CN101914630B - Method for determining biomass of hyphae of edible fungi in substrate by detecting DNA (Deoxyribonucleic Acid) concentration - Google Patents
Method for determining biomass of hyphae of edible fungi in substrate by detecting DNA (Deoxyribonucleic Acid) concentration Download PDFInfo
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Abstract
The invention relates to a method for determining the biomass of hyphae of edible fungi in a substrate by detecting the DNA (Deoxyribonucleic Acid) concentration, comprising the following steps of: (1) culturing pure hyphae of the edible fungi; (2) filtering, washing, freezing and fully drying the hyphae; (3) gradually weighing the hyphae, extracting DNA and then establishing an associated standard curve; (4) extracting the DNA from the relevant substrate and then determining the background level of the DNA in the substrate; (5) mixing the hyphae with the substrate, extracting the DNA, determining DNA amount, and comparing the result with the results obtained in the steps (3) and (4) to determine the influence of the substrate on DNA extraction; and (6) extracting the DNA from a sample to be detected, determining the DNA amount, and then comparing the result with the standard curve, the substrate background and the substrate influence to determine the biomass of the hyphae of the edible fungi in the sample containing the substrate. The method has the advantages of simplicity, short consumed time, high efficiency and accuracy and can be used for stably monitoring the biomass of the hyphae of the edible fungi in a relevant culture mode or growing environment for a long term by adopting the determination of a detecting system.
Description
Technical field
The invention belongs to the method field of measuring the hypha of edible fungus living weight, particularly relate to a kind of through detecting the method for hypha of edible fungus living weight in the DNA concentration determination culture substrate.
Background technology
Living weight is a basic parameter of all microorganism growth situations, and the mensuration of living weight is to confirm the basis of growth kinetics with the further investigation microorganism metabolism controlling of microbial cultivation process.Because the mixed characteristic of most mikrobes and its growth matrix is difficult to directly measure its living weight, especially mycelia gos deep into culture medium inside in tame edible mushrooms, can't carry out separation determination.
Existing microorganism biological quantity measuring method has four big types: counting is directly separated in (1) from substratum, like nephelometry, direct-counting method etc.; (2) infer living weight through detecting Metabolic activity, like the metabolism of oxygen and carbonic acid gas, born of the same parents laccase method etc. outward; (3) measure in the organism content of some particular matter and know its living weight by inference, like Regitex FA analytical method, ergosterol method, purine analytical method, nitrogen and protein etc.; (4) utilize some phenomenon estimation living weight relevant with thalli growth.These measuring methods all have limitation separately; Require the pure culture state like (1); Remaining method is the estimation that the microbial activities in the fixed system or special composition are carried out; Other interference base confrontation result's influence in can not removal system, thereby can not accurately quantitatively also can't learn the limit of error of estimation.Present method is motivated; In the DNA amount that makes up kind fungi to be measured with extract on the basis that influences with DNA in the typical curve of mycelia amount and the corresponding base confrontation leaching process; Adopt and directly measure the DNA amount that contains the matrix sample, thereby obtain the hypha of edible fungus biomass parameters in the unit mass testing sample quickly and easily.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method through hypha of edible fungus living weight in the detection DNA concentration determination culture substrate, and this method is simple, and is consuming time few, can be quick, detect the living weight of hypha of edible fungus in the culture medium accurately.
Of the present invention a kind of through detecting the method for hypha of edible fungus living weight in the DNA concentration determination culture substrate, comprising:
(1) cultivates the standard mycelia
Get edible mushrooms slant culture to be measured, smash after the activation, insert in the fresh standard liquid culture medium, shaking table is cultured to mycelia stationary phase;
(2) mycelia is handled
Filter mycelia, aseptic water washing, be refrigerated to solid-state, thorough drying;
(3) make up typical curve
Mycelia after step (2) processing is ground rapidly, and gradient takes by weighing and is placed in the container, extracts DNA with the CTAB method, and NANODROP 1000 ultraviolet spectrophotometers are confirmed the DNA amount, makes up extraction DNA and measures and extract the criteria associated curve with the mycelia amount;
(4) corresponding matrix dna level is confirmed
Get corresponding matrix, extract DNA with the CTAB method, NANODROP 1000 ultraviolet spectrophotometers are confirmed the DNA amount, confirm matrix DNA background level;
(5) cultivate mycelia and mix, confirm the influence of corresponding base confrontation DNA extraction with matrix
The mycelia that takes by weighing after step (2) is handled mixes with above-mentioned matrix, extracts DNA with the CTAB method, and the NANODROP1000 ultraviolet spectrophotometer is confirmed the DNA amount, with result's comparison of (3) and (4), confirms the influence of this kind matrix to DNA extraction;
(6) the confirming of hypha of edible fungus living weight in the testing sample
Sample thief (culture of this edible mushrooms in matrix) lyophilize; Grind; Batch scale is taken in the container, extracts DNA with the CTAB method, and NANODROP 1000 ultraviolet spectrophotometers are confirmed the DNA amount; Reference standard curve, matrix background and matrix contributive rate are confirmed the living weight of the hypha of edible fungus in the culture medium.
Mycelia in the said step (1) is mushroom 135 mycelia.
In the said step (1); Tame edible mushrooms slant strains is got in the acquisition of edible mushrooms standard mycelia to be measured, and switching (is according to circumstances selected the PDB substratum of commercially producing for use after the activation in fresh standard liquid culture medium; The Gao Shi substratum; Stable components such as Cha Shi substratum, the standard liquid culture medium of good reproducibility), shaking table is cultured to mycelia stationary phase.
Standard liquid culture medium in the said step (1) is the PDB substratum.
Mycelia water cut after the processing in the said step (2) is near zero, and DNA is difficult for degraded under the refrigerated condition.
Said step (2) is used the freeze drier thorough drying.
Extract DNA, 5 repetitions of every processing with the CTAB method in the said step (3).
Making up typical curve in the said step (3) is the benchmark of sample determination; Extract DNA and NANODROP1000 ultraviolet spectrophotometer in the CTAB method and confirm to be provided with in the step of DNA amount the contrast between the mycelia that repetition and different batches cultivate, in order to confirm the limit of error of whole detection architecture.
In the said step (4); Tame edible mushrooms can carry out artificial culture by different prescriptions with different agricultural waste materials; These culture medium staples are major ingredient (wood chip, straw, cotton seed hulls, bagasse, corn cob, wheat straw etc.) and auxiliary material (wheat bran, rice bran, soya-bean cake etc.); Sometimes also increase added ingredientss such as sugar and inorganic salt as required; Different raw materials possibly cause the DNA background difference of culture medium with prescription, need in step (4), confirm the dna level with the culture medium of testing sample same recipe here, the influence that is brought by the matrix background in the elimination actual measurement.
Corresponding matrix in the said step (4) is got wood chip and is soaked earlier 12-24h, mixes with wheat bran again, and the adjustment water cut is sterilized to 50%-60%, be refrigerated to then solid-state, thorough drying again.
In the said step (5), the process preliminary experiment settles the standard and cultivates mycelia and matrix blending ratio (trying one's best near the data of testing sample), confirms the influence of matrix to DNA extraction.
The mycelia in the said step (5) and the mass ratio of corresponding matrix are 1: 4 or 3: 7.
Through detect DNA concentration confirm the method for hypha,hyphae living weight can realize to hypha,hyphae living weight in the different substrates accurately quantitatively, the used time of this method is short, the linear lag is high, and hypha,hyphae living weight in the matrix what judge fast.Reusable once setting up to particular types hypha,hyphae amount with the typical curve that extracts the DNA amount, confirm that different substrates then makes the hypha biomass of measuring particular types fungi in the different substrates become possibility to the influence of extracting DNA.
The present invention is through detecting the method for hypha,hyphae living weight in the DNA concentration determination matrix; Having solved living weight measures inaccurate, limit of error and is difficult to problems such as assessment; What of fungal biomass in the matrix are this method can judge fast, and accurately represent with MU (mg).Detection system is once confirming and can carrying out long-term, stable monitoring to the fungal biomass in corresponding training mode or the growing environment.
Beneficial effect
(1) method of the present invention is simple; In the DNA amount that makes up kind fungi to be measured with extract on the basis that influences with DNA in the typical curve of mycelia amount and the corresponding base confrontation leaching process; Adopt and directly measure the DNA amount that contains the matrix sample, thereby obtain the fungal biomass parameter of unit mass testing sample quickly and easily;
(2) the invention solves living weight measures inaccurate, limit of error and is difficult to problems such as assessment.
Description of drawings
First substandard of Fig. 1 is cultivated mycelia and is measured the related curve of measuring with DNA;
Second batch of type culture mycelia of Fig. 2 measured the related curve with the DNA amount;
Fig. 3 cultivates pure mycelia for the 3rd batch and measures the related curve of measuring with DNA;
Fig. 4 different batches pure culture mycelia and dna content relation.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
The present invention is that embodiment sets forth concrete steps with mushroom mycelium living weight in the mushroom 135 mycelium culture things of confirming the wood chip conventional substrate:
(1) cultivates the standard mycelia of mushroom 135 with standard P DB liquid nutrient medium
After the 135 mycelia activation of the mushroom on the solid PDA substratum; Smash 30S at interval with homogeneous appearance high and low shift, access contains in the 250mL triangular flask of the fresh standardized liquid PDB of 90mL (U.S. company BD production) substratum rotating speed 120rpm; Under 25 ℃, cultivate 14d to mycelia stationary phase.Every other day cultivate a batch of mycelia, each batch cultivated 5 bottles, cultivates 3 batches altogether, for making up typical curve.
(2) mycelia is handled
Filter mycelia with non-woven fabrics, blot excessive moisture with aseptic thieving paper again behind the aseptic water washing, be refrigerated to solid-stately in-20 ℃ of refrigerators, change thorough drying in the freeze drier (Heto Power Dry PL3000) over to.
(3) make up typical curve
Freeze dried mycelia is ground rapidly at normal temperatures, and gradient accurately takes by weighing mycelia (2mg, 3mg, 4mg, 5mg, 6mg, 7mg), and is divided in the 2mL centrifuge tube, and the CTAB method is extracted DNA, 5 repetitions of every processing.
Get the CTAB 800 μ L of 65 ℃ of preheatings, add in the centrifuge tube, 65 ℃ of water-bath 1h, or shake up mixed solution; 12000rpm, 4 ℃ of centrifugal 20min; Get in the centrifuge tube that supernatant changes another 2mL over to, add isopyknic phenol of precooling (4 ℃): chloroform (1: 1), abundant mixing, 12000rpm, 4 ℃ of centrifugal 10min; Get in the centrifuge tube that supernatant changes another 2mL over to, with isopyknic chloroform of precooling (4 ℃): primary isoamyl alcohol (24: 1) extracts twice, abundant mixing, 12000rpm, 4 ℃ of centrifugal 10min; Get in the centrifuge tube that supernatant changes 1.5mL over to, (3mol/L pH52), and adds the Virahol of 500 μ L precoolings (20 ℃) to add the NaAc of 60 μ L; Leave standstill 20min in-20 ℃ and make the DNA deposition, 12000rpm, 4 ℃ of centrifugal 10min abandon supernatant; Add 70% ethanol of 1mL precooling (4 ℃) again, turn upside down 12000rpm gently; 4 ℃ of centrifugal 10min discard ethanol, the dry 10min (can not heat) of Vacuumdrier.Add the distilled water (containing 1 μ L RNase) of 50 μ L, beat gently and make resolution of precipitate, 37 ℃ of water-bath 1h remove RNA.
Confirm the amount of DNA with NANODROP 1000 ultraviolet spectrophotometers, make up and extract the DNA amount and extract criteria associated curve with the mycelia amount.
1) first substandard mushroom 135 mycelia situation:
First substandard of table 1 is cultivated the DNA concentration that mycelia is extracted
| Sample number | 1 | 2 | 3 | 4 | 5 |
| Mycelia heavy (g) | 0.0019 | 0.0019 | 0.0020 | 0.0020 | 0.0019 |
| DNA concentration (ng/ μ L) | 236.2 | 210.6 | 241.9 | 247.1 | 225.4 |
| |
6 | 7 | 8 | 9 | 10 |
| Mycelia heavy (g) | 0.0029 | 0.0030 | 0.0030 | 0.0030 | 0.0029 |
| DNA concentration (ng/ μ L) | 342.4 | 372.5 | 363.6 | 358.8 | 332.3 |
| Sample number | 11 | 12 | 13 | 14 | 15 |
| Mycelia heavy (g) | 0.0041 | 0.0040 | 0.0039 | 0.0039 | 0.0039 |
| DNA concentration (ng/ μ L) | 523.3 | 481.5 | 441.1 | 457.8 | 450.0 |
| Sample number | 16 | 17 | 18 | 19 | 20 |
| Mycelia heavy (g) | 0.0050 | 0.0050 | 0.0049 | 0.0049 | 0.0051 |
| DNA concentration (ng/ μ L) | 636.8 | 639.6 | 582.5 | 565.4 | 672.5 |
| Sample number | 21 | 22 | 23 | 24 | 25 |
| Mycelia heavy (g) | 0.0059 | 0.0061 | 0.0059 | 0.0060 | 0.0060 |
| DNA concentration (ng/ μ L) | 683.4 | 776.5 | 695.4 | 755.0 | 744.5 |
| Sample number | 26 | 27 | 28 | 29 | 30 |
| Mycelia heavy (g) | 0.0070 | 0.0069 | 0.0070 | 0.0069 | 0.0070 |
| DNA concentration (ng/ μ L) | 856.8 | 831.1 | 828.4 | 824.6 | 825.5 |
First substandard of table 2 is cultivated the mycelia dna content
| Sample number | 1 | 2 | 3 | 4 | 5 | MV |
| 2mg mycelia DNA measures (μ g) | 12.43 | 11.08 | 12.10 | 12.36 | 11.86 | 11.97 |
| Error (%) | 3.89 | 7.37 | 1.08 | 3.25 | 0.86 | —— |
| |
6 | 7 | 8 | 9 | 10 | MV |
| 3mg mycelia DNA measures (μ g) | 17.71 | 18.63 | 18.18 | 17.94 | 17.19 | 17.93 |
| Error (%) | 1.22 | 3.88 | 1.40 | 0.06 | 4.13 | —— |
| Sample number | 11 | 12 | 13 | 14 | 15 | MV |
| 4mg mycelia DNA measures (μ g) | 25.53 | 24.08 | 22.62 | 23.48 | 23.08 | 23.76 |
| Error (%) | 7.46 | 1.35 | 4.78 | 1.17 | 2.86 | —— |
| Sample number | 16 | 17 | 18 | 19 | 20 | MV |
| 5mg mycelia DNA measures (μ g) | 31.84 | 31.98 | 29.72 | 28.85 | 32.97 | 31.07 |
| Error (%) | 2.48 | 2.93 | 4.35 | 7.16 | 6.10 | —— |
| Sample number | 21 | 22 | 23 | 24 | 25 | MV |
| 6mg mycelia DNA measures (μ g) | 34.75 | 38.19 | 35.36 | 37.75 | 37.23 | 36.65 |
| Error (%) | 5.20 | 4.19 | 3.53 | 2.99 | 1.56 | —— |
| Sample number | 26 | 27 | 28 | 29 | 30 | MV |
| 7mg mycelia DNA measures (μ g) | 42.84 | 42.16 | 41.42 | 41.83 | 41.28 | 41.90 |
| Error (%) | 2.23 | 0.60 | 1.15 | 0.18 | 1.50 | —— |
Known by table 2: first batch limit of error is between 0.06%-7.46%.
2) second batch of standard mushroom 135 mycelia situation:
The DNA concentration that second batch of type culture mycelia of table 3 extracted
| Sample number | 1 | 2 | 3 | 4 | 5 |
| Mycelia heavy (g) | 0.0021 | 0.0021 | 0.0020 | 0.0021 | 0.0021 |
| DNA concentration (ng/ μ L) | 287.3 | 262.4 | 244.6 | 285.9 | 282.4 |
| |
6 | 7 | 8 | 9 | 10 |
| Mycelia heavy (g) | 0.0030 | 0.0031 | 0.0031 | 0.0030 | 0.0029 |
| DNA concentration (ng/ μ L) | 370.1 | 413.1 | 431.0 | 375.3 | 327.0 |
| Sample number | 11 | 12 | 13 | 14 | 15 |
| Mycelia heavy (g) | 0.0041 | 0.0040 | 0.0040 | 0.0041 | 0.0040 |
| DNA concentration (ng/ μ L) | 552.8 | 528.0 | 534.7 | 548.6 | 531.0 |
| Sample number | 16 | 17 | 18 | 19 | 20 |
| Mycelia heavy (g) | 0.0049 | 0.0050 | 0.0050 | 0.0051 | 0.0049 |
| DNA concentration (ng/ μ L) | 603.0 | 598.8 | 631.5 | 673.5 | 590.4 |
| Sample number | 21 | 22 | 23 | 24 | 25 |
| Mycelia heavy (g) | 0.0059 | 0.0060 | 0.0060 | 0.0059 | 0.0060 |
| DNA concentration (ng/ μ L) | 724.7 | 767.7 | 712.8 | 690.0 | 763.9 |
| Sample number | 26 | 27 | 28 | 29 | 30 |
| Mycelia heavy (g) | 0.0069 | 0.0071 | 0.0071 | 0.0070 | 0.0069 |
| DNA concentration (ng/ μ L) | 789.6 | 848.6 | 883.3 | 827.5 | 827.8 |
Second batch of type culture mycelia of table 4 dna content
| Sample number | 1 | 2 | 3 | 4 | 5 | MV |
| 2mg mycelia DNA measures (μ g) | 13.68 | 12.50 | 12.23 | 13.61 | 13.45 | 13.09 |
| Error (%) | 4.49 | 4.57 | 6.60 | 3.98 | 2.70 | —— |
| |
6 | 7 | 8 | 9 | 10 | MV |
| 3mg mycelia DNA measures (μ g) | 18.51 | 19.99 | 20.85 | 18.77 | 16.91 | 19.01 |
| Error (%) | 2.63 | 5.17 | 9.73 | 1.27 | 11.01 | —— |
| Sample number | 11 | 12 | 13 | 14 | 15 | MV |
| 4mg mycelia DNA measures (μ g) | 26.97 | 26.40 | 26.74 | 26.76 | 26.55 | 26.68 |
| Error (%) | 1.06 | 1.06 | 0.20 | 0.29 | 0.50 | —— |
| Sample number | 16 | 17 | 18 | 19 | 20 | MV |
| 5mg mycelia DNA measures (μ g) | 30.77 | 29.94 | 31.58 | 33.01 | 30.12 | 31.08 |
| Error (%) | 1.02 | 3.68 | 1.58 | 6.21 | 3.09 | —— |
| Sample number | 21 | 22 | 23 | 24 | 25 | MV |
| 6mg mycelia DNA measures (μ g) | 36.85 | 38.39 | 35.64 | 35.08 | 38.20 | 36.83 |
| Error (%) | 0.05 | 4.22 | 3.23 | 4.74 | 3.70 | —— |
| Sample number | 26 | 27 | 28 | 29 | 30 | MV |
| 7mg mycelia DNA measures (μ g) | 40.05 | 41.83 | 43.54 | 41.38 | 41.99 | 41.76 |
| Error (%) | 4.09 | 0.18 | 4.27 | 0.92 | 0.55 | —— |
Known by table 4: second batch limit of error is between 0.05%-11.01%.
3) the mushroom 135 mycelia situation of the 3rd batch of standard P DB liquid culture:
The DNA concentration that the 3rd batch of type culture mycelia of table 5 extracted
| Sample number | 1 | 2 | 3 | 4 | 5 |
| Mycelia heavy (g) | 0.0021 | 0.0019 | 0.0019 | 0.0021 | 0.0020 |
| DNA concentration (ng/ μ L) | 248.1 | 238.2 | 245.9 | 284.0 | 255.0 |
| |
6 | 7 | 8 | 9 | 10 |
| Mycelia heavy (g) | 0.0031 | 0.0030 | 0.0031 | 0.0030 | 0.0029 |
| DNA concentration (ng/ μ L) | 394.6 | 348.3 | 391.3 | 344.9 | 310.5 |
| Sample number | 11 | 12 | 13 | 14 | 15 |
| Mycelia heavy (g) | 0.0041 | 0.0040 | 0.0041 | 0.0040 | 0.0040 |
| DNA concentration (ng/ μ L) | 483.2 | 472.6 | 498.7 | 438.8 | 477.7 |
| Sample number | 16 | 17 | 18 | 19 | 20 |
| Mycelia heavy (g) | 0.0051 | 0.0050 | 0.0049 | 0.0049 | 0.0049 |
| DNA concentration (ng/ μ L) | 628.2 | 591.8 | 564.4 | 537.9 | 564.0 |
| Sample number | 21 | 22 | 23 | 24 | 25 |
| Mycelia heavy (g) | 0.0061 | 0.0061 | 0.0059 | 0.0059 | 0.0060 |
| DNA concentration (ng/ μ L) | 767.2 | 759.4 | 691.7 | 702.9 | 741.4 |
| Sample number | 26 | 27 | 28 | 29 | 30 |
| Mycelia heavy (g) | 0.0069 | 0.0071 | 0.0069 | 0.0069 | 0.0071 |
| DNA concentration (ng/ μ L) | 806.8 | 856.8 | 805.5 | 793.3 | 836.3 |
The 3rd batch of type culture mycelia dna content of table 6
| Sample number | 1 | 2 | 3 | 4 | 5 | MV |
| 2mg mycelia DNA measures (μ g) | 11.81 | 12.55 | 12.94 | 13.52 | 12.75 | 12.72 |
| Error (%) | 7.10 | 1.29 | 1.77 | 6.35 | 0.26 | —— |
| |
6 | 7 | 8 | 9 | 10 | MV |
| 3mg mycelia DNA measures (μ g) | 19.09 | 17.42 | 18.93 | 17.25 | 16.06 | 17.75 |
| Error (%) | 7.57 | 1.88 | 6.67 | 2.84 | 9.52 | —— |
| Sample number | 11 | 12 | 13 | 14 | 15 | MV |
| 4mg mycelia DNA measures (μ g) | 23.57 | 23.63 | 24.33 | 21.94 | 23.89 | 23.47 |
| Error (%) | 0.43 | 0.68 | 3.65 | 6.52 | 1.77 | —— |
| Sample number | 16 | 17 | 18 | 19 | 20 | MV |
| 5mg mycelia DNA measures (μ g) | 30.79 | 29.59 | 28.80 | 27.44 | 28.78 | 29.08 |
| Error (%) | 5.89 | 1.75 | 0.98 | 5.63 | 1.05 | —— |
| Sample number | 21 | 22 | 23 | 24 | 25 | MV |
| 6mg mycelia DNA measures (μ g) | 37.73 | 37.35 | 35.17 | 35.74 | 37.07 | 36.61 |
| Error (%) | 3.06 | 2.01 | 3.94 | 2.38 | 1.25 | —— |
| Sample number | 26 | 27 | 28 | 29 | 30 | MV |
| 7mg mycelia DNA measures (μ g) | 40.92 | 42.24 | 40.86 | 40.24 | 41.23 | 41.10 |
| Error (%) | 0.42 | 2.77 | 0.58 | 2.09 | 0.31 | —— |
Known by table 6: the 3rd batch limit of error is between 0.26%-9.52%.
The dna content of the standard mycelia of three batches of cultivations is concluded like table 7.
The dna content of table 7 different batches type culture mycelia
| Mycelia amount (mg) | 2 | 3 | 4 | 5 | 6 | 7 |
| First batch of DNA amount (μ g) | 11.97 | 17.93 | 23.76 | 31.07 | 36.65 | 41.90 |
| Second batch of DNA amount (μ g) | 13.09 | 19.01 | 26.68 | 31.08 | 36.83 | 41.76 |
| The 3rd batch of DNA amount (μ g) | 12.72 | 17.75 | 23.47 | 29.08 | 36.61 | 41.10 |
Three batches typical curves are concluded like Fig. 4:
Analyze: can find out that from Fig. 4 and table 7 have the good linear relation in the useful range between each batch type culture mycelia amount and its dna content, the facies relationship number average is greater than 0.99; 3 different typical curve coincidence factor height of cultivating batch mycelia show that the influence of hold in the present invention different batches mycelia and operation is less; Statistics makes up 90 data of typical curve can know that its limit of error is at 0.05%-13.74%, less than 14%; The DNA amount that every mg mycelia is extracted in the mushroom 135 type culture mycelia is about 6.11 μ g.Therefore, this typical curve can become the benchmark of confirming mushroom 135 hypha biomass through the detection dna content.
(4) the conventional culture medium DNA of mushroom 135 wood chips background assay
(soaked overnight of 0.2-0.3 * 0.5cm) is mixed with the 15g wheat bran again, adjusts water cut to 60% to get the 85g wood chip by the conventional culture material prescription of mushroom 135 wood chips; (121 ℃ of autoclavings; 1.5h), the cooling back is refrigerated to solid-state in-20 ℃ of refrigerators, change freeze drier (Heto Power Dry PL3000) thorough drying over to; After getting an amount of culture material and fully grinding set by step (3) method extract DNA, extract the result and see table 8 and table 9.
The DNA concentration of table 8 pure culture material
| Sample number | 1 | 2 | 3 | 4 | 5 |
| Culture material heavy (g) | 0.0099 | 0.0099 | 0.0101 | 0.0101 | 0.0101 |
| DNA concentration (ng/ μ L) | 126.9 | 120.7 | 134.4 | 145.4 | 161.5 |
| |
6 | 7 | 8 | 9 | 10 |
| Culture material heavy (g) | 0.0201 | 0.0201 | 0.0200 | 0.0200 | 0.0199 |
| DNA concentration (ng/ μ L) | 330.0 | 303.4 | 249.4 | 266.5 | 235.6 |
The dna content of table 9 pure culture material
| Sample number | 1 | 2 | 3 | 4 | 5 | MV |
| 10mg pure culture material DNA measures (μ g) | 6.41 | 6.10 | 6.65 | 7.20 | 7.00 | 6.87 |
| Error (%) | 6.71 | 11.27 | 3.16 | 4.77 | 16.37 | —— |
| |
6 | 7 | 8 | 9 | 10 | MV |
| 20mg pure culture material DNA measures (μ g) | 16.42 | 15.09 | 12.47 | 13.33 | 11.84 | 13.83 |
| Error (%) | 18.72 | 9.15 | 9.83 | 3.65 | 14.40 | —— |
Known by table 9: the average dna content of every mg pure culture material is 0.689 μ g.
(5) mixing of mycelia and culture material, confirm the influence of matrix to DNA extraction
The conventional culture material of freeze dried type culture mushroom 135 mycelia and freeze dried mushroom 135 wood chips [with (4)] is mixed in two kinds of ratios (40mg mycelia+160mg culture material and 60mg mycelia+140mg culture material); Fully grind; Accurately take by weighing 10mg, 20mg mixture respectively; Extract DNA by (3) method, each handles 5 repetitions, experimental result such as table 10 and table 11.
Table 1040mg type culture mycelia and 160mg culture material blended DNA concentration
| Sample number | 1 | 2 | 3 | 4 | 5 |
| Compound heavy (g) | 0.0099 | 0.0100 | 0.0099 | 0.0101 | 0.0100 |
| DNA concentration (ng/ μ L) | 389.3 | 378.4 | 349.2 | 352.2 | 358.1 |
| |
6 | 7 | 8 | 9 | 10 |
| Compound heavy (g) | 0.0200 | 0.0201 | 0.0201 | 0.0200 | 0.0199 |
| DNA concentration (ng/ μ L) | 745.3 | 744.5 | 796.9 | 718.2 | 779.0 |
Table 1140mg type culture mycelia and 160mg culture material blended dna content
| Sample number | 1 | 2 | 3 | 4 | 5 | MV |
| 10mg compound DNA measures (μ g) | 19.66 | 18.92 | 17.64 | 17.44 | 17.91 | 18.31 |
| Error (%) | 7.37 | 3.32 | 3.69 | 4.78 | 2.22 | —— |
| |
6 | 7 | 8 | 9 | 10 | MV |
| 20mg compound DNA measures (μ g) | 37.27 | 37.04 | 39.65 | 35.91 | 39.15 | 37.80 |
| Error (%) | 1.42 | 2.01 | 4.88 | 5.00 | 3.56 | —— |
Known by table 11: the limit of error of 40mg type culture mycelia and 160mg culture material blended dna content is between 1.42%-7.37%.
Table 1260mg type culture mycelia and 140mg culture material blended DNA concentration
| Sample number | 1 | 2 | 3 | 4 | 5 |
| Compound heavy (g) | 0.0100 | 0.0099 | 0.0101 | 0.0101 | 0.0100 |
| DNA concentration (ng/ μ L) | 479.6 | 462.4 | 457.0 | 458.7 | 435.4 |
| |
6 | 7 | 8 | 9 | 10 |
| Compound heavy (g) | 0.0200 | 0.0200 | 0.0199 | 0.0201 | 0.0200 |
| DNA concentration (ng/ μ L) | 955.9 | 945.0 | 974.5 | 972.2 | 932.9 |
Table 1340mg type culture mycelia and 160mg culture material blended dna content relation
| Sample number | 1 | 2 | 3 | 4 | 5 | MV |
| 10mg compound DNA measures (μ g) | 23.98 | 23.35 | 22.62 | 22.71 | 21.77 | 22.89 |
| Error (%) | 4.78 | 2.04 | 1.15 | 0.78 | 4.88 | —— |
| |
6 | 7 | 8 | 9 | 10 | MV |
| 20mg compound DNA measures (μ g) | 47.80 | 47.25 | 48.97 | 48.37 | 46.65 | 47.81 |
| Error (%) | 0.02 | 1.16 | 2.44 | 1.18 | 2.43 | —— |
Known by table 13: the limit of error of 60mg type culture mycelia and 140mg culture material blended dna content is between 0.02%-4.88%.
Conclude two kinds of blending ratio results such as table 14.
Table 14 mushroom 135 bacterium rod culture medium and standard mycelia blended dna content relation
| Mix mycelia amount (mg) | 2 | 3 | 4 | 6 |
| Dna content (μ g) | 18.31 | 22.89 | 37.80 | 47.81 |
The result of (4) and (5) is compared, confirm of the influence of this kind matrix DNA extraction.
That is: matrix is to contributive rate=(theoretical value-measured value)/theoretical value of DNA extraction
The pure mycelia dna content of theoretical value=type culture mushroom * pure mycelia and corresponding matrix compound percentages+corresponding matrix dna content * (pure mycelia of 1-and corresponding matrix compound percentages)
The measured value of measured value=mycelia and corresponding substrate mixture.
Take a sample respectively 10mg, the pairing matrix influence of 20mg mixture of two kinds of blending ratios sees table 15.
Table 15 matrix is to the influence of dna content
| Mix mycelia amount (mg) | 2 | 3 | 4 | 6 |
| Contributive rate | 0.03 | -0.01 | 0.06 | 0.03 |
Can be known by table 15: mushroom 135 mycelium culture things are 0.03 to the average influence rate of mushroom 135 mycelia DNA extraction.(6) the confirming of mushroom 135 hypha biomass in the testing sample
Mushroom 135 hypha biomass confirms in the mushroom 135 triangular flask cultures
Press the conventional culture material prescription of mushroom 135 wood chips [with (4) method], make 5 bottles of triangular flask (250mL, charge is 120-130g) culture materials; Insert fresh mushroom 135 mycelia, in 25 ℃ of thermostat containers, cultivate, treat that mycelia is covered with material (cultivating 18 days) after; Get the culture mixing in mushroom 135 triangular flasks, take by weighing 15g, be refrigerated to solid-state in-20 ℃ of refrigerators; Change freeze drier (Heto Power Dry PL3000) thorough drying over to, after fully grinding, accurately take by weighing 10mg, 20mg lyophilized products; The method of (3) is extracted DNA set by step, and each handles 5 repetitions, experimental result such as table 16 and table 17.
The DNA concentration of table 16 mushroom 135 triangular flask cultures
| Sample number | 1 | 2 | 3 | 4 | 5 |
| Lyophilized products heavy (g) | 0.0099 | 0.0100 | 0.0101 | 0.0100 | 0.0100 |
| DNA concentration (ng/ μ L) | 154.1 | 145.0 | 143.9 | 147.9 | 151.7 |
| |
6 | 7 | 8 | 9 | 10 |
| Lyophilized products heavy (g) | 0.0200 | 0.0199 | 0.0200 | 0.0199 | 0.0200 |
| DNA concentration (ng/ μ L) | 277.8 | 281.6 | 290.7 | 278.9 | 290.1 |
The dna content of table 17 mushroom 135 triangular flask cultures
| Sample number | 1 | 2 | 3 | 4 | 5 | MV |
| 10mg lyophilized products DNA measures (μ g) | 7.78 | 7.25 | 7.12 | 7.40 | 7.59 | 7.43 |
| Error (%) | 4.79 | 2.39 | 4.09 | 0.44 | 2.12 | —— |
| |
6 | 7 | 8 | 9 | 10 | MV |
| 20mg lyophilized products DNA measures (μ g) | 13.89 | 14.15 | 14.54 | 14.02 | 14.51 | 14.22 |
| Error (%) | 2.31 | 0.48 | 2.22 | 1.44 | 2.01 | —— |
The living weight that contains mushroom 135 mycelia when according to table 17 result and step (3), (4), (5) result relatively, knowing mushroom 135 mycelium culture to 18 day in every mg dried culture is 0.00701mg.The error that mushroom 135 cultures are measured does not exceed the limit of error of typical curve, so inferior error at measurment is less than 14% between 0.44%-4.79%.
The result can know in view of the above, and this method is used in and measures the hypha of edible fungus living weight in the mixed-matrix.
Claims (8)
1. the method through hypha of edible fungus living weight in the detection DNA concentration determination culture substrate comprises the following steps:
(1) cultivates the standard mycelia
Get edible mushrooms slant culture to be measured, smash after the activation, insert in the fresh standard liquid culture medium, shaking table is cultured to mycelia stationary phase; Described standard liquid culture medium is the PDB substratum;
(2) mycelia is handled
Filter above-mentioned mycelia, aseptic water washing, freezing and thorough drying;
(3) make up typical curve
Mycelia after step (2) processing is ground rapidly, and gradient takes by weighing and is placed in the container, extracts DNA with the CTAB method, and NANODROP 1000 ultraviolet spectrophotometers are confirmed the DNA amount, makes up extraction DNA and measures and extract the criteria associated curve with the mycelia amount;
(4) corresponding matrix dna level is confirmed
Get corresponding matrix, extract DNA with the CTAB method, NANODROP 1000 ultraviolet spectrophotometers are confirmed matrix DNA background level;
(5) cultivate mycelia and mix, confirm the influence of corresponding base confrontation DNA extraction with matrix
The mycelia that takes by weighing after step (2) is handled mixes with above-mentioned matrix, extracts DNA with the CTAB method, and NANODROP 1000 ultraviolet spectrophotometers are confirmed the DNA amount, with result's comparison of step (3) and (4), confirms the influence of this kind matrix to DNA extraction;
(6) the confirming of hypha of edible fungus living weight in the testing sample
Get testing sample, thorough drying is ground; Quantitatively weigh the back and extract DNA with the CTAB method; NANODROP 1000 ultraviolet spectrophotometers are confirmed DNA amount, and reference standard curve, matrix background and matrix influence confirm to contain the living weight of hypha of edible fungus in the sample of matrix.
2. according to claim 1 a kind of through detecting the method for hypha of edible fungus living weight in the DNA concentration determination culture substrate, it is characterized in that: the mycelia in the said step (1) is mushroom 135 mycelia.
3. according to claim 1 a kind of through detecting the method for hypha of edible fungus living weight in the DNA concentration determination culture substrate; It is characterized in that: the mycelia water cut after the processing in the said step (2) is near zero, and DNA is difficult for degraded under the refrigerated condition.
4. according to claim 1 a kind of through detecting the method for hypha of edible fungus living weight in the DNA concentration determination culture substrate, it is characterized in that: said step (2) is used the freeze drier thorough drying.
5. according to claim 1 a kind of through detecting the method for hypha of edible fungus living weight in the DNA concentration determination culture substrate, it is characterized in that: extract DNA, 5 repetitions of every processing with the CTAB method in the said step (3).
6. according to claim 1 a kind of through detecting the method for hypha of edible fungus living weight in the DNA concentration determination culture substrate; It is characterized in that: corresponding matrix comprises major ingredient and auxiliary material in the said step (4); Wherein major ingredient is wood chip, straw, cotton seed hulls, bagasse, corn cob or wheat straw, and auxiliary material is wheat bran, rice bran or soya-bean cake.
7. according to claim 1 or 6 described a kind of methods through hypha of edible fungus living weight in the detection DNA concentration determination culture substrate; It is characterized in that: described corresponding matrix is got wood chip and is soaked 12-24h earlier; Mix with wheat bran, the adjustment water cut is sterilized to 50%-60% again; Be refrigerated to then solid-state, thorough drying again.
8. according to claim 1 a kind of through detecting the method for hypha of edible fungus living weight in the DNA concentration determination culture substrate, it is characterized in that: the mycelia in the said step (5) and the mass ratio of corresponding matrix are 1:4 or 3:7.
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| P. Morgan等.automated image analysis method to determine fungal biomass in soils and on solid matrices.《soil biol. biochem》.1999,第23卷(第7期),609-616. * |
| 路秀玲等.固态发酵中生物量的测定方法.《天津轻工业学院学报》.2000,(第4期),57-62. * |
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