CN1873404A - Quick detection method for estimating capability of culture medium for separating and recycling microbe groups - Google Patents
Quick detection method for estimating capability of culture medium for separating and recycling microbe groups Download PDFInfo
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- CN1873404A CN1873404A CN 200510037264 CN200510037264A CN1873404A CN 1873404 A CN1873404 A CN 1873404A CN 200510037264 CN200510037264 CN 200510037264 CN 200510037264 A CN200510037264 A CN 200510037264A CN 1873404 A CN1873404 A CN 1873404A
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Abstract
The invention relates to the method used polymerase chain reaction-degeneration gradient gel electrophoresis to evaluate reclaiming separated germ group with different culture medium or condition according to DGGE electrophoresis pattern and spectral band similarity coefficient. It can screen out better culture medium, and has the advantages of fast, simple, and exact process compared with the traditional method. And it can be used to rapidly analyze environment microbial population diversity and community dynamic change.
Description
[affiliated technical field]
The present invention relates to a kind of method for quick that utilizes polymerase chain reaction-denaturing gradient gel electrophoresis (it is abbreviated as PCR-DGGE PCR-DENATURINGGRADIENT GEL ELECTROPHORESIS) to estimate different nutrient culture media or condition of culture recovery separate microorganism monoid ability.
[background technology]
Microbial resources are very abundant in the physical environment, yet up to the present, microorganism that can ARTIFICIAL CULTURE is very limited, less than 1% of total amount.Because the deficiency of condition of culture and cultural method, many in environment dominant bacterium also under lab be not separated up to now, or what separate is not dominant bacteria in the environment.Therefore the ability of estimating different nutrient culture media and condition of culture recovery separate microorganism monoid is very important.
When estimating nutrient culture media and reclaim the effect of separate microorganism monoid ability, whether the microbe groups that is separated to is consistent with the microbe groups in the environmental sample is a key problem.In the past for different nutrient culture media and condition of culture reclaim separate microorganism monoid ability assessment method be based upon traditional plate count and morphologic observation technical.But because the microbe colony form is fairly simple, some is more similar, only can't accuracy of judgement with microscope, be easy to generate erroneous judgement, and be inaccurate so carry out colony counting only according to morphological analysis.And according to physiological and biochemical property to the cultured microorganism evaluation of classifying because workload is big, consuming time, also almost be impossible.Therefore there is very big drawback in the method for traditional estimating capability of culture medium for separating and recycling microbe groups.
[summary of the invention]
The objective of the invention is to by utilizing the PCR-DGGE technology that different nutrient culture media or condition of culture separating and recycling microbe groups ability are carried out fast detecting.
Method for quick of the present invention may further comprise the steps:
(1) selects the sample be used to test;
(2) separate with purification of samples in microorganism: specimen preparation is become 10
-1～10
-5Different dilutabilitys is coated with flat board respectively on different nutrient culture media, thermophilic is cultivated down or under the different condition;
(3) extract and purification of samples in the genomic DNAs of all microorganisms: take by weighing the 10.0g sample, put into and fill 10 sterile glass beads (triangular flask of diameter 3mm～5mm) adds 15mL extraction damping fluid (100mmol/L Tris-HCl, 100mmol/L EDTA-Na
2, 200mmol/L NaCl, 1%PVP, 2%CTAB, pH 8.0); At 180r/min, the 30min that vibrates under 37 ℃ of conditions adds 2mL20%SDS then and continues vibration 10min; The centrifugal 15min of 6000r/min behind 65 ℃ of water-bath 1h, collect supernatant, add 0.5 times of volume PEG-NaCl solution (wherein containing 30%PEG and 1.6mol/L NaCl), leave standstill 2h under the room temperature behind the mixing, the centrifugal 20min of 10000r/min, precipitation suspends again with 2mL TE; Add 4mol/L NaAC (pH5.4) to final concentration 0.3mol/L, with phenol/chloroform/isoamylol solution (25: 24: 1) extracting once, add 0.6 times of volume isopropanol precipitating 2h, the centrifugal 20min of 12000r/min, after the precipitation drying, dissolve with 200 μ l TE, use Wizard@DNA clean-up system (Promega) purifying again after, place-20 ℃ of preservations, stand-by;
(4) flat board that extracts under different nutrient culture media and the condition of culture reclaims the total DNA of bacterium: dull and stereotyped with the washing of 10ml sterilized water respectively, collect thalline, and extract the total DNA of thalline then;
(5) the 16SrDNA V3 district of all samples carries out pcr amplification: get 10 times of damping fluids, 5 μ l, and dNTP (10mM) 1 μ l, F341-357 (GC) (10 μ M) 1 μ l, R534-518 (10 μ M) 1 μ l, Taq (5U/ μ l) 0.4 μ l, template DNA 2 μ l supply ddH
2O to 50 μ l; Response procedures is: 94 ℃ of pre-sex change of 5min; 94 ℃ of 1min, 60 ℃ of 30s, 72 ℃ of 2min, 10 circulations; 94 ℃ of 30S, 60～55 ℃ of 30s (0.5 ℃/circulation), 72 ℃ of 2min, 10 circulations; 94 ℃ of 30S, 55 ℃ of 30s, 72 ℃ of 2min, 15 circulations; 72 ℃ of 7min; Amplified production adopts 1.5% agarose analytical control;
(6) pcr amplification product is carried out polyacrylamide gel (DGGE) electrophoresis: after the PCR product concentrates, adopt D-Code sudden change detection system that sample is carried out DGGE and analyze; Used polyacrylamide concentration is 8%, and denatured gradient is 35%～60% (100% denaturant is a 7mol/L urea, 40% formamide), 180V, 60 ℃ of constant temperature, electrophoresis 4.0h among 0.5 * TAE, GoldView dyeing 30min, the UVI imaging system is taken pictures; Help to determine the brightness peak of what and band of sample electrophoresis band with the UVIBand analysis software;
(7) DGGE bands of a spectrum likeness coefficient (Cs) is analyzed: the similarity of using the DGGE finger-print of the different samples of Sorenson paired comparisons; Cs=2j/ (a+b), wherein a, b represent two DNA band numbers in the comparison other, j represents identical band quantity among a and the b;
(8) evaluation method: analyze according to DGGE electrophoresis pattern and bands of a spectrum likeness coefficient (Cs), different nutrient culture media of comprehensive evaluation or condition of culture reclaim separate microorganism monoid ability; General each band can be regarded as a microbe groups in the DGGE electrophoresis pattern, the intensity of band can reflect the number of the quantity of this monoid, electrophoretic band is many more, bands of a spectrum likeness coefficient (Cs) is high more, illustrates that the ability of the microbe groups in this kind nutrient culture media or the condition of culture recovery sample separation is better;
Utilize method for quick of the present invention, can filter out the nutrient culture media that can better separate the microbe groups in the recovery sample intuitively, compare with the method that physiological and biochemical property is identified with traditional usefulness form, has quick, easy, accurate, repeated advantages of higher, be that conventional art is incomparable, provide a kind of evaluation method of molecular level for developing new nutrient culture media and culture technique, this detection method can also be used for express-analysis environmental microorganism population diversity and communities dynamics variation in addition.
[description of drawings]
The DGGE finger-print of tomato pedotheque under 4 kinds of different nutrient culture media (soil soaks juice for nutrient broth, YG, root exudates) and different condition of culture relatively.Wherein L1-L4 is: nutrient broth, and YG, soil soaks juice, and 4 kinds of nutrient culture media such as root exudates are at 28 ℃ of DGGE collection of illustrative plates of cultivating 2 days; L6-L9 is: nutrient broth, and YG, soil soaks juice, and 4 kinds of nutrient culture media such as root exudates are at the DGGE collection of illustrative plates of 20 ℃ of cultivations after 12 days.L5 is the DGGE collection of illustrative plates of tomato rhizosphere soil.From the DGGE electrophoresis result as can be seen, the electrophoresis pattern of different samples has very big difference: 4 kinds of nutrient culture media are more approaching at 28 ℃ of DGGE finger-prints of cultivating 12 days, and the bacteria flora that is recovered to is more similar; And the band number average of each sample is many in the DGGE of 20 ℃ of cultivations finger-print, and is more in the monoid that sample was separated to of 28 ℃ of cultivations, more approaches the bacterium monoid in the rhizosphere soil.
[embodiment]
Embodiment 1:
A kind of quick detection kit of estimating capability of culture medium for separating and recycling microbe groups: comprise 10 times of damping fluids, dNTP (10mM), F341-357 (GC) (10 μ M), R534-518 (10 μ M), Taq (5U/ μ l) and ddH
2O.
This detection kit is used to estimate the method for different capability of culture medium for separating and recycling microbe groups:
(1) select the environmental sample be used to test: soil picks up from Da Feng test base, academy of agricultural sciences, Guangdong Province, at the tomato fruiting period, chooses tomato plant by five point samplings, and its root system is dug out together with soil, and natural air drying ground the 1mm sieve;
(2) bacterium in separation and the purifying environmental sample: adopt nutrient broth, YG (yeast glucose agar medium), root exudates, 4 kinds of nutrient culture media of soil maceration extract; Soil sample is fully vibrated preparation 10
-1～10
-5Different dilution samples are coated with flat board respectively on 4 kinds of nutrient culture media, place under two kinds of different temperatures and cultivate; Cultivated 2 days for 28 ℃, cultivated 12 days for 20 ℃;
(3) genomic DNA of all microorganisms in extraction and the purifying environmental sample: take by weighing the 10.0g soil sample, put into and fill 10 sterile glass beads (triangular flask of diameter 3mm～5mm), add 15mL and extract damping fluid (100mmol/L Tris-HCl, 100mmol/L EDTA-Na
2, 200mmol/L NaCl, 1%PVP, 2%CTAB, pH 8.0).At 180r/min, the 30min that vibrates under 37 ℃ of conditions adds 2mL 20%SDS then and continues vibration 10min; The centrifugal 15min of 6000r/min behind 65 ℃ of water-bath 1h, collect supernatant, add 0.5 times of volume PEG-NaCl solution (wherein containing 30%PEG and 1.6mol/L NaCl), leave standstill 2h under the room temperature behind the mixing, the centrifugal 20min of 10000r/min, precipitation suspends again with 2mL TE; Add 4mol/L NaAC (pH5.4) to final concentration 0.3mol/L, with phenol/chloroform/isoamylol (25: 24: 1) extracting once, add 0.6 times of volume isopropanol precipitating 2h, the centrifugal 20min of 12000r/min, after the precipitation drying, dissolve with 200 μ lTE, use Wizard@DNA clean-up system (Promega) purifying again after, place-20 ℃ of preservations, stand-by;
(4) extract the dull and stereotyped total DNA of bacterium that reclaims: dull and stereotyped with the washing of 10ml sterilized water respectively, collect thalline; Adopt the CTAB method to extract the total DNA of thalline;
(5) the 16SrDNA V3 district pcr amplification of all samples: reaction system is 10 times of damping fluids, 5 μ l, dNTP (10mM) 1 μ l, each 1 μ l of primers F 341-357 (GC) (10 μ M) and R534-518 (10 μ M), Taq (5U/ μ l) 0.4 μ l, template DNA 2 μ l supply ddH
2O to 50 μ l; Response procedures is 94 ℃ of pre-sex change of 5min; 94 ℃ of 1min, 60 ℃ of 30s, 72 ℃ of 2min, 10 circulations; 94 ℃ of 30S, 60～55 ℃ of 30s (0.5 ℃/circulation), 72 ℃ of 2min, 10 circulations; 94 ℃ of 30S, 55 ℃ of 30s, 72 ℃ of 2min, 15 circulations; 72 ℃ of 7min; Amplified production adopts 1.5% agarose analytical control;
(6) pcr amplification product is carried out polyacrylamide gel (DGGE) electrophoresis: after the PCR product concentrates, adopt D-Code sudden change detection system that sample is carried out DGGE and analyze; Used polyacrylamide concentration is 8%, and denatured gradient is 35%～60% (100% denaturant is a 7mol/L urea, 40% formamide), 180V, 60 ℃ of constant temperature, electrophoresis 4.0h among 0.5 * TAE, GoldView dyeing 30min, the UVI imaging system is taken pictures; Help to determine the brightness peak of what and band of sample electrophoresis band with the UVIBand analysis software;
DGGE bands of a spectrum likeness coefficient (Cs): with the similarity of the more different sample DGGE of Sorenson paired comparisons likeness coefficient (Cs) finger-print; Cs=2j/ (a+b), wherein a, b represent two DNA band numbers in the comparison other, j represents identical band quantity among a and the b.Result's (seeing Table 1) shows: tomato rhizosphere sample is with lower at nutrient broth, YG, soil maceration extract, the root exudates nutrient culture media similarity index of 28 ℃ of cultivations, the Cs value is respectively 0.21,0.10,0.20,0.11, is respectively 0.34,0.43,0.36,0.44 with 4 kinds of nutrient culture media similarity indexes 20 ℃ of cultivations.As seen root exudates and YG nutrient culture media can more be isolated the bacterium monoid of tomato rhizosphere under 20 ℃ of cultivations, are better than other nutrient culture media and condition of culture.
The Cs value of the different samples of table 1
Table 1 Cs values of different samples
| Lanes compared | L1 | L2 | L3 | L4 | L5 | L6 | L7 | L8 | L9 |
| L1 L2 L3 L4 L5 L6 L7 L8 L9 | 1.00 0.79 0.76 0.77 0.21 0.33 0.26 0.19 0.21 | 1.00 0.91 0.88 0.10 0.27 0.20 0.12 0.15 | 1.00 0.79 0.20 0.32 0.29 0.18 0.25 | 1.00 0.11 0.23 0.16 0.06 0.11 | 1.00 0.37 0.43 0.36 0.44 | 1.00 0.56 0.33 0.38 | 1.00 0.51 0.58 | 1.00 0.53 | 1.00 |
L1-L4: nutrient broth, YG, soil soaks juice, root exudates; 28 ℃ of cultivations.
L6-L9: nutrient broth, YG, soil soaks juice, root exudates; 20 ℃ of cultivations.
L5: rhizosphere soil
DGGE electrophoresis pattern and bands of a spectrum likeness coefficient (Cs) are integrated the size that 4 kinds of different nutrient culture media of assay reclaim separate microorganism monoid abilities: the YG nutrient culture media carries out the cultivation of long period under 20 ℃ of lower cultivation temperature, produce more, more representative bacteriums than nutrient broth medium; And it is maximum based on the nutrient culture media of root exudates from the bacteria flora that the tomato rhizosphere is recovered to.
Claims (2)
1. the method for quick of an estimating capability of culture medium for separating and recycling microbe groups may further comprise the steps:
(1) selects the sample be used to test;
(2) separate with the purifying environmental sample in microorganism: specimen preparation is become 10
-1～10
-5Different dilutabilitys is coated with flat board respectively on different nutrient culture media, thermophilic is cultivated down or under the different condition;
(3) genomic DNA of all microorganisms in extraction and the purifying environmental sample: take by weighing the 10.0g sample, put into and fill 10 sterile glass beads (triangular flask of diameter 3mm～5mm), add 15mL and extract damping fluid (100mmol/L Tris-HCl, 100mmol/L EDTA-Na
2, 200mmol/L NaCl, 1%PVP, 2%CTAB, pH8.0); At 180r/min, the 30min that vibrates under 37 ℃ of conditions adds 2mL 20%SDS then and continues vibration 10min; The centrifugal 15min of 6000r/min behind 65 ℃ of water-bath 1h, collect supernatant, add 0.5 times of volume PEG-NaCl solution (wherein containing 30%PEG and 1.6mol/L NaCl), leave standstill 2h under the room temperature behind the mixing, the centrifugal 20min of 10000r/min, precipitation suspends again with 2mL TE; Add 4mol/L NaAC (pH5.4) to final concentration 0.3mol/L, with phenol/chloroform/isoamylol solution (25: 24: 1) extracting once, add 0.6 times of volume isopropanol precipitating 2h, the centrifugal 20min of 12000r/min, after the precipitation drying, dissolve with 200 μ l TE, use Wizard@DNA clean-up system (Promega) purifying again after, place-20 ℃ of preservations, stand-by;
(4) flat board that extracts under different nutrient culture media and the condition of culture reclaims the total DNA of bacterium: dull and stereotyped with the washing of 10ml sterilized water respectively, collect thalline; Extract the total DNA of thalline then;
(5) the 16SrDNA V3 district of all samples carries out pcr amplification: get 10 times of damping fluids, 5 μ l, dNTP (10mM) 1 μ l, F341-357 (GC) (10 μ M) 1 μ l, R534-518 (10 μ M) 1 μ l, Taq (5U/ μ l) 0.4 μ l, template DNA 2 μ l supply ddH2O to 50 μ l.Response procedures is 94 ℃ of pre-sex change of 5min; 94 ℃ of 1min, 60 ℃ of 30s, 72 ℃ of 2min, 10 circulations; 94 ℃ of 30S, 60～55 ℃ of 30s (0.5 ℃/circulation), 72 ℃ of 2min, 10 circulations; 94 ℃ of 30S, 55 ℃ of 30s, 72 ℃ of 2min, 15 circulations; 72 ℃ of 7min.Amplified production adopts 1.5% agarose analytical control;
(6) pcr amplification product is carried out the DGGE electrophoresis: polyacrylamide concentration is 8%, denatured gradient is that 35%～60% (100% denaturant is a 7mol/L urea, 40% formamide), 180V, 60 ℃ of constant temperature, 0.5 electrophoresis 4.0h among the * TAE, GoldView dyeing 30min, the UVI imaging system is taken pictures; Help to determine the brightness peak of what and band of sample electrophoresis band with the UVIBand analysis software;
(7) DGGE bands of a spectrum likeness coefficient (Cs) is analyzed: the similarity of using the DGGE finger-print of the different samples of Sorenson paired comparisons; Cs=2j/ (a+b), wherein a, b represent two DNA band numbers in the comparison other, j represents identical band quantity among a and the b.
2. the described detection method of claim 1 can be used for estimating fast the recovery separating power of different nutrient culture media to microbe groups in the various environment.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286618A (en) * | 2011-07-22 | 2011-12-21 | 上海市农业科学院 | Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) technology applied to safety evaluation of genetically modified crop rhizosphere soil micro ecosystem |
| TWI595095B (en) * | 2015-05-13 | 2017-08-11 | Nat Univ Chung Hsing | Gel electrophoresis gel preparation method, and the prepared solid-type coagulation Gel electrophoresis gel, and mobile gel electrophoresis gel |
-
2005
- 2005-09-16 CN CNB2005100372645A patent/CN100507546C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286618A (en) * | 2011-07-22 | 2011-12-21 | 上海市农业科学院 | Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) technology applied to safety evaluation of genetically modified crop rhizosphere soil micro ecosystem |
| TWI595095B (en) * | 2015-05-13 | 2017-08-11 | Nat Univ Chung Hsing | Gel electrophoresis gel preparation method, and the prepared solid-type coagulation Gel electrophoresis gel, and mobile gel electrophoresis gel |
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