CN103882097A - Identification method of bacillus subtilis with high protease output - Google Patents
Identification method of bacillus subtilis with high protease output Download PDFInfo
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- CN103882097A CN103882097A CN201210561060.1A CN201210561060A CN103882097A CN 103882097 A CN103882097 A CN 103882097A CN 201210561060 A CN201210561060 A CN 201210561060A CN 103882097 A CN103882097 A CN 103882097A
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- bacterial strain
- pcr amplification
- identification method
- primer
- bacillus subtilis
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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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator 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
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses an identification method of bacillus subtilis with a high protease output. The method includes: inoculating a bacterial strain to be identified onto a skim milk powder plate culture medium, culturing at 35 DEG C for 24 h, performing Gram staining, performing microscopic examination, separately extracting a DNA template from 1.0 mL of the bacterial strain to be identified by adoption of a genome DNA extraction solvent kit, designing and synthesizing PCR amplification primers, with the upstream primer P1 being 5'-AGAGTTTGATCCTGGCTCAG-3' and the downstream primer P2 being 5'-AGTAAGGAGGTGATCCAACCGCA-3', and performing PCR amplification of 16SrDNA. The method is high in bacterial strain selectivity, scientific in identification method, short in time and period and high in efficiency.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the authentication method of bacterial strain.
Background technology
The enzyme of catalytic proteins hydrolysis has many types, and important have stomach en-, trypsinase, kethepsin, papoid, subtilisin and a Bacillus licheniformis proteolytic enzyme etc.Proteolytic enzyme has strict selectivity to acted on reaction substrate, and a kind of proteolytic enzyme only can act on peptide bond certain in protein molecule, the peptide bond being formed as trypsinase catalytic hydrolysis basic aminoacids.Proteolytic enzyme is extensively present in pluck, plant stem-leaf, fruit and microorganism.Microbial protease, mainly by mould, bacterium, is secondly produced by yeast, actinomycetes.
The method ubiquity of prior art qualification subtilis is low to bacterial strain selectivity, and authentication method is science not, and the time cycle is long, inefficient problem.
Summary of the invention
The above-mentioned defect existing in order to overcome prior art field, the object of the invention is to, and the authentication method of a kind of high proteinase yield subtilis is provided, solve prior art low to bacterial strain selectivity, authentication method is science not, and the time cycle is long, inefficient problem.
The authentication method of high proteinase yield subtilis provided by the invention, comprises the following steps:
One, Morphological Identification, get inoculation to be measured on skim-milk plate culture medium 35 DEG C cultivate after 24h, gramstaining, microscopy;
Two, PCR qualification, gets respectively bacterial strain 1.0ml genome DNA extracting reagent kit to be measured and extracts DNA profiling, the synthetic pcr amplification primer of design, upstream primer P1:5 ＇-AGAGTTTGATCCTGGCTCAG-3 ＇; Downstream primer P2:5 ＇-AGTAAGGAGGTGATCCAACCGCA-3 ＇, carries out the pcr amplification of 16S rDNA, and PCR reaction conditions is: 94 DEG C of denaturation 10min, then 94 DEG C of sex change 1min, 54 DEG C of annealing 1min, 72 DEG C are extended 2min, totally 25 circulations, last 72 DEG C are extended 10min.
The authentication method of high proteinase yield subtilis provided by the invention, its beneficial effect is, strong to bacterial strain selectivity, authentication method science, the time cycle is short, efficiency is high.
Embodiment
Below in conjunction with an embodiment, the authentication method of high proteinase yield subtilis provided by the invention is described in detail.
Embodiment
The authentication method of the high proteinase yield subtilis of the present embodiment, comprises the following steps:
One, Morphological Identification, get inoculation to be measured on skim-milk plate culture medium 35 DEG C cultivate after 24h, gramstaining, microscopy;
Two, PCR qualification, gets respectively bacterial strain 1.0ml genome DNA extracting reagent kit to be measured and extracts DNA profiling, the synthetic pcr amplification primer of design, upstream primer P1:5 ＇-AGAGTTTGATCCTGGCTCAG-3 ＇; Downstream primer P2:5 ＇-AGTAAGGAGGTGATCCAACCGCA-3 ＇, carries out the pcr amplification of 16S rDNA, and PCR reaction conditions is: 94 DEG C of denaturation 10min, then 94 DEG C of sex change 1min, 54 DEG C of annealing 1min, 72 DEG C are extended 2min, totally 25 circulations, last 72 DEG C are extended 10min.
After PCR product purification, connect cloning vector and transform, enzyme is delivered to the order-checking of Shanghai Invitrogen company after cutting qualification, and the row that check order submit to GenBank to carry out homology comparison.Institute's calling sequence is:
agagtttgat?cctggctcag?gacgaacgct?ggcggcgtgc?ctaatacatg?caagtcgagc?ggacagatgg?gagcttgctc?cctgatgtta?gcggcggacg?ggtgagtaac?acgtgggtaa?cctgcctgta?agactgggat?aactccggga?aaccggggct?aataccggat?gcttgtttga?accgcatggt?tcagacataa?aaggtggctt?cggctaccac?ttacagatgg?acccgcggcg?cattagctag?ttggtgaggt?aacggctcac?caaggcaacg?atgcgtagcc?gacctgagag?ggtgatcggc?cacactggga?ctgagacacg?gcccagactc?ctacgggagg?cagcagtagg?gaatcttccg?caatggacga?aagtctgacg?gagcaacgcc?gcgtgagtga?tgaaggtttt?cggatcgtaa?agctctgttg?ttagggaaga?acaagtgccg?ttcaaatagg?gcggcacctt?gacggtacct?aaccagaaag?ccacggctaa?ctacgtgcca?gcagccgcgg?taatacgtag?gtggcaagcg?ttgtccggaa?ttattgggcg?taaagggctc?gcaggcggtt?tcttaagtct?gatgtgaaag?cccccggctc?aaccggggag?ggtcattgga?aactggggaa?cttgagtgca?gaagaggaga?gtggaattcc?acgtgtagcg?gtgaaatgcg?tagagatgtg?gaggaacacc?agtggcgaag?gcgactctct?ggtctgtaac?tgacgctgag?gagcgaaagc?gtggggagcg?aacaggatta?gataccctgg?tagtccacgc?cgtaaacgat?gagtgctaag?tgttaggggg?tttccgcccc?ttagtgctgc?agctaacgca?ttaagcactc?cgcctgggga?gtacggtcgc?aagactgaaa?ctcaaaggaa?ttgacggggg?cccgcacaag?cggtggagca?tgtggtttaa?ttcgaagcaa?cgcgaagaac?cttaccaggt?cttgacatcc?tctgacaatc?ctagagatag?gacgtcccct?tcgggggcag?agtgacaggt?ggtgcatggt?tgtcgtcagc?tcgtgtcgtg?agatgttggg?ttaagtcccg?caacgagcgc?aacccttgat?cttagttgcc?agcattcagt?tgggcactct?aaggtgactg?ccggtgacaa?accggaggaa?ggtggggatg?acgtcaaatc?atcatgcccc?ttatgacctg?ggctacacac?gtgctacaat?gggcagaaca?aagggcagcg?aaaccgcgag?gttaagccaa?tcccacaaat?ctgttctcag?ttcggatcgc?agtctgcaac?tcgactgcgt?gaagctggaa?tcgctagtaa?tcgcggatca?gcatgccgcg?gtgaatacgt?tcccgggcct?tgtacacacc?gcccgtcaca?ccacgagagt?ttgtaacacc?cgaagtcggt?gaggtaacct?tttaggagcc?agccgccgaa?ggtgggacag?atgattgggg?tgaagtcgta?acaaggtagc?cgtatcggaa?ggtgcggttg?gatcacctcc?ttact。
Claims (1)
1. an authentication method for high proteinase yield subtilis, is characterized in that: comprise the following steps:
One, Morphological Identification, get inoculation to be measured on skim-milk plate culture medium 35 DEG C cultivate after 24h, gramstaining, microscopy;
Two, PCR qualification, gets respectively bacterial strain 1.0ml genome DNA extracting reagent kit to be measured and extracts DNA profiling, the synthetic pcr amplification primer of design, upstream primer P1:5 ＇-AGAGTTTGATCCTGGCTCAG-3 ＇; Downstream primer P2:5 ＇-AGTAAGGAGGTGATCCAACCGCA-3 ＇, carries out the pcr amplification of 16S rDNA, and PCR reaction conditions is: 94 DEG C of denaturation 10min, then 94 DEG C of sex change 1min, 54 DEG C of annealing 1min, 72 DEG C are extended 2min, totally 25 circulations, last 72 DEG C are extended 10min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016118864A1 (en) * | 2015-01-23 | 2016-07-28 | Novozymes A/S | Bacillus strains improving health and performance of production animals |
| US11331351B2 (en) | 2015-01-23 | 2022-05-17 | Novozymes A/S | Bacillus strains improving health and performance of production animals |
| US11473052B2 (en) | 2015-01-23 | 2022-10-18 | Novozymes A/S | Bacillus subtilis subspecies |
-
2012
- 2012-12-21 CN CN201210561060.1A patent/CN103882097A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016118864A1 (en) * | 2015-01-23 | 2016-07-28 | Novozymes A/S | Bacillus strains improving health and performance of production animals |
| CN107567493A (en) * | 2015-01-23 | 2018-01-09 | 诺维信公司 | Improve the health of production animal and the Bacillus strain of performance |
| AU2016209132B2 (en) * | 2015-01-23 | 2021-07-01 | Novozymes A/S | Bacillus strains improving health and performance of production animals |
| US11166989B2 (en) | 2015-01-23 | 2021-11-09 | Novozymes A/S | Bacillus strains improving health and performance of production animals |
| CN107567493B (en) * | 2015-01-23 | 2021-11-16 | 诺维信公司 | Bacillus strains for improving the health and performance of production animals |
| US11331351B2 (en) | 2015-01-23 | 2022-05-17 | Novozymes A/S | Bacillus strains improving health and performance of production animals |
| US11473052B2 (en) | 2015-01-23 | 2022-10-18 | Novozymes A/S | Bacillus subtilis subspecies |
| US11801272B2 (en) | 2015-01-23 | 2023-10-31 | Novozymes A/S | Bacillus strains improving health and performance of production animals |
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Application publication date: 20140625 |