CN112680437A - Method for rapidly releasing gram-positive strain intracellular DNA - Google Patents
Method for rapidly releasing gram-positive strain intracellular DNA Download PDFInfo
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Abstract
The invention discloses a method for quickly releasing gram-positive strain intracellular DNA, which is implemented by ddH2O, ET and ETS buffer and the physical temperature changing method rapidly prepare PCR amplification templates through single colonies, directly skip the steps of strain amplification and DNA extraction, are rapid and convenient, and greatly reduce the detection period, the detection cost and the labor output.
Description
Technical Field
The invention belongs to the technical field of aquaculture, and particularly relates to a method for rapidly releasing gram-positive strain intracellular DNA.
Background
Bacillus (Bacillus), a genus of bacteria, gram-positive bacteria, are capable of forming spores (endospores). The bacillus can secrete various extracellular proteases, hydrolases, antibacterial peptides and the like, can effectively degrade organic matters in the water body and inhibit the proliferation of pathogenic bacteria in the culture water body, and can also efficiently assimilate inorganic nitrogen, hydrogen sulfide and other substances which have toxic action on economic animals in the culture water body to realize the water quality purification effect. Meanwhile, the method provides favorable conditions for clinical application in the fields of industrial production and aquaculture due to the characteristics of strong vitality, high tolerance degree, spore production and the like. The bacillus is one of the most widely applied strains in the aspects of water quality purification, intestinal flora regulation and the like in the field of aquaculture as a heterotrophic microorganism strain.
In recent years, with the rapid development of aquaculture industry in China, bacillus microecological preparation products are used in a large amount in the culture process of economic fishes, shrimps, crabs, shellfish and the like, and the application effect of the bacillus microecological preparation products is accepted by a plurality of researchers and farmers. In the preparation and application processes of the bacillus microecological preparation, the pollution evaluation and the product effect evaluation are carried out by regularly monitoring the target strains in the corresponding processes. The bacillus serving as gram-positive bacteria can not break the wall of the strain only by heating, and an effective wall-breaking gram-positive bacteria kit is not available temporarily, so that wall breaking and DAN release can be performed on the gram-positive bacteria only by a liquid nitrogen grinding mode. This process requires a significant investment in time costs, reagent costs, labor costs, and the like.
The invention passes only ddH2O, ET and ETS, and the DNA release of gram-positive bacteria is realized by respectively carrying out repeated freeze thawing, thereby reducing the detection cost, shortening the detection period and providing a feasible method for the rapid identification of gram-positive bacteria.
The conventional bacterial culture identification process is still remained in the conventional bacterial culture identification process, and the evaluation process relates to the steps of massive selection and amplification of single colonies and subsequent DNA extraction and identification, and mainly comprises the following steps:
(1) performing plate coating and single colony culture on samples such as the body surface, intestinal tracts and the like of economic animals in a water body, and picking up the samples for 24-48h after colonies grow into usable inoculating loops;
(2) the picked single colony was subjected to amplification culture using a bacterial flask, OD600More than 1.0 (24-48 h);
(3) collecting thalli in the bacterial liquid and performing DNA extraction by using a bacterial DNA extraction kit (2-4 h);
(4) carrying out PCR amplification and gel electrophoresis detection on the extracted DNA by using a 16S universal primer (3-5 h);
(5) sent to a DNA nucleic acid sequence detection company and subjected to sequencing analysis.
(6) Using NCBI _ BLAST: https:// blast.ncbi.nlm.nih.gov/blast.cgi online software compares and analyzes the 16S sequence obtained by sequencing to determine the strain name.
The conventional 16S strain identification technology used at the present stage has the characteristics of high sensitivity, strong specificity, simple operation and the like, and is widely applied to various industries. However, this detection method has an effect only on gram-negative bacteria detection, and cannot directly detect gram-positive bacteria, and the preparation of DNA templates involved in the identification of gram-positive bacteria has become an important resistance for gram-positive bacteria detection.
Therefore, the identification of strains in the aquaculture industry at present is difficult to popularize and popularize due to the characteristics of high detection frequency, long detection period, large sample quantity, high detection cost, large manual investment and the like, and reagents and kits which have no special effect on wall breaking of gram-positive bacteria and DNA extraction in the market at present are available. Therefore, the conventional DNA extraction steps and identification methods are not suitable for identifying gram-positive bacteria.
Disclosure of Invention
The object of the present invention is to provide a method for the rapid release of intracellular DNA from gram-positive bacteria by the addition of dd H2O, ET and ETS buffer and the physical temperature changing method rapidly prepare PCR amplification templates through single colonies, directly skip the steps of strain amplification and DNA extraction, are rapid and convenient, and greatly reduce the detection period, the detection cost and the labor output.
The above object of the present invention can be achieved by the following technical solutions: a method for rapidly releasing intracellular DNA of a gram-positive bacterial species comprising the steps of:
(1) selecting a sample to be detected, and performing flat plate coating or scribing by adopting a solid culture medium;
(2) sealing the flat plate by using a sealing film, and placing the flat plate in an incubator for culture;
(3) taking out the plate, picking out single colony and inoculating in the medium containing ddH2O in Eppendorf tube, blowing off single colony, putting in ice-water mixture, putting in freezer, and cooling to obtain ddH2Condensing O into ice, standing, rapidly placing in a hot water bath to rapidly increase the temperature, and then standing;
(4) removal of ddH containing Single colony2Transferring O into an Eppendorf tube containing ET solution, placing the Eppendorf tube in an ice-water mixture, placing the Eppendorf tube in a freezing device to quickly reduce the temperature of the Eppendorf tube, standing the Eppendorf tube after the solution in the test tube is condensed into ice, quickly placing the Eppendorf tube in a hot water bath to quickly increase the temperature of the Eppendorf tube, and then standing the Eppendorf tube;
(5) adding sodium dodecyl sulfate into the Eppendorf tube containing the ET solution in the step (4), placing the tube in an ice-water mixture, placing the tube in a freezing device to quickly reduce the temperature of the tube, standing the tube after the solution in the tube is condensed into ice, quickly placing the tube in a hot water bath to quickly increase the temperature of the tube, and then standing the tube;
(6) taking the supernatant to obtain the gram-positive strain intracellular DNA.
In the above method for rapidly releasing intracellular DNA of gram-positive bacterial species:
preferably, the sample to be detected in the step (1) is a gram-positive bacterium microecological preparation or a water sample of an aquaculture water body.
Preferably, the sample to be detected in the step (1) can also be an aquatic product, and the aquatic product preferably adopts the body surface, intestinal contents and the like of the aquatic product, and is preferably scraped by an inoculating loop.
Preferably, the solid medium in step (1) is LB solid medium or TCBS solid medium.
Different solid culture media, preferably LB solid culture media or TCBS solid culture media, can be adopted according to different samples to be tested for culturing single colonies.
Preferably, in the step (2), the culture is carried out in an incubator at 28-37 ℃ for 12-24 h.
More preferably, the step (2) is carried out in an incubator at 32 ℃ for 12 hours.
Preferably, the plate is taken out in the step (3), and a single colony is selected and inoculated in a medium containing 30-50 muL ddH2O in an Eppendorf tube, blowing off single colonies, placing the single colonies in an ice-water mixture for 3-5 minutes, placing the mixture in a refrigerating device, rapidly reducing the temperature of the mixture, and waiting for ddH in a test tube2And (3) after the O is condensed into ice, standing for 5-10 min, quickly placing in a hot water bath, quickly raising the temperature to 95-100 ℃, and then standing for 10-20 min.
Preferably, the Eppendorf tube is a 1.5mLEppendorf tube.
Preferably, the ddH containing a single colony is taken out in step (4)2And transferring the O into an Eppendorf tube containing 30-50 mu L of ET solution, placing the Eppendorf tube in an ice water mixture for 3-5 minutes, placing the Eppendorf tube in a freezing device to reduce the temperature of the Eppendorf tube quickly, standing the Eppendorf tube for 5-10 minutes after the solution in the tube condenses into ice, placing the Eppendorf tube in a hot water bath quickly to increase the temperature of the Eppendorf tube to 95-100 ℃, and standing the Eppendorf tube for 10-20 minutes.
Preferably, the ET solution in step (4) comprises EDTA and Tris-Cl, wherein the final concentration of the EDTA and the Tris-Cl is 0.02 mol/L.
Preferably, in the step (5), sodium dodecyl sulfate is added into the Eppendorf tube containing the ET solution in the step (4) to enable the final concentration to be 3-4%. More preferably, the final concentration of Sodium Dodecyl Sulfate (SDS) is 3.33%, and the final concentration is a mass volume concentration such as that 3-4 g of Sodium Dodecyl Sulfate (SDS) is added into 100mL of water.
Preferably, in the step (5), sodium dodecyl sulfate is added into the Eppendorf tube containing the ET solution in the step (4), the tube is placed in an ice-water mixture for 3-5 minutes, then the tube is placed in a freezing device to rapidly reduce the temperature of the tube, after the solution in the tube is condensed into ice, the tube is placed still for 5-10 minutes, and then the tube is placed in a hot water bath to rapidly increase the temperature of the tube by 95-100 ℃, and then the tube is placed still for 10-20 minutes.
Preferably, the temperature is rapidly reduced in the steps (3) to (5) by placing the mixture in a freezer at a temperature of-20 ℃ or lower.
Wherein the refrigerating device below-20 deg.C can be refrigerator at-20 deg.C or refrigerator at-80 deg.C.
Preferably, the supernatant obtained in step (6) is further placed in a PCR tube containing a PCR reaction solution as a PCR template, and then placed in a PCR instrument for PCR amplification, wherein the denaturation temperature in the early non-cyclic reaction is 95 ℃ and the denaturation time is 10 min.
Compared with the prior art, the invention has the following advantages:
(1) the method of the invention can be directly realized by adding dd H2O, ET and ETS buffer and the physical temperature changing method rapidly prepare PCR amplification templates through single colonies, directly skip the steps of amplification culture and DNA extraction of the single colonies, and the existing kit has no ideal wall breaking effect on gram-positive bacteria;
the differences between the process of the present invention and the conventional process of the background art are shown in table 1 below;
TABLE 1 differences between the process of the invention and the conventional process of the background art
(2) The method of the invention provides convenience for industrial strain detection and product evaluation, greatly reduces labor force output, detection period, detection cost and the like, provides an important support means for preparation, scientific application and scientific evaluation of the microecological preparation, and simultaneously provides a feasible and easily popularized method for mass detection of strains in aquaculture water and intestinal flora in the aquaculture industry.
Drawings
FIG. 1 is a 16S rDNA PCR amplification and gel electrophoresis test chart showing the DNA of Bacillus prepared by the method for rapidly releasing intracellular DNA of gram-positive bacteria in example 1 as a template;
m: DL5000 marker; 1-6 are all gram-positive bacteria: 1: bacillus velezensis (Bacillus velezensis); 2: bacillus subtilis; 3: bacillus licheniformis (Bacillus licheniformis); 4: bacillus amyloliquefaciens (Bacillus amyloliquefaciens); 5: bacillus megaterium (Bacillus coaguluns); 6: bacillus firmus; 7 is negative control;
FIG. 2 is a 16S rDNA PCR amplification and gel electrophoresis detection map of Bacillus DNA prepared by the method for rapidly releasing gram-positive bacterial intracellular DNA of example 2 as a template;
m: DL5000 marker; 1: bacillus subtilis; 2: bacillus amyloliquefaciens (Bacillus amyloliquefaciens); 3: escherichia coli (Escherichia coli); 4: vibrio parahaemolyticus (Vibrio parahaemolyticus); 5: bacillus velezensis (Bacillus velezensis); 6: bacillus subtilis (Bacillus firmus), 7 is a negative control.
Detailed Description
Example 1
The method for rapidly releasing gram-positive bacterial intracellular DNA provided by the embodiment comprises the following steps:
(1) selecting 6 bacillus (gram positive bacteria) microecological preparation products, and coating a flat plate by using an LB solid culture medium (10 g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 15g/L of agar powder), wherein each sample is coated with one flat plate;
(2) sealing the flat plate by using a sealing film, and culturing in an incubator at 32 ℃ for 12 h;
(3) solid culture plates of 6 samples were removed, and a single colony of each sample was inoculated to a medium containing 50. mu.L ddH2O in an Eppendorf tube, blowing off single colonies, placing the single colonies in an ice-water mixture for 3-5 minutes, then placing the mixture in a freezing device to rapidly reduce the temperature of the mixture, and waiting for ddH in a test tube2After the O is condensed into ice, standing for 5-10 min, quickly placing in a hot water bath, quickly raising the temperature to 100 ℃, and standing for 10-20 min;
(4) removal of ddH containing Single colony2Transferring O into a 1.5-mL LEppendorf tube containing 50-mL ET (final concentration 0.02mol/L EDTA; final concentration 0.02mol/L Tris-Cl) solution, placing in an ice-water mixture for 3-5 minutes, then placing in a freezing device to rapidly reduce the temperature, standing for 5-10 minutes after the solution in the tube condenses into ice, rapidly placing in a hot water bath to rapidly increase the temperature to 100 ℃, and standing for 10-20 minutes;
(5) adding 50 mu L of 10% SDS into an Eppendorf tube containing an ET solution to prepare an ETS (final concentration of 0.02mol/L EDTA; final concentration of 0.02mol/L Tris-Cl; final concentration of 3.33% SDS) solution, placing the ETS solution in an ice-water mixture for 3-5 minutes, then placing the ETS solution in a freezing device to rapidly reduce the temperature of the ETS solution, standing for 5-10 minutes after the solution in the test tube is condensed into ice, rapidly placing the ETS solution in a hot water bath to rapidly increase the temperature of the ETS solution to 100 ℃, and standing for 10-20 minutes;
(6) then taking the supernatant to prepare a DNA template;
the prepared DNA template was placed in a reaction solution containing PCR (20. mu.L system: PCR mix 10. mu.L; template 2. mu.L; universal primers each 1. mu.L; ddH)2O6. mu.L) in a PCR tube as a PCR template; placing the mixture in a PCR instrument for PCR amplification, wherein the early-stage denaturation (95 ℃) time is changed into 10min (the denaturation temperature in the non-cyclic reaction is 5min originally), and the PCR reaction conditions are as follows: 10min at 95 ℃; 35 cycles of reaction are as follows: 30s at 95 ℃, 2min at 60 ℃ and 2min at 72 ℃; extending for 10min at 72 ℃, and storing at 4 ℃. The general primer is as follows: 16S rRNA-F: AGAGTTTGATCCTGGCTCAG, 16S rRNA-R: GGTTACCTTGTTACGACTT.
The other steps are carried out according to conventional methods.
The gel electrophoresis detection chart is shown in figure 1, wherein 1-6 are gram-positive bacteria, and 7 is a negative control; m: DL5000 marker; 1: bacillus velezensis (Bacillus velezensis); 2: bacillus subtilis; 3: bacillus licheniformis (Bacillus licheniformis); 4: bacillus amyloliquefaciens (Bacillus amyloliquefaciens); 5: bacillus megaterium (Bacillus coaguluns); 6: bacillus subtilis (Bacillus firmus), 7 is a negative control.
This result illustrates that: the method can effectively release the gram-positive bacteria intracellular DNA, and the release rate is 100 percent.
Example 2
Different from the embodiment 1, the sample to be detected is the aquaculture water body.
Step (3) waiting for ddH in the test tube2Condensing O into ice, standing for 10min, rapidly placing in 100 deg.C water bath to rapidly raise its temperature to 100 deg.C, and standing for 20 min;
and (4) standing for 10min after the solution in the test tube is condensed into ice, quickly placing in a hot water bath, quickly raising the temperature to 100 ℃, and standing for 20 min.
And (5) standing for 10min after the solution in the test tube is condensed into ice, quickly placing in a hot water bath, quickly raising the temperature to 100 ℃, and standing for 20 min.
The test results were similar to those of example 1, except that gram-negative bacteria were detected in addition to gram-positive bacteria, because the culture water contained gram-positive bacteria as well as gram-negative bacteria, and dd H in this application was not generally used2O, ET, ETS buffer and a physical temperature changing mode, gram-negative bacteria can be detected, dd H is adopted2O, ET, ETS buffer and a physical temperature changing mode, gram-positive bacteria can be further detected, as shown in figure 2, wherein 3: coli (Escherichia coli) and 4: vibrio parahaemolyticus (Vibrio parahaemolyticus) is a gram-negative bacterium, and the remainder are gram-positive bacteria.
The gel electrophoresis detection chart is shown in FIG. 2, wherein 1-6 are all single colony detection, and 7 is a negative control; m: DL5000 marker; 1: bacillus subtilis; 2: bacillus amyloliquefaciens (Bacillus amyloliquefaciens); 3: escherichia coli (Escherichia coli); 4: vibrio parahaemolyticus (Vibrio parahaemolyticus); 5: bacillus velezensis (Bacillus velezensis); 6: bacillus subtilis (Bacillus firmus), 7 is a negative control.
The results in examples 1-2 show that: the method can effectively release the gram-positive bacteria intracellular DNA, and the release rate is 100 percent.
However, the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (10)
1. A method for rapidly releasing gram-positive bacterial intracellular DNA is characterized by comprising the following steps:
(1) selecting a sample to be detected, and performing flat plate coating or scribing by adopting a solid culture medium;
(2) sealing the flat plate by using a sealing film, and placing the flat plate in an incubator for culture;
(3) the plate was removed, and a single colony was picked and inoculated in a medium containing ddH2O in Eppendorf tube, blowing off single colony, putting in ice-water mixture, putting in freezer, and cooling to obtain ddH2Condensing O into ice, standing, rapidly placing in a hot water bath to rapidly increase the temperature, and then standing;
(4) removal of ddH containing Single colony2Transferring O into an Eppendorf tube containing ET solution, placing the Eppendorf tube in an ice-water mixture, placing the Eppendorf tube in a freezing device to quickly reduce the temperature of the Eppendorf tube, standing the Eppendorf tube after the solution in the test tube is condensed into ice, quickly placing the Eppendorf tube in a hot water bath to quickly increase the temperature of the Eppendorf tube, and then standing the Eppendorf tube;
(5) adding sodium dodecyl sulfate into the Eppendorf tube containing the ET solution in the step (4), placing the tube in an ice-water mixture, placing the tube in a freezing device to quickly reduce the temperature of the tube, standing the tube after the solution in the tube is condensed into ice, quickly placing the tube in a hot water bath to quickly increase the temperature of the tube, and then standing the tube;
(6) taking the supernatant to obtain the gram-positive strain intracellular DNA.
2. The method for the rapid release of intracellular DNA of gram-positive bacteria according to claim 1, wherein: the sample to be detected in the step (1) is a gram-positive bacterium microecological preparation or a culture water body water sample; the solid culture medium in the step (1) is an LB solid culture medium or a TCBS solid culture medium.
3. The method for the rapid release of intracellular DNA of gram-positive bacteria according to claim 1, wherein: and (3) placing the mixture in an incubator at 28-37 ℃ for culturing for 12-24 h in the step (2).
4. The method for the rapid release of intracellular DNA of gram-positive bacteria according to claim 1, wherein: taking out the plate in the step (3), selecting a single colony and inoculating the single colony to ddH containing 30-50 mu L2In an Eppendorf tube of O, blowing off single colonies, placing the single colonies in an ice-water mixture for 3-5 minutes, and then placing the mixture in a refrigerating device for rapid reductionLow temperature, wait for ddH in the tube2And (3) after the O is condensed into ice, standing for 5-10 min, quickly placing in a hot water bath, quickly raising the temperature to 95-100 ℃, and then standing for 10-20 min.
5. The method for the rapid release of intracellular DNA of gram-positive bacteria according to claim 1, wherein: step (4) of removing ddH containing a single colony2And transferring the O into an Eppendorf tube containing 30-50 mu L of ET solution, placing the Eppendorf tube in an ice water mixture for 3-5 minutes, placing the Eppendorf tube in a freezing device to reduce the temperature of the Eppendorf tube quickly, standing the Eppendorf tube for 5-10 minutes after the solution in the tube condenses into ice, placing the Eppendorf tube in a hot water bath quickly to increase the temperature of the Eppendorf tube to 95-100 ℃, and standing the Eppendorf tube for 10-20 minutes.
6. The method for the rapid release of intracellular DNA of gram-positive bacteria according to claim 1 or 5, wherein: the ET solution in the step (4) comprises EDTA and Tris-Cl, wherein the final concentration of the EDTA and the Tris-Cl is 0.02 mol/L.
7. The method for the rapid release of intracellular DNA of gram-positive bacteria according to claim 1, wherein: and (5) adding sodium dodecyl sulfate into the Eppendorf tube containing the ET solution in the step (4) to enable the final concentration to be 3-4%.
8. The method for the rapid release of intracellular DNA of gram-positive bacteria according to claim 1, wherein: and (5) adding sodium dodecyl sulfate into the Eppendorf tube containing the ET solution in the step (4), placing the tube in an ice-water mixture for 3-5 minutes, placing the tube in a freezing device to rapidly reduce the temperature of the tube, standing for 5-10 minutes after the solution in the tube is coagulated into ice, rapidly placing the tube in a hot water bath to rapidly increase the temperature of the tube to 95-100 ℃, and then standing for 10-20 minutes.
9. The method for the rapid release of intracellular DNA of gram-positive bacteria according to claim 1, wherein: and (5) putting the mixture in a refrigerating device below-20 ℃ in the steps (3) to (5) to rapidly reduce the temperature of the mixture.
10. The method for the rapid release of intracellular DNA of gram-positive bacteria according to claim 1, wherein: and (3) further placing the supernatant in the step (6) into a PCR tube containing a PCR reaction solution as a PCR template, and then placing the PCR tube into a PCR instrument for PCR amplification, wherein the denaturation temperature in the early-stage non-cyclic reaction is 95 ℃, and the denaturation time is 10 min.
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