitable for PCR amplification according to claim 1, wherein the step S1 comprises the following steps:
step S1 (a): performing high-pressure sterilization treatment on the sampling spatula and the sampling centrifuge tube;
step S1 (b): placing the feces into a sterilized centrifuge tube by holding a sterilized sampling spatula, immediately covering the centrifuge tube after filling the centrifuge tube with 1/5-1/2 of the volume, putting the centrifuge tube into dry ice for freezing, and then preserving the centrifuge tube in an environment of an ultralow temperature refrigerator at-80 ℃ to obtain a feces sample.
3. The method of claim 1, wherein the PBS buffer in step S2 has a pH of 7.1-7.3 and Tris-HCl has a pH of 7.9-8.1.
4. The method for extracting genomic DNA from fecal microorganisms suitable for PCR amplification according to any one of claims 1 to 3, wherein the total volume of the PCR amplification reaction system is 25. Mu.L when the genomic DNA from microorganisms extracted by the extraction method is used in the PCR amplification reaction, the PCR amplification reaction system comprises: 2-3U Taq polymerase, dATP, dCTP, dGTP and dTTP substrates in the concentration range of 180-220nM, and primers in the concentration range of 180-220 nM.
5. The method for extracting genomic DNA from fecal microorganisms suitable for PCR amplification according to claim 4, wherein the thermal cycling conditions of the PCR amplification reaction are as follows: preheating at 90-100deg.C for 1.5-2.5min, performing 30-45 cycles, and extending at 70-75deg.C for 4-6min.
6. The method of claim 5, wherein the cycling comprises: after denaturation at 90-100℃for 25-30s, annealing for 18-22s and extension at 70-75℃for 0.8-1.2min/Kb product length.
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