CN107475242B - Method for extracting mould spore DNA by mechanical crushing method - Google Patents
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Abstract
The invention relates to a method for extracting mould spore DNA by a mechanical crushing method, which comprises the following steps: adding glass beads into mould spore liquid, performing mechanical crushing treatment, and then performing DNA extraction. The invention uses a mechanical method to crush the mould spore wall, extracts and purifies DNA by combining a column purification method after crushing, detects the quality of the extraction effect through real-time fluorescence quantitative PCR and gel electrophoresis, and establishes a technical system for accurately extracting the mould spore DNA according to the detection result. The invention has the advantages of high DNA extraction efficiency, convenient and controllable operation, no toxicity and no harm. The invention firstly makes the mould spore accurate to 102DNA extraction is carried out on the spores, the mass research of the mould spores from a large number of cells can be turned to the accurate research of single cells or a small number of cells, the extracted DNA has good quality, the subsequent sequencing can be met, and the reliable research basis and the powerful guarantee are provided for the subsequent molecular biology research.
Description
Technical Field
The invention relates to a method for extracting mould spore DNA by a mechanical crushing method.
Background
Although many methods for extracting DNA are mature, the conventional methods for extracting DNA have limitations on fungi with complex cell wall components, and the abundant substances such as polysaccharides, pigments, nucleases and the like in the fungi make the extraction and separation of DNA more difficult, especially the extraction of DNA containing harder spore walls such as mold spores is more limited.
On the other hand, due to the development of sequencing technology, the traditional large sample cell sequencing mode is changed, and the analysis of one cell population is converted into the analysis of single cells, so that more valuable researches can be provided for us, such as different positions of a mold culture dish, and the genetic compositions of molds in different positions are different due to different reasons of nutrient substances, mold quantity and the like. The traditional method requires a large amount of samples to extract DNA, so that the existing method for extracting the DNA of the mould spores cannot meet the sequencing condition.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for extracting the DNA of the mould spores by a mechanical crushing method.
The invention uses a mechanical method to crush the mould spore wall, extracts and purifies DNA by combining a column purification method after crushing, detects the quality of the extraction effect through real-time fluorescence quantitative PCR and gel electrophoresis, and establishes a technical system for accurately extracting the mould spore DNA according to the detection result.
The technical scheme of the invention is as follows:
a method for extracting mould spore DNA by a mechanical crushing method comprises the following steps: the mold spore liquid is taken, glass beads (hereinafter referred to as beads) are added, mechanical crushing treatment is carried out, and then DNA extraction is carried out.
Unless otherwise specified, the mechanical crushing according to the invention is mainly referred to as grinding treatment.
Furthermore, the number of spores in the mold spore liquid is more than or equal to 1 × 102And each m L.
Furthermore, the number of spores in the mold spore liquid is more than or equal to 5 × 107And each m L.
Further, the number of glass beads used in the mechanical disruption treatment process was 10 to 50, the size (diameter, the same applies hereinafter) of the glass beads was 1 to 3mm, and the mechanical disruption treatment (grinding) time was (1 × 5) min- (5 × 5) min, based on 1m L mold spore liquid.
Wherein (1 × 5) min refers to mechanical crushing (grinding) for 5min, then taking out and placing on ice, cooling for at least 30s, and performing the operation for 1 time, and (5 × 5) min refers to mechanical crushing (grinding) for 5min, then taking out and placing on ice, cooling for at least 30s, and performing the operation for 5 times.
In one embodiment of the present invention, the number of glass beads is 40, the size of glass beads is 1-3mm, and the mechanical disruption treatment (grinding) time is 5min based on 1m L mould spore liquid.
In one embodiment of the present invention, the size of the glass beads is 3mm based on 1m L mould spore liquid, the mechanical disruption treatment (grinding) time is 5min, and the number of the glass beads is 10-50, preferably 30.
In one embodiment of the present invention, the number of glass beads is 30, the size of glass beads is 3mm, and the mechanical disruption treatment (grinding) time is (1 × 5) min- (5 × 5) min, preferably 3 × 5min, based on 1m L mould spore liquid.
In a preferred embodiment of the present invention, the number of spores in the mold spore liquid is 5 to 5 × 10 based on 1m L of the mold spore liquid7Pieces/m L, the number of glass beads is 30, the size of the glass beads is 3mm, and the mechanical crushing treatment (grinding) time is (2 × 5) min.
In another preferred embodiment of the present invention, when the number of spores in the mold spore liquid is not less than 1 × 10/1 m L/2 m2At L pieces/m, the number of glass beads is 20, the size of the glass beads is 3mm, and the mechanical crushing treatment (grinding) time is (2 × 5) min.
Specifically, the DNA extraction comprises the following steps:
based on 1ml of mechanically crushed (ground) mold spore liquid,
(1) adding 20 mu L protease K solution into the tube, and mixing uniformly;
(2) adding 220 μ L buffer solution GB, shaking for 15sec, standing at 70 deg.C for 10min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;
(3) adding 220 mu L absolute ethyl alcohol, fully shaking and mixing for 15sec, wherein flocculent precipitate may appear, and centrifuging briefly to remove water drops on the inner wall of the tube cover;
(4) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12,000rpm for 30sec, pouring off waste liquid, and placing an adsorption column CB3 into the collecting pipe;
(5) adding 500 mu L buffer solution GD into an adsorption column CB3, centrifuging at 12,000rpm for 30sec, pouring waste liquid, and putting the adsorption column CB3 into a collection tube;
(6) adding 600 mu L of rinsing liquid PW into an adsorption column CB3, centrifuging at 12,000rpm for 30sec, pouring waste liquid, and putting the adsorption column CB3 into a collecting pipe;
(7) repeating the operation step (6);
(8) placing adsorption column CB3 back into the collecting tube, centrifuging at 12,000rpm for 2min, and pouring off waste liquid; placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
(9) transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 mu L elution buffer TE into the middle part of the adsorption membrane, standing for 5min at room temperature, centrifuging for 2min at 12,000rpm, and collecting the solution into the centrifuge tube.
The fluorescent quantitative PCR program was set up as follows: denaturation at 1.95 deg.C for 5 min; at 95 deg.C, 30s, 60 deg.C, 30 s; 68 ℃ for 30 s; after 39 cycles, 65 ℃ to 95 ℃ and increment0.5 ℃. The PCR reaction system is as follows: 10.75. mu.l of water, 11.25. mu.l of realtime mix, 0.5. mu.l of each primer, and 2. mu.l of DNA template. The primers are Asp-F (CTGTCCGAGCGTCATTGAT) and ITS-R (TCCTCCGCTTATTGAT).
Further, the mould spores are aspergillus niger spores.
The strains and reagents used in the invention are commercially available.
The technology for extracting DNA by a mould spore mechanical method provides a powerful guarantee for the precise research of mould spores, and the difference among mould spore individuals can be researched after the mould spore DNA with high quality and high efficiency is accurately extracted. The optimized mould spore DNA extraction method obtained by the research not only has the advantages of high efficiency, convenient and easy operation, no toxicity, no harm and the like, but also has the most important advantage that the extracted DNA has good quality, thereby providing a reliable research basis and powerfully ensuring the subsequent molecular biology research.
The invention has the following advantages:
1. has high DNA extraction efficiency.
2. Has the advantages of convenient operation and easy control.
3. Has the advantages of no toxicity and no harm.
4. For the first time, the mould spores are accurate to 102DNA extraction was performed on individual spores.
5. The mold spores can be diverted from population studies of a large number of cells to precise studies of single cells or a small number of cells.
6. The extracted DNA has good quality, can meet the requirement of subsequent sequencing, and provides reliable research foundation and powerful guarantee for the subsequent molecular biology research.
Drawings
FIG. 1 shows the quantitative determination results of bead size single factor assays;
FIG. 2 shows the quantitative determination results of the single-factor test of the number of beads;
FIG. 3 shows the result of quantitative determination of single-factor test of grinding time;
FIG. 4 is a diagram of agarose gel electrophoresis of a large number of spore DNA physical disruption orthogonal tests;
FIG. 5 shows the quantitative determination results of the physical orthogonal assay of a large number of spore DNAs;
FIG. 6 shows the results of small amounts of spore DNA by semi-nested PCR;
FIG. 7 shows the linear dependence of a small spore gradient dilution;
FIG. 8 is a diagram of agarose gel electrophoresis of a small number of spore DNA gradients;
FIG. 9 shows the results of semi-nested PCR quantification of small amounts of spore DNA in an orthogonal assay;
FIG. 10 is an agarose gel electrophoresis image of a small amount of spore DNA orthogonal assay.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention, those skilled in the art will recognize that the techniques or conditions described in the literature of the art, or in accordance with the specifications for the product, those reagents or equipment not known to the manufacturer, are conventional products commercially available from normal suppliers.
Examples
1. Experimental Material
The following experiments used mould spores mainly of aspergillus niger, the species aspergillus niger purchased from shanghai lucky biology corporation, stored in 0.9% sterile sodium chloride solution containing 15% glycerol, the freeze-dried strain was used for 0 generation, and the experiments were mainly used for 3 rd and 4 th generations. Culturing is carried out according to conventional methods in the art.
2. The following experiments were performed using a mechanical disruption method for mold spores: a mold spore solution is taken, glass beads (hereinafter referred to as beads) are added, mechanical crushing (grinding) treatment is carried out, and then DNA extraction is carried out.
3. The DNA extraction method used in the following experiments:
(1) adding 20 mu L protease K solution into the tube, and mixing uniformly;
(2) adding 220 μ L buffer solution GB, shaking for 15sec, standing at 70 deg.C for 10min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;
(3) adding 220 mu L absolute ethyl alcohol, fully shaking and mixing for 15sec, wherein flocculent precipitate may appear, and centrifuging briefly to remove water drops on the inner wall of the tube cover;
(4) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12,000rpm for 30sec, pouring off waste liquid, and placing an adsorption column CB3 into the collecting pipe;
(5) adding 500 mu L buffer solution GD into an adsorption column CB3, centrifuging at 12,000rpm for 30sec, pouring waste liquid, and putting the adsorption column CB3 into a collection tube;
(6) adding 600 mu L of rinsing liquid PW into an adsorption column CB3, centrifuging at 12,000rpm for 30sec, pouring waste liquid, and putting the adsorption column CB3 into a collecting pipe;
(7) repeating the operation step (6);
(8) placing adsorption column CB3 back into the collecting tube, centrifuging at 12,000rpm for 2min, and pouring off waste liquid; placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
(9) transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 mu L elution buffer TE into the middle part of the adsorption membrane, standing for 5min at room temperature, centrifuging for 2min at 12,000rpm, and collecting the solution into the centrifuge tube.
The fluorescent quantitative PCR program was set up as follows: denaturation at 1.95 deg.C for 5 min; at 95 deg.C, 30s, 60 deg.C, 30 s; 68 ℃ for 30 s; after 39 cycles, 65 ℃ to 95 ℃ and increment0.5 ℃. The PCR reaction system is as follows: 10.75. mu.l of water, 11.25. mu.l of realtime mix, 0.5. mu.l of each primer, and 2. mu.l of DNA template. The primers are Asp-F (CTGTCCGAGCGTCATTGAT) and ITS-R (TCCTCCGCTTATTGAT).
4. Specific experimental scheme
4.1 Experimental materials, supra.
4.2 study of spore wall breaking effectiveness by physical method
Before the physical method orthogonal test is started, three factors with high correlation with the crushing effect, namely the size of beads, the number of the beads and the grinding time of the beads, are selected, and the influence and the effectiveness of the three factors on DNA extraction are researched, wherein the concentration of the bacteria liquid obtained in the test is 5 × 107Each of L m, 1ml of each test, and a grinder operating specification of 28 frequency/s.
(1) Size of the beads
In the bead size single factor test, there are 1mm, 2mm and 3mm size beads, respectively, the number of beads is fixed to 40 per group, and the grinding time is fixed to 5 min. No. 6 is a stock solution control directly used for subsequent extraction treatment of the kit without physical crushing, and the specific parameters are as shown in the following table for carrying out single-factor tests.
Bead size single factor test
After the bacteria liquid is physically crushed, a certain amount of bacteria liquid is left to be directly counted by a blood counting chamber, and other bacteria liquid is directly subjected to subsequent DNA extraction, and the specific operation is the same as the above.
(2) Number of beads
According to the experimental result of (1), the crushing effect is best when the bead size is determined to be 3mm, so that the fixed bead size is 3mm, and the fixed grinding time is still 5min to study the single-factor test. No. 6 is a stock solution control directly used for subsequent extraction treatment of the kit without physical crushing, and the specific parameters are as shown in the following table for carrying out single-factor tests.
Single factor test for bead number
After the bacterial liquid is physically crushed, a certain amount of bacterial liquid is reserved and is directly counted by a blood counting chamber, other bacterial liquids are directly subjected to subsequent DNA extraction, the same as the above step (1), then the purity of the extracted DNA solution is measured and agarose gel electrophoresis is verified, the RT-PCR reaction is performed on the extracted DNA product, and the reaction system is the same as the above step (1).
(3) Time of grinding
According to the experimental results of (2) and (3), the fixed bead size was 3mm and the number of beads was 30, and a single factor test of the milling time was performed. Considering that the physical method generates heat by friction, and the overhigh temperature can influence the quality and quantity of nucleic acid extraction, the nucleic acid is taken out and placed on ice to be cooled for 30s after being ground for 5min in the method, wherein No. 6 is the stock solution for directly carrying out the extraction treatment of the subsequent kit without physical crushing, and the specific parameters refer to the following table to carry out the single-factor test.
Single factor test of grinding time
After the bacterial liquid is physically crushed, a certain amount of bacterial liquid is reserved and is directly counted by a blood counting chamber, other bacterial liquids are directly subjected to subsequent DNA extraction, the same as the above step (1), then the purity of the extracted DNA solution is measured and agarose gel electrophoresis is verified, the RT-PCR reaction is performed on the extracted DNA product, and the reaction system is the same as the above step (1).
4.3 orthogonal method to study the influence of various factors on the extraction of a large amount of spore DNA by a physical method
In order to obtain a physical method multifactor test and a high-efficiency test design of an optimal horizontal combination, an orthogonal test method is used for testing in the research. Based on the 4.2 physical single-factor test result, a three-factor three-level orthogonal test is designed for testing, wherein No. 10 is a stock solution control which is directly used for subsequent extraction treatment of the kit without physical crushing, and the specific parameters refer to the following table for carrying out the orthogonal test.
Physical orthogonal assay for large numbers of spore DNA extractions
After the bacteria liquid is physically crushed, a certain amount of bacteria liquid is reserved and is directly counted by a blood counting plate, other bacteria liquid is directly subjected to subsequent DNA extraction, the purity of the extracted DNA solution is measured and verified by agarose gel electrophoresis, the extracted DNA product is subjected to RT-PCR reaction, and the reaction system is the same as the above.
4.4 threshold test for extraction of Small amounts of spore DNA
Threshold tests were performed prior to a small amount of spore DNA extraction. The primers were replaced by a pair of semi-nested primers, the first semi-nested PCR with pfkA-qian-F (TCAGTGACGTACTGGTTGGC) and pfkA-zhong-R (AATGACGAATCCACGCTGGT), and the second semi-nested PCR with pfkA-qian-F and pfkA-qian-R (CCACCAGGTCAAGACACCC).
Are respectively provided with 1 × 1041, 1 × 1031, 1 × 1021, 1 × 101The first two groups are obtained by diluting high-concentration bacterial liquid, and the specific method is that the concentration is 5 × 10 per ml7The spore suspension is diluted in gradient to 2.5 × 104Pieces/m L, 2.5 × 103Respectively taking 400 mu L bacterial liquid to each m LCentrifuging to obtain 1 × 1041, 1 × 103Spore solution, since 1 × 1021, 1 × 101The concentration of the spore solution is too low, and the error caused by the gradient dilution method is too large, so the method for picking single cells is used for the first time. The specific method is that spore bacteria liquid with certain concentration is sucked and dropped on a sterilized glass slide by a clean gun head, the glass slide is placed under a microscope, and a certain amount of spores are sucked into 0.05% (v/v) Tween 80 and 0.9% sterile NaCl solution, so that the spores can be better dissolved in the solution.
In the threshold test of extraction of the small amount of spore DNA, the pretreatment process adopts the optimal method obtained in the orthogonal test of extraction of the large amount of spore DNA to process the sample, namely 3mm, 30 and 5min × 2.
After physical crushing, directly absorbing bacterial liquid to perform a first round of semi-nested PCR reaction, and taking a quantitative product after the first round of semi-nested PCR reaction as a DNA template of a second round of semi-nested PCR. The specific systems of the first semi-nested PCR and the second semi-nested PCR are shown in the following table, and the samples are added and put into the fluorescent quantitative PCR for reaction.
Semi-nested PCR system
Because the length of the first semi-nested PCR fragment is longer, the extension time is prolonged to 1min, and the specific program is as follows: denaturation at 95 ℃ for 5 min; at 95 °, 30s, 60 °, 30 s; 68 ℃ and 1 min; after 40 cycles, 65 ° to 95 ° increment0.5 °. The second semi-nested PCR procedure was: denaturation at 95 ℃ for 5 min; at 95 °, 30s, 60 °, 30 s; 68 DEG, 30 s; after 40 cycles, 65 ° to 95 ° increment0.5 °. After the second semi-nested PCR, the size of the gel running by combining the dissolution peak and gel electrophoresis is determined to determine whether the target gene is present.
4.5 investigating the Effect of the physical orthogonal method on the extraction of DNA from a small number of spores
After the minimum threshold value of extracting a small amount of spore DNA is obtained, taking the bacteria liquid with the minimum concentration as the extraction concentration of the small amount of spore DNA, sucking 400 mu L bacteria liquid into a centrifugal tube, and performing tests according to the three-factor three level of the physical orthogonal test of the large amount of spore DNA, wherein the test parameters are shown in the table below.
Physical orthogonal assay for extraction of small amounts of spore DNA
After the bacterial liquid is physically broken, the bacterial liquid is centrifuged at 12,000rpm for 2min, and the supernatant is taken as a DNA template of the first round of semi-nested PCR. And (3) taking the quantitative product of the semi-nested PCR after the first round of reaction as a DNA template of the second round of semi-nested PCR, and determining whether the product is the target gene or not by combining a dissolution peak and gel electrophoresis gel running size after the second round of semi-nested PCR. The reaction system and the reaction procedure of the semi-nested PCR are the same as 4.4.
5. Results of the experiment
5.1 study on spore wall breaking effectiveness by physical method
(1) Size of beads
In the single-factor test of bead size, the remaining spore count and spore disruption wall obtained by counting the disrupted solution with a hemocytometer were as follows (initial concentration of 5 × 10)7Pieces/m L), the quantitative detection results of spore DNA extraction by each group of beads are shown in the following table and figure 1, in figure 1, RFU is relative fluorescence unit, Cycle is Cycle number, Temperature is Temperature, Celsius is centigrade, d (RFU)/dt is derivation, Melt peak is dissolution peak.
Wall breaking rate of bead size single factor test
Note that the upper letters in the table are the results of the duncan's new repolarization test (α ═ 0.05), and the different letters represent significant differences between the two sets of data.
Quantitative detection result of bead size single-factor test
Note that the upper letters in the table are the results of the duncan's new repolarization test (α ═ 0.05), and the different letters represent significant differences between the two sets of data.
From the above analysis, it is known that the bead size factor of physical disruption has a significant influence on the extraction efficiency. When the number of the beads is fixed to 40, and the grinding time is fixed to 5min, when the size of the beads in the group 2 is 3mm, the crushing effect is best, the wall breaking rate reaches 64.20%, and the stock solution in the group 6 is also crushed in the extraction of the kit, and the dissolving temperature is 89.5-90 ℃.
(2) Number of beads
In the single-factor test of the number of beads, the remaining spore count and the spore-disrupted wall obtained by counting the wall-disrupted solution with a hemocytometer were as follows (initial concentration: 5 × 10)7Pieces/m L), the results of quantitative detection of spore DNA extraction by each set of beads are shown in the table below and fig. 2.
Wall breaking rate of bead quantity single factor test
Note that the upper letters in the table are the results of the duncan's new repolarization test (α ═ 0.05), and the different letters represent significant differences between the two sets of data.
Quantitative detection result of single-factor test of bead quantity
Note that the upper letters in the table are the results of the duncan's new repolarization test (α ═ 0.05), and the different letters represent significant differences between the two sets of data.
From the above analysis, it can be seen that the number of physically disrupted beads has a significant effect on the extraction efficiency. When the size of the beads is fixed to be 3mm and the grinding time is fixed to be 5min, and the number of the beads in the 3 rd group is 30, the crushing effect is best, the wall breaking rate reaches 66.87%, and the stock solution in the 6 th group is also crushed in the extraction of the kit, and the dissolving temperature is 89.5-90 ℃.
(3) Grinding time
In the single-factor test of the milling time, the remaining spore count and the spore-broken wall obtained by counting the wall-broken solution with a hemocytometer were as follows (initial concentration of 5 × 10)7Pieces/m L), the results of quantitative detection of spore DNA extraction by each set of beads are shown in the table below and fig. 3.
Wall breaking rate of grinding time single factor test
Note that the upper letters in the table are the results of the duncan's new repolarization test (α ═ 0.05), and the different letters represent significant differences between the two sets of data.
Grinding time single-factor test quantitative detection result
Note that the upper letters in the table are the results of the duncan's new repolarization test (α ═ 0.05), and the different letters represent significant differences between the two sets of data.
From the above analysis, it can be seen that the grinding time factor of physical crushing has significant influence on the extraction efficiency, when the size of the beads is fixed to 3mm and the number of the beads is fixed to 30, the crushing effect is best when the grinding time of group 3 is 5min × 3, the wall-breaking rate reaches 68.42%, the stock solution of group 6 is also crushed in the extraction of the kit, and the dissolution temperature is 89.5-90 ℃.
5.2 orthogonal method for studying influence of various factors on extraction of a large number of spore DNAs by physical method
In a large quantity of spore DNA extraction experiments, orthogonal experiment results are analyzed in three aspects of concentration, purity and integrity, wherein the concentration is taken as a main condition to optimize indexes, and the purity and the integrity are taken as auxiliary conditions. The concentration scale is reflected in the Cq value of the quantitative determination, the purity is reflected by the value of the measurement A260/280 and the integrity is reflected by the agarose gel electrophoresis.
The agarose gel electrophoresis image of the DNA product is shown in FIG. 4, and the clear band and no obvious tailing phenomenon can be seen from FIG. 4, which shows that the integrity of the DNA is better.
The results of quantitative determination and specific numerical values of DNA purity are shown in the following table and FIG. 5, and it can be seen that in 9 orthogonal experiments of the physical disruption method, the Cq value of the 4 th group is the lowest, i.e., the DNA concentration is the highest, the 10 th group stock solution is also partially disrupted in the extraction of the kit, all purities are high, and the sample dissolution temperature is stabilized at 89.5-90 ℃.
Quantitative detection result and DNA purity of massive spore DNA physical crushing orthogonal test
Note that the upper letters in the table are the results of the duncan's new repolarization test (α ═ 0.05), and the different letters represent significant differences between the two sets of data.
When orthogonal analysis is performed according to the above results, the sum K (A) of the test indicators corresponding to a level of factor A1, i.e., 1mm of beads, is equal to 38.67, the average value K (A) is equal to 12.89, the sum K (A) of the test indicators corresponding to a level of factor A2, i.e., 3mm of beads, is equal to 31.03, the average value K (A) is equal to 10.35, the sum K (A) of the test indicators corresponding to a level of factor A3, i.e., 2mm of beads, is equal to 48.53, the average value K (A) is equal to 16.18, when the Cq value is inversely proportional to the nucleic acid content, the sum K (A) of the test indicators is greater than K (A), i.e., 3mm is the level of factor A, the sum K (B) of the test indicators corresponding to a level of factor B1, i.e., 20, the sum K (B) of the test indicators corresponding to 41, when the average value K (B) is greater than 13.k (A), the optimal value K (A) is equal to a level, when the sum K (K) of the test indicators corresponding to a level of factor B, the test indicators corresponding to a level, the time K (C) is equal to 30.3 mm, the average value K) is equal to 30.3, when the test indicators corresponding to a level, the test indicators, the time of factor C, the sum K (C) is equal to a level, the average value K) is equal to 30.3 mm, the test indicators corresponding to a value K (C, the test indicators, the sum K (C, when the test indicators corresponding to a value K) is equal to a value, the time level, the time of the test indicators, the sum K (C, the optimal value K) is equal to a value of factor K (C, the test indicators, the sum K (C) is equal to a value, the sum K (C, the optimal, the sum K (C) of factor C, when the test indicators, the number of factor B (C) is equal to a value, the test indicators, the sum K (C) is equal to 30.3.3.3 mm, the sum K) is equal to a value, when the test indicators.
In terms of purity, all samples have the purity of 1.8-1.9, and the purity is relatively high, so that downstream experiments can be performed conveniently. In the aspect of integrity, agarose gel electrophoresis shows that the DNA has better integrity and no serious tailing phenomenon.
5.3 threshold test for extraction of Small amounts of spore DNA
In the threshold test for extracting DNA from a small number of spores, the quantitative results of hemi-nested PCR are shown in the following table and FIG. 6 (the first two are the first and the second are the second).
Semi-nested PCR quantitative result of a small amount of spore DNA
As can be seen from the Cq values of the first round of semi-nested PCR quantification results, the Cq values gradually increased as the number of spores decreased, i.e., the DNA concentration decreased as the number of spores decreased. As can be seen from the solvent peak plot on the right, both the positive and test samples have two dissolution peaks, one at the dissolution temperature of the target product (88.5-89.5 ℃) and one at the negative dissolution temperature (79-79.5 ℃). As can be seen from the second round of semi-nested PCR quantification, the DNA concentration is linearly related to the Cq value, and in the right dissolution temperature, the dissolution temperatures of 1, 2, 3 and stock solutions are 87 ℃ while that of sample No. 4 is 90 ℃.
As can be seen from fig. 7, the linear relationship between the group 4 data and the group 3 data is not good, the correlation coefficient R2 of the standard curve with the numerical value of the group 3 data is 0.9998, and the regression equation is: y is-4.3975 x +41.55, the linear correlation is good. Agarose gel electrophoresis run-up validation of the quantified products is shown in FIG. 8.
As can be seen from FIG. 8, the samples No. 1, 2, 3 and the positive sample have a significant band at the primer size of 303bp, and No. 4 and the negative sample have no band, and the concentration of the extracted small amount of spore DNA was defined as 1 × 10 in combination with the above results2And (4) spores.
5.4 investigating the Effect of the physical orthogonal method on the extraction of DNA from a small number of spores
In the physical orthogonal test of extraction of a small amount of spore DNA, the quantitative results of the hemi-nested PCR are shown in the following table and FIG. 9 (the first two are the first round of hemi-nested PCR, and the second two are the second round of hemi-nested PCR).
Semi-nested PCR quantitative result of small amount of spore DNA orthogonal test
As can be seen from the Cq values of the first round of semi-nested PCR quantification results, the Cq values between samples were not very different. As can be seen from the solvent peak diagram on the right, the dissolution peaks of the positive and test samples are relatively regular, with a large dissolution peak at 78.5 ℃ and a small peak at about 88.5 ℃. As can be seen from the Cq values of the second semi-nested PCR quantification result, in 9 groups of data of the orthogonal test, the Cq value of the 4 th group is the lowest, namely the highest DNA concentration, and then 6, 8, 1, 2, 3, 7, 5 and 9 are sequentially carried out, and all the sample dissolution temperatures are stabilized at 86.5-87 ℃. The agarose gel electrophoresis pattern of the quantified product is shown in FIG. 10, and the size of the bands 1-9 is consistent with the size of the target product as seen from the gel running result.
The results of the orthogonal analysis are further determined to indicate that the sum of the test indicators corresponding to a factor A1 level, i.e., 1mm of beads, K (A) equals 36.08, the average value of K (A) equals 12.02, the sum of the test indicators corresponding to a factor A2 level, i.e., 3mm of beads, K (A) equals 31.28, the average value of K (A) equals 10.43, the sum of the test indicators corresponding to a factor A3 level, i.e., 2mm of beads, K (A) equals 36.01, K (A) equals 12.00, Cq is inversely proportional to the nucleic acid content, so that each level has an effect on the test indicators of K (A) > K (A), i.e., 3mm of the optimal level of the factor A, K (B) equals 34.36, K (B) equals 11.45, the sum of the test indicators corresponding to a factor B1 level, i.e., 20, K (K) equals 34.36, K (B) equals 11.45, the factor B2 level, the number of beads, the factor B2 level, the optimal level, K (C) equals 20, K) equals 11.k equals 11.5, K (K) equals 11.5, K (C, K) equals 3.k equals 3.5, the sum of the test indicators corresponding to the average value of the test indicators, C (C) is equal to the time of the test indicator C, the sum of the test indicator, the test indicator corresponding to the sum of the optimal level, the number of the test indicators, K (C) of the number of the test indicators, K) of the test indicators, K (C) of the sum of the number of the optimal level, K (C) of the number of the test indicators is equal to the number of the test indicators, K (C) of the number of the test indicators, the number of the test indicators, K (C, K (C) of the number of.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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<120> method for extracting mould spore DNA by mechanical crushing method
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Claims (3)
1. A method for extracting mould spore DNA by a mechanical crushing method is characterized by comprising the following steps: adding glass beads into mould spore liquid, performing mechanical crushing treatment, and then performing DNA extraction;
when the number of spores in the mold spore liquid is more than or equal to 5 × 10 calculated by 1m L mold spore liquid7At the rate of L pieces/m, the number of the glass beads is 30, and the glass isThe size of the glass beads is 3mm, and the mechanical crushing treatment time is (2 × 5) min;
when the number of spores in the mold spore liquid is 1 × 10 calculated by 1m L mold spore liquid2When the number of the glass beads is L/m, the number of the glass beads is 20, the size of the glass beads is 3mm, and the mechanical crushing treatment time is (2 × 5) min;
wherein (2 × 5) min refers to mechanical crushing for 5min, taking out, placing on ice, cooling for at least 30s, and performing the operation for 2 times;
the mechanical crushing is a grinding treatment.
2. The method of claim 1, wherein the DNA extraction comprises the steps of: based on 1ml of mould spore liquid after mechanical crushing treatment,
(1) adding 20 mu L protease K solution into the tube, and mixing uniformly;
(2) adding 220 μ L buffer solution GB, shaking for 15sec, standing at 70 deg.C for 10min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;
(3) adding 220 mu L absolute ethyl alcohol, fully shaking and uniformly mixing for 15sec, and centrifuging briefly to remove water drops on the inner wall of the tube cover;
(4) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, placing the adsorption column CB3 into a collecting pipe, centrifuging at 12,000rpm for 30sec, pouring the waste liquid, and placing the adsorption column CB3 into the collecting pipe;
(5) adding 500 mu L buffer solution GD into an adsorption column CB3, centrifuging at 12,000rpm for 30sec, pouring waste liquid, and putting the adsorption column CB3 into a collection tube;
(6) adding 600 mu L of rinsing liquid PW into an adsorption column CB3, centrifuging at 12,000rpm for 30sec, pouring waste liquid, and putting the adsorption column CB3 into a collecting pipe;
(7) repeating the operation step (6);
(8) placing adsorption column CB3 back into the collecting tube, centrifuging at 12,000rpm for 2min, and pouring off waste liquid; placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
(9) transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 mu L elution buffer TE into the middle part of the adsorption membrane, standing for 5min at room temperature, centrifuging for 2min at 12,000rpm, and collecting the solution into the centrifuge tube.
3. The method according to claim 1 or 2, wherein the mould spores are aspergillus niger spores.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586333A (en) * | 2016-01-07 | 2016-05-18 | 中国人民解放军第二军医大学 | Quick extraction method for total DNA of yeast-like fungi for nucleic acid amplification |
| WO2017129814A1 (en) * | 2016-01-30 | 2017-08-03 | Safeguard Biosystems Holdings Ltd. | Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586333A (en) * | 2016-01-07 | 2016-05-18 | 中国人民解放军第二军医大学 | Quick extraction method for total DNA of yeast-like fungi for nucleic acid amplification |
| WO2017129814A1 (en) * | 2016-01-30 | 2017-08-03 | Safeguard Biosystems Holdings Ltd. | Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms |
Non-Patent Citations (3)
| Title |
|---|
| 一种经济快速提取丝状真菌基因组DNA的方法;陈锋菊等;《生命科学研究》;20100430;第14卷(第2期);第122-124页 * |
| 玻璃珠法提取基因组DNA;戈海泽等;《天津医科大学学报》;20061231;第12卷(第2期);第313-314页 * |
| 玻璃珠破碎毕赤酵母条件的优化;卢俊文等;《生物加工过程》;20160131;第14卷(第1期);第30-35页 * |
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Inventor after: Xu Wentao Inventor after: Luo Yunbo Inventor after: Huang Kunlun Inventor after: Tian Lanfang Inventor after: He Xiaoyun Inventor after: Guo Mingzhang Inventor before: Luo Yunbo Inventor before: Huang Kunlun Inventor before: Xu Wentao Inventor before: Tian Lanfang Inventor before: He Xiaoyun Inventor before: Guo Mingzhang |