CN112553354A - Molecular biological method for rapidly detecting gentamicin high-yield strain - Google Patents
Molecular biological method for rapidly detecting gentamicin high-yield strain Download PDFInfo
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- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 title claims abstract description 22
- 229930182566 Gentamicin Natural products 0.000 title claims abstract description 22
- 229960002518 gentamicin Drugs 0.000 title claims abstract description 22
- 238000010170 biological method Methods 0.000 title claims description 8
- 238000012216 screening Methods 0.000 claims abstract description 61
- 108091092878 Microsatellite Proteins 0.000 claims abstract description 35
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 23
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims abstract description 22
- 231100000350 mutagenesis Toxicity 0.000 claims abstract description 16
- 238000002703 mutagenesis Methods 0.000 claims abstract description 16
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- 239000000126 substance Substances 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 abstract description 14
- 241000187722 Micromonospora echinospora Species 0.000 abstract description 9
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- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
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- 206010029155 Nephropathy toxic Diseases 0.000 description 1
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- 239000008049 TAE buffer Substances 0.000 description 1
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- HGEVZDLYZYVYHD-UHFFFAOYSA-N acetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound CC(O)=O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O HGEVZDLYZYVYHD-UHFFFAOYSA-N 0.000 description 1
- 229940126574 aminoglycoside antibiotic Drugs 0.000 description 1
- 239000002647 aminoglycoside antibiotic agent Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- 229910052734 helium Inorganic materials 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
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- NKAAEMMYHLFEFN-UHFFFAOYSA-M monosodium tartrate Chemical compound [Na+].OC(=O)C(O)C(O)C([O-])=O NKAAEMMYHLFEFN-UHFFFAOYSA-M 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 231100000262 ototoxicity Toxicity 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
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- 238000011218 seed culture Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a molecular biology method for rapidly detecting a gentamicin high-yield strain, which firstly adopts a method of combining lithium chloride and ARTP to mutate a production strain, and can generate a large amount of positive mutant strains; then, a microsatellite marking technology is adopted, an agarose gel electrophoresis method is combined, strain DNA to be detected is used as a template, a microsatellite mark is used as a primer, a large number of specific bands are amplified through polymerase chain reaction, and colonies without the bands are removed through detection of agarose gel electrophoresis. The invention realizes large-scale primary screening of mutant strains, avoids detection of an enzyme-labeling instrument, and avoids influence on data caused by unstable derived products and easy decomposition. Meanwhile, the screening quantity is enlarged, and the screening efficiency is greatly improved. Can carry out the screening on micromonospora purpurea mutagenesis bacterial strain on a large scale, and is easy and simple to handle, and the screening scope increases, and screening efficiency improves, alleviates later stage screening dynamics.
Description
Technical Field
The invention relates to a molecular biological method for rapidly detecting a gentamicin high-yield strain. Belongs to the technical field of strain breeding.
Background
Gentamicin (GM) is a multi-component aminoglycoside antibiotic, has ototoxicity and nephrotoxicity, is still widely used clinically, and has a large demand. Gentamicin was originally isolated and purified from Micromonospora purpurea (Micromonospora purpurea) and Micromonospora echinospora (Micromonospora echinospora) by Weinstein et al, U.S. company, in 1963[1]. In 1966, the Genencomia strain producing gentamicin was isolated by Wang Yue et al, a scientist in China, and then mass production was carried out. Gentamicin is obtained by fermentation of micromonospora purpurea, and the strains decline per se in continuous subculture, so that the yield of gentamicin is reduced year by year. Screening of high-yield strains in various laboratories is always performed, traditional natural breeding is performed, time and labor are consumed, and a method combining normal-temperature plasma physical mutagenesis (ARTP) and lithium chloride chemical mutagenesis is used in the research, so that more high-yield strains are obtained. Lithium chloride mutagenesis can result in the conversion of AT base pairs to GC base pairs and may also result in the deletion of bases. ARTP is in high purity N2Under the protection of the (B), the generated high-energy particles can act on cell walls or cell membranes to change the structure and permeability of the cell walls or the cell membranes so as to cause gene damage, the microorganisms start an SOS repair mechanism, the DNA of the microorganisms can be damaged in a diversity way, the positive mutation rate is high, mutant strains with better genetic stability can be easily obtained, and toxic substances are not generated[2]。
In the screening of high-yield strains of micromonospora purpurea, measures such as natural breeding, mutation breeding, genetic engineering breeding and the like are widely adopted. A large number of high-yield strains can be produced by using a plurality of breeding means with high positive mutation rate. In the screening process of the gentamicin producing bacteria, an enzyme labeling instrument detection technology is mainly adopted, gentamicin does not have an absorption group, the derivative treatment of gentamicin is carried out by using o-phthalaldehyde, the preparation process of a derivative reagent needs to be protected from light, the derivative reagent needs to be prepared and used on site, derivative products are unstable, visible light is easy to decompose, heat is easy to decompose, detection is needed in time, and otherwise, the result is low, so that the detection is influenced. The method needs to sequentially cultivate the seed bottle pore plate and the fermentation pore plate, needs about 5 days before and after the cultivation, is limited by time, and has a larger increase of screening quantity than the traditional seed bottle screening, but still is limited. The invention shortens the screening time, uses the microsatellite molecular marker technology to carry out PCR amplification of specific segments on the strains, finds out the strains with specific amplification bands through agarose gel electrophoresis detection, and then carries out re-screening detection. The method has short time, avoids the limitation of unstable products, and has more accurate results.
Disclosure of Invention
The invention aims to overcome the defects of an enzyme-linked immunosorbent assay detection technology and provide a molecular biological method for rapidly detecting a gentamicin high-yield strain.
In order to achieve the purpose, the invention adopts the following technical scheme:
a molecular biological method for rapidly detecting a gentamicin high-yield strain comprises the following specific steps:
(1) firstly, mutagenizing an original strain to obtain a large number of mutant strains, and then sequencing the original strain and the mutant strains serving as strains to be detected to obtain a plurality of pairs of different microsatellite markers;
(2) then, taking a strain to be detected as template DNA, marking the microsatellite obtained in the step (1) as a primer, and carrying out colony PCR amplification to amplify a large number of specific strips;
(3) detecting by agarose gel electrophoresis, removing colonies without bands, and determining primary screening microsatellite markers;
(4) and (3) additionally taking an original strain, carrying out mutagenesis to obtain a large number of mutant strains, carrying out colony PCR amplification by using the primary screening microsatellite markers obtained in the step (3) as primers, and screening out strains which have specific bands in the mutant strains and have no specific bands in the original strain, namely high-yield strains.
Preferably, in the step (1), the mutagenesis is performed by a method combining room temperature plasma physical mutagenesis (ARTP) and lithium chloride chemical mutagenesis.
Preferably, the specific method of step (3) is: and (3) removing primers which have no band or a band from both the mutant strain and the original strain through agarose gel electrophoresis detection, and leaving the mutant strain with the band, wherein the original strain has no band, or the mutant strain has no band, and the original strain has the band, namely the primary screening microsatellite marker is determined.
The invention has the beneficial effects that:
the invention adopts a method of combining lithium chloride and ARTP (ARTP is an active particle, under the protection of high-purity nitrogen or helium, acts on the cell wall or cell membrane of microorganism, so that the permeability of the cell wall or cell membrane is changed, the diversity damage of gene is caused, and the DNA of the microorganism is mutated), so as to mutate the production strain, and can produce a large amount of positive mutant strains, and although the pore plate screening can screen more strains than a seed bottle, a large amount of strains are still not selected. Microsatellite markers (SSRs), short for SSRs, are short repetitive sequences consisting of 2-6 base pairs, have the characteristics of high polymorphism, wide distribution and the like, and become a useful genetic marker[3]. One of the most effective markers currently used for distinguishing different species, different groups and different genotype individuals[4]. The microsatellite molecular marker has the advantages of co-dominance, good repeatability, high polymorphism and the like, is widely applied to genetic breeding of animals and plants, and has less application in the field of microorganisms.
The invention adopts the microsatellite marking technology and combines the agarose gel electrophoresis method, takes the strain DNA to be detected as a template, the microsatellite marking as a primer, amplifies a large number of specific bands by Polymerase Chain Reaction (PCR), removes the bacterial colony without the bands by the detection of the agarose gel electrophoresis, and carries out high-flux strain screening on the bacterial colony with the bands again. Therefore, the early detection of the high-yield strains is realized, most harmful mutant strains are removed, beneficial mutant strains are left, the detection quantity can be increased, the detection base number is enlarged, and the detection probability is increased. Finally, 1 pair of microsatellite markers is screened out, bands can be amplified in mutant strains, bands cannot be amplified in original strains, the detection rate reaches 92.47%, and the method can be used for rapid detection of high-yield strains.
The invention realizes large-scale preliminary screening of mutant strains, enlarges screening quantity and greatly improves screening efficiency. Can carry out the screening on micromonospora purpurea mutagenesis bacterial strain on a large scale, and is easy and simple to handle, and the screening scope increases, and screening efficiency improves, alleviates later stage screening dynamics. The microsatellite marker technology reduces the blindness of pore plate screening, strains are mutagenized in the early stage to obtain a large amount of mutant strains, a large amount of negative mutations are removed through microsatellite marker screening, and positive mutant individuals are left to increase the accuracy.
The screening work is an important link of strain screening, and a screening method with high screening speed, convenience and good effect should be used according to factors such as mutation rate, false positive and the like. A plurality of samples are obtained during primary screening, and a simple and accurate detection method is adopted; a precise detection method may be used during rescreening. The invention applies the microsatellite marker to the screening of the mutant strains of the gentamicin producing strain for the first time, the method is carried out on a 96-hole plate, 96 strains can be detected on one plate, the detection is finished in about 3 hours, and the method is simple, convenient and efficient.
In the screening process of the gentamicin producing bacteria, an enzyme labeling instrument detection technology is mainly adopted, gentamicin does not have an absorption group, the derivative treatment of gentamicin is carried out by using o-phthalaldehyde, the preparation process of a derivative reagent needs to be protected from light, the derivative reagent needs to be prepared and used on site, derivative products are unstable, visible light is easy to decompose, heat is easy to decompose, detection is needed in time, and otherwise, the result is low, so that the detection is influenced. The method needs to sequentially cultivate the seed bottle pore plate and the fermentation pore plate, needs about 5 days before and after the cultivation, is limited by time, and has a larger increase of screening quantity than the traditional seed bottle screening, but still is limited. The invention shortens the screening time, uses the microsatellite molecular marker technology to carry out PCR amplification of specific segments on the strains, finds out the strains with specific amplification bands through agarose gel electrophoresis detection, and then carries out re-screening detection. The method has short time, avoids the limitation of unstable products, and has more accurate results.
Detailed Description
The present invention will be further illustrated by the following examples, which are intended to be merely illustrative and not limitative.
Firstly, experimental materials:
1. the original micromonospora purpurea strain is a sand soil pipe storage strain of the applicant, the strain type is identified by 16sRNA sequencing before use, and the high-yield strain is a strain obtained by the applicant through a high-throughput strain breeding technology.
2. 0.9% physiological saline: weighing 0.90g NaCl in a beaker, adding a certain amount of purified water for dissolving, taking a 100ml volumetric flask, and fixing the volume of the purified water to the scale.
3. 20% lithium chloride: weighing 20.00g of lithium chloride in a beaker, adding a certain amount of purified water for dissolving, taking a 100ml volumetric flask, and fixing the volume of the purified water to the scale.
4. 45% NaOH: 90.00g of NaOH was weighed, 110.00g of purified water was added, and the mixture was stirred until dissolved.
5. 0.4M boric acid (pH 10.4): 6.183g of boric acid is weighed in a beaker, a certain amount of purified water is added for dissolving, a 250ml volumetric flask is taken, and the volume of the purified water is fixed to the scale.
6. Ortho-phthalaldehyde derivatizing agent (OPA): 2.50g of o-phthalaldehyde was weighed into a beaker, 12.5ml of methanol was measured, dissolved by ultrasonic waves, added with 237.5ml of 0.4M boric acid (pH 10.4) and 5ml of thioglycolic acid, mixed well, and adjusted to 10.4 with 45% NaOH. In the preparation process, attention is paid to avoiding light and avoiding direct irradiation of sunlight, and the preparation is required to be used at present.
7. Preparing a standard substance: weighing 100.0mg gentamicin standard, adding ddH2O is metered to 25ml, 4mg/ml solution is prepared, and the solution is diluted to 3.2mg/ml, 2.4mg/ml, 1.6mg/ml and 0.8 mg/ml.
8. Preparing a mobile phase: methanol: water phase: acetic acid 710: 240: 50 (volume ratio), aqueous phase: 5.50g of sodium heptanesulfonate are added to 240ml of ultrapure water.
9. Culture medium (%, mass%):
separating a culture medium: 1.2 parts of agar strips, 0.75 part of soluble starch, 0.05 part of sodium chloride, 0.05 part of magnesium sulfate, 0.03 part of dipotassium phosphate, 0.1 part of potassium nitrate and 0.1 part of calcium carbonate, and the pH value is 7.6-7.8. Sterilizing with high pressure steam at 121 deg.C for 25 min.
Slant culture medium, well plate slant culture medium: 1.2 parts of agar strips, 1.2 parts of bran, 0.75 part of soluble starch, 0.05 part of sodium chloride, 0.05 part of magnesium sulfate, 0.1 part of potassium nitrate, 0.03 part of dipotassium hydrogen phosphate, 1.0 part of calcium carbonate and 7.6-7.8 parts of pH. Sterilizing with high pressure steam at 121 deg.C for 25 min.
Seed culture medium, seed bottle well plate culture medium: 0.625 of soluble starch, 0.5 of peptone, 1.88 of soybean cake powder, 0.1 of glucose, 1.5 of corn flour, 0.06 of potassium nitrate, 0.625 of calcium carbonate, 0.288 mu g of cobalt chloride and 7.0-7.2 of pH. Sterilizing with high pressure steam at 121 deg.C for 30 min.
Fermentation medium, fermentation orifice plate medium: 3.7 parts of corn starch, 0.4 part of peptone, 3.5 parts of soybean cake powder, 0.2 part of glucose, 1.15 parts of corn flour, 0.0185 part of potassium nitrate, 0.1 part of ammonium sulfate, 0.3 part of calcium carbonate, 2.4 mu g of cobalt chloride and 7.0-7.2 parts of pH. Sterilizing with high pressure steam at 121 deg.C for 30 min.
10. The primer is obtained by sequencing of the company Limited in the engineering and biological engineering.
11. 0.5M EDTA (pH 8.0): 186.10g of EDTA-Na were weighed2Adding 800mL of distilled water, heating and stirring until the distilled water is completely dissolved, adjusting the pH value to 8.0 by NaOH, taking a 1000mL volumetric flask, adding purified water to reach a constant volume of 1000mL, and sterilizing in an autoclave.
12. 50 × TAE buffer: 242.00g of Tris base is weighed, 100mL of 0.5M EDTA (pH8.0) and 57.1mL of glacial acetic acid are weighed, a 1000mL volumetric flask is taken, and purified water is added to the volumetric flask to reach 1000 mL.
13. 1.0% agarose gel: 0.80g of agarose was weighed, 80ml of 1 XTAE was added, and the mixture was heated in a microwave oven until dissolved.
Secondly, obtaining high-yield strains:
the high-yield mutant strain is 10 mutant strains finally obtained by physically and chemically mutagenizing an original strain by a high-throughput strain screening technology and a lithium chloride-ARTP technology before the applicant, wherein the yield is improved by 20-30%, the C1 component is improved by 15-30%, and the total impurities are reduced by 5-8%.
Thirdly, screening microsatellite markers related to high-yield genes:
1. selecting 10 high-yield mutant strains and 10 original strains, and sending the strains to a biological company for sequencing to obtain 137 pairs of differential microsatellite markers.
2. Screening of microsatellite markers and optimization of conditions: colony PCR amplification was performed using the 20 strains as template DNAs and 137 pairs of microsatellite markers as primers. The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 40s, annealing for 30s, and extension at 72 ℃ for 60s for 30 cycles; extending for 5min at 72 ℃, and storing at 4 ℃. The annealing temperature was set to 55 ℃ and PCR amplification was carried out using the following reaction system (Table 1) and the above procedure; if the primers of the bands are not amplified at the temperature of 55 ℃, reducing the annealing temperature and then carrying out PCR amplification; if the annealing temperature of the primer with unclear bands is optimized. The 25 μ l system is shown in the following table (table 1):
the presence of the product was checked by electrophoresis on a 1% agarose gel, and primers were removed which had no or a band in both the mutant and original strains, leaving either the mutant strain with the band in the original strain, or the mutant strain with the microsatellite marker in the original strain. 1 amplified product in the mutant strain and no amplified product in the original strain, which is GM-73, was finally obtained, and the sequence is shown in Table 2.
TABLE 125. mu.l PCR reaction System
TABLE 2 GM-73 primer information
3. Another 15 original strains and 15 high-producing mutant strains were taken and PCR-amplified. It was found that no amplification product was found in all of the 10 original strains by amplification with GM-73, and that the occurrence of microsatellite marker amplification products in the mutant strains was more than 90%.
And fourthly, screening and verifying by using the screened marks:
1. preparation of spore suspension: taking a rigid inclined plane, and digging 2cm under aseptic condition2Adding spore block into glass bead-containing conical flask, adding 10ml 0.9% physiological saline, shaking at 220rpm and 35.5 deg.C for 20min, filtering, and packaging into 50ml centrifugal containerIn the tube, ready for use.
2. ARTP + LiCl mutagenesis: adding 250 μ l of 20% lithium chloride and 250 μ l of purified water into 500 μ l of spore suspension, shaking for 5min, subjecting 20 μ l of spore suspension to mutagenesis for 60s by a mutagenic instrument, adding 980 μ l of physiological saline, and storing in a sterilized centrifuge tube for use.
3. Diluting and coating dishes: simultaneously, equal amounts of non-mutagenized spore suspension and mutagenized spore liquid are taken and coated on a flat plate, the flat plate is cultured for 10 days at the temperature of 35.5 ℃, the colony count is counted, and the inactivation rate is calculated.
The inactivation rate is ═ 100%
4. Colony PCR: under aseptic conditions, single colonies are picked by using sterilized toothpicks, streaked on a plate, and preserved, marked with 1, 2 and 3. PCR amplification was performed and specific bands were detected using 1% agarose gel electrophoresis.
5. Inoculation orifice plate, fermentation orifice plate and inclined plane orifice plate: and sequentially carrying out pore plate screening on the bacterial strains subjected to colony PCR. Culturing in 24-well seed bottle pore plate at 35.5 deg.C for 2 days, inoculating the well-grown seed bottle pore plate into fermentation pore plate and slant pore plate, and sequentially culturing for 3 days and 10 days.
Detection by a microplate reader: acidifying with 20% sulfuric acid to pH of 1.5-2.0, shaking at 220rpm for 20min, mixing, and centrifuging at 4000rpm for 20 min. 20. mu.L of the supernatant was aspirated, 160. mu.L of OPA and 820. mu.L of purified water were added, and water bath was kept at 60 ℃ in the dark for 15 minutes. Immediately absorbing 200 mu L of sample, adding the sample into a quartz plate, and detecting the absorbance by an enzyme-linked immunosorbent assay.
6. And (5) comparing the numbers of the strains screened in the step (4) with those screened in the step (5).
7. Re-screening the seed bottles and the fermentation bottles: selecting strains with specific bands and strains with higher absorbance, transferring the strains from a slant hole plate, culturing at 35.5 deg.C for 9 days, inoculating into a bottle and fermenting in a bottle.
Liquid phase detection: acidifying the fermentation liquid with concentrated sulfuric acid to pH 1.5-2.0, and shakingHomogenizing, standing for 20min, centrifuging at 4000rpm, sucking 1ml supernatant and 1ml each standard, adding 800 μ l OPA and 700 μ l methanol, water bathing at 60 deg.C for 15min, and filtering with 0.45 μm filter membrane. Use of C18And (3) carrying out chromatography column, detecting the wavelength of 330nm, the column temperature of 30 ℃ and the flow rate of 1.0ml/min, testing the components and the potency on a machine, and calculating the units and the components of the test sample.
Fifthly, experimental results:
1. after primary screening of 10 pairs of original strains and 10 pairs of high-yield strains and secondary screening of 15 pairs of original strains and 15 pairs of high-yield strains, GM-73 is screened out to have a specific band in the high-yield strains and have no specific band in the original strains.
2. Screening and verifying by using a screened microsatellite marker, carrying out mutation by ARTP and lithium chloride, diluting and coating butterfly, and then picking up 480 single colonies in total, (1) carrying out PCR amplification by using a 96-well plate, and picking out 10 strains with specific bands in total; (2) and simultaneously, screening by using a 24-hole plate, screening by using an enzyme-labeling instrument, and picking the first 10 higher strains. The 20 strains were subjected to seed bottle inspection and liquid phase detection. The data are shown in tables 3 and 4. 371,442 were found to be screened in the microsatellite marker screen and not in the well plate screen; 3. 359 were screened in the well plate screen and not screened in the microsatellite marker screen. The control strain titer was 1147 and C1 was 27.45%. By comparing the two strains, the strains screened by the microsatellite marker technology are high-yield strains, so the microsatellite marker screening method can be used for strain screening.
TABLE 3 comparison of GM-73 marker screening with well plate screening
TABLE 4 comparison of GM-73 marker screening with well plate liquid phase data
Although the present invention has been described with reference to the specific embodiments, it is not intended to limit the scope of the present invention, and various modifications and variations can be made by those skilled in the art without inventive changes based on the technical solution of the present invention.
[ REFERENCE ] to
[1]Weinstein MJ,Luedemann GM,Oden EM,et al.Gentamicin,new antibiotic complex fromMicromonospora.JMed Chem,1963,6(7):463–464；
[2] The method comprises the following steps of (1) obtaining a high-yield beta-glucosidase strain [ J/OL ] by recursive ARTP-UV compound mutagenesis screening: 1-10;
[3] wudan, application of microsatellite technology in identification of mink individuals [ J ]. biochemistry, 2019, 5 (01): 60-62, 65;
[4] application of microsatellite technology in somatic cell cloning animal studies [ J ] livestock ecology report, 2006 (04): 14-17.
Claims (3)
1. A molecular biological method for rapidly detecting a gentamicin high-yield strain is characterized by comprising the following specific steps:
(1) firstly, mutagenizing an original strain to obtain a large number of mutant strains, and then sequencing the original strain and the mutant strains serving as strains to be detected to obtain a plurality of pairs of different microsatellite markers;
(2) then, taking a strain to be detected as template DNA, marking the microsatellite obtained in the step (1) as a primer, and carrying out colony PCR amplification to amplify a large number of specific strips;
(3) detecting by agarose gel electrophoresis, removing colonies without bands, and determining primary screening microsatellite markers;
(4) and (3) additionally taking an original strain, carrying out mutagenesis to obtain a large number of mutant strains, carrying out colony PCR amplification by using the primary screening microsatellite markers obtained in the step (3) as primers, and screening out strains which have specific bands in the mutant strains and have no specific bands in the original strain, namely high-yield strains.
2. The molecular biological method of claim 1, wherein in step (1), the mutagenesis is performed by a method combining room temperature plasma physical mutagenesis and lithium chloride chemical mutagenesis.
3. The molecular biological method according to claim 1, wherein the specific method of step (3) is: and (3) removing primers which have no band or a band from both the mutant strain and the original strain through agarose gel electrophoresis detection, and leaving the mutant strain with the band, wherein the original strain has no band, or the mutant strain has no band, and the original strain has the band, namely the primary screening microsatellite marker is determined.
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