CN101956018A - Method for identifying brevibacillus laterosporus in microbial fertilizer - Google Patents
Method for identifying brevibacillus laterosporus in microbial fertilizer Download PDFInfo
- Publication number
- CN101956018A CN101956018A CN2010105226612A CN201010522661A CN101956018A CN 101956018 A CN101956018 A CN 101956018A CN 2010105226612 A CN2010105226612 A CN 2010105226612A CN 201010522661 A CN201010522661 A CN 201010522661A CN 101956018 A CN101956018 A CN 101956018A
- Authority
- CN
- China
- Prior art keywords
- swimming lane
- bacillus brevis
- primer
- side spore
- brevibacillus laterosporus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides technology for simply and rapidly detecting and identifying brevibacillus laterosporus in a microbial fertilizer. In the technology, a specific primer for the brevibacillus laterosporus is designed, a PCR identification method for the brevibacillus laterosporus is constructed, and the brevibacillus laterosporus in the microbial fertilizer can be detected and identified according to a PCR amplification result of sample genomic DNA.
Description
Technical field
The present invention relates to biological technical field,, detect the method for side spore bacillus brevis (Brevibacillus laterosporus) in the microbial fertilizer in particular to utilizing suitable primer to react by PCR, and used in the method primer.
Technical background
Side spore bacillus brevis is the bacterium that occurring in nature extensively distributes, and existence is all arranged in soil, ocean, animal, the insect.Along with going deep into that bacillus brevis is studied, found that it can produce the multiple meta-bolites that application potential is arranged, regulate material as insecticidal proteins, antibacterial substance, antitumor immune.In addition, side spore bacillus brevis also is found gradually, studies and develop in multiple functions such as dissolving inorganic phosphorus, degrading organic phosphor, pest control, raising crop defence capabilities.
In view of the multifunctionality of side spore bacillus brevis, existing a lot of enterprises use it for microbial fertilizer production, and have obtained good effect.Be the result of use of the microbial fertilizer product of guaranteeing to contain side spore bacillus brevis, need to guarantee the quantity of side spore bacillus brevis in quality, the especially product of product.Plate count detection method key commonly used is accurately to discern effective bacterium, and traditional bacterial classification identification of using at present is that operation steps is loaded down with trivial details based on morphological specificity and every Physiology and biochemistry method, and detection time is long, and poor specificity.Therefore, press for research set up fast, accurately, sensitive detects novel method, with the accuracy of the quality examination of satisfying side spore bacillus brevis product and ageing.
PCR method has fast, accurately and sensitive characteristics, obtained application in the microbial fertilizer field, close the specific PCR method that people such as David once set up and be used for the evaluation and the detection of Rhodopseudomonas palustris, and obtained good effect.For with the round pcr broader applications in the microbial fertilizer detection range, the inventor by grope repeatedly to put into practice set up a kind of PCR-based technology, detect the method for side spore bacillus brevis in the microbial fertilizer fast, accurately, delicately.This method is applicable to the evaluation and the detection of side spore bacillus brevis.The present invention realizes by following technical scheme.
Summary of the invention
The object of the present invention is to provide a kind of method that the special primer round pcr is identified side spore bacillus brevis in the microbial fertilizer of using.The authentication method of identifying side spore bacillus brevis by the special primer round pcr is provided in an embodiment preferred of the present invention.In a further preferred embodiment, described side spore bacillus brevis is a pure strain.Another object of the present invention is to the special primer that provides used in the above-mentioned authentication method.
Particularly, the present invention is based on known side spore bacillus brevis 16S rDNA nucleotide sequence, designed special primer corresponding to above-mentioned bacterial classification.By optimizing the specificity that conditions such as magnesium ion concentration in the PCR reaction and annealing temperature improve PCR method, and set up the PCR authentication method of side spore bacillus brevis, be used for the evaluation of microbial fertilizer product side spore bacillus brevis according to optimum result.
The PCR method of identifying side spore bacillus brevis of this patent invention is:
Upstream primer L25:5 '-TGAAGCGAAACGGAAAG-3 ';
Downstream primer R322:5 '-CGTCAAGGTGCTACCTTATT-3 '.
PCR reaction system: 10 * buffer 2.0 μ L
Mg
2+(25mmol/L) 0.8μL
dNTPS(10mmol/L) 0.8μL
Upstream primer L25 (10 μ mol/L) 0.3 μ L
Downstream primer R322 (10 μ mol/L) 0.3 μ L
Taq?E(2.5U/μL) 0.2μL
Template DNA 2.0 μ L
Supply ddH
2O 20 μ L
Pcr amplification parameter: 95 ℃ of 5min
72℃ 10min。
Description of drawings
The figure experimental result (photo) that to be application side spore bacillus brevis special primer carry out PCR to reference culture as shown.Wherein, each swimming lane specify as follows:
Swimming lane 1: standard molecular weight: 100bp standard molecular weight
Swimming lane 2: side spore bacillus brevis (Brevibibacillus laterosporus1.2012)
Swimming lane 3: side spore bacillus brevis (Brevibibacillus laterosporus10249)
Swimming lane 4: side spore bacillus brevis (Brevibibacillus laterosporus1.2738)
Swimming lane 5: side spore bacillus brevis (Brevibibacillus laterosporus3005)
Swimming lane 6: side spore bacillus brevis (Brevibibacillus laterosporus3006)
Swimming lane 7: side spore bacillus brevis (Brevibibacillus laterosporus3007)
Swimming lane 8: short genus bacillus (Brevibacillus brevis)
Swimming lane 9: soil bacillus brevis (Brevibacillus agri)
Swimming lane 10: Paenibacillus polymyxa (Paenibacillus polymyxa)
Swimming lane 11: gel-shaped series bacillus (Paenibacillus mucilaginosus)
Swimming lane 12: fixed nitrogen series bacillus (Paenibacillus azotofixans)
Swimming lane 13: soil series bacillus (Paenibacillus edaphicus)
Swimming lane 14: subtilis (Bacillus subtilis)
Swimming lane 15: Bacillus licheniformis (Bacillus licheniformis)
Swimming lane 16: bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
Swimming lane 17: bacillus pumilus (Bacillus pumilus)
Swimming lane 18: bacillus megaterium (Bacillus megaterium)
Swimming lane 19: bacillus cereus (Bacillus cereus)
Swimming lane 20: Bacillus circulans (Bacillus circulans)
Swimming lane 21: Pseudomonas fluorescens (Pseudomonas fluorescens)
Swimming lane 22: Pseudomonas stutzeri (Pseudomonas stutzeri)
Swimming lane 23: pseudomonas putida (Pseudomonasputida)
Swimming lane 24: Pseudomonas chlororaphis (Pseudomonas chlororaphis)
Swimming lane 25: blown-ball Azotobacter (Azotobacter chroococcum).
Embodiment
Below in conjunction with accompanying drawing specific embodiments of the present invention are further specified.Those skilled in the art should understand, and following embodiment scheme is that exemplary technical scheme is with explanation the present invention.
Embodiment
PCR method by application side spore bacillus brevis Auele Specific Primer is to the detection of reference culture.
(1) material
The test bacterium: the listed bacterial strain of following table 1 is a reference culture to be measured
Table 1
(2) primer design and synthetic
16S rDNA nucleotide sequence design side spore bacillus brevis Auele Specific Primer according to side spore bacillus brevis.Described primer is as shown in table 2.Described primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Table 2
| Kind | Primer | Nucleotide sequence |
| Side spore bacillus brevis | Upstream primer L25 | 5′-TGAAGCGAAACGGAAAG-3′ |
| Side spore bacillus brevis | Downstream primer R322 | 5′-CGTCAAGGTGCTACCTTATT-3′ |
(3) DNA extraction: get 1.5mL bacterium liquid in the Eppendorf pipe, the centrifugal 4min of 9000r/min collects thalline ,-20 ℃ of preservations.The thalline collected with 1 * TE damping fluid washing thalline 2 times, is added 500 μ L GUTC damping fluids again, mixes, leave standstill 20min after, every pipe adds 20 μ L～30 μ L diatomite adsorption liquid, 15min is placed in vibration evenly back room temperature.The centrifugal 30s of 13000r/min again, abandon supernatant liquor after, add 200 μ L GUTC damping fluids, mix the back room temperature and place 10min, the centrifugal 30s of 13000r/min again.Lavation buffer solution washs above-mentioned diatomite precipitation 3 times, each 300 μ L.Alcohol washing diatomite with 200 μ L 70% precipitates 1 time again, and the centrifugal 2min of 13000r/min abandons supernatant liquor, drying.Add diatomaceous amount according to every pipe at last, add 20 μ L～30 μ L, 1 * TE damping fluid to every pipe, thorough mixing is even on the vortex vibrator, behind 65 ℃ of insulation 30min, the centrifugal 5min of 13000r/min, carefully supernatant liquor is moved to another numbered Eppendorf pipe then, this solution is the DNA sample that extracts from bacterial strain.The DNA sample is preserved in-20 ℃.
(4) PCR detects:
(4.1) PCR mixture preparation
Press prescription shown in the table 3, in the 200uLPCR pipe, add reagent and dna solution, be prepared into the PCR mixed solution.
Table 3
| Composition | Volume |
| 10×buffer | 2μL |
| Mg 2+(25mmol/L) | 0.8μL |
| dNTPs(10mmol/L) | 0.8μL |
| Upstream primer L25 (10 μ mol/L) | 0.3μL |
| Downstream primer R322 (10 μ mol/L) | 0.3μL |
| Taq?E(2.5U/μL) | 0.2μL |
| Template DNA | 2.0μL |
| Supply ddH 2O | 20μL |
(4.2) put into the PCR instrument by the 200uLPCR pipe of mixed solutions of 4.1 preparations and carry out the PCR reaction containing.Concrete steps are, 95 ℃ carry out thermally denature 5min after, finish following 30 circulations: 95 ℃ of thermally denature 40s, 61 ℃ of annealing/renaturation 40s, 72 ℃ prolong reaction 40s.Last 72 ℃ prolong reaction 10min.
(4.3) electrophoresis
After finishing pcr amplification, get 5 μ L reaction mixtures, after mixing with 1 μ L sample-loading buffer, on 1% sepharose of EB dyeing 30min, carry out electrophoresis, 140V voltage stabilizing electrophoresis 40min.
(4.4) observe
After finishing electrophoresis, by observations under the gel images analyser.
(4.5) result
This experimental result (photo) as shown in the figure.
According to result shown in the photo, a specific band that size is 296bp has all appearred in 6 strain side spore bacillus brevis, and other test bacterium then driftlessness band produce, and this illustrates that special primer of the present invention can detect and identify side spore bacillus brevis reliably.
Claims (1)
1. the Auele Specific Primer of a side spore bacillus brevis (Brevibacillus laterosporus) is right, it is characterized in that described primer to being made up of upstream primer and downstream primer,
Upstream primer L25 5 '-TGAAGCGAAACGGAAAG-3 ';
Downstream primer R322 5 '-CGTCAAGGTGCTACCTTATT-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010522661.2A CN101956018B (en) | 2010-10-28 | 2010-10-28 | Method for identifying brevibacillus laterosporus in microbial fertilizer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010522661.2A CN101956018B (en) | 2010-10-28 | 2010-10-28 | Method for identifying brevibacillus laterosporus in microbial fertilizer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101956018A true CN101956018A (en) | 2011-01-26 |
| CN101956018B CN101956018B (en) | 2015-01-07 |
Family
ID=43483603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010522661.2A Expired - Fee Related CN101956018B (en) | 2010-10-28 | 2010-10-28 | Method for identifying brevibacillus laterosporus in microbial fertilizer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101956018B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016135766A1 (en) | 2015-02-26 | 2016-09-01 | Università Degli Studi Di Sassari | Markers for the detection of brevibacillus laterosporus and related methods and kits |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1490398A (en) * | 2003-08-13 | 2004-04-21 | 云南大学 | Bacillus pumilus for killing nematoda and its preparation and use |
| CN1580242A (en) * | 2004-05-18 | 2005-02-16 | 北京生太平生物工程技术有限公司 | Laterosporo short bacillus, and its fermentating process and use |
-
2010
- 2010-10-28 CN CN201010522661.2A patent/CN101956018B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1490398A (en) * | 2003-08-13 | 2004-04-21 | 云南大学 | Bacillus pumilus for killing nematoda and its preparation and use |
| CN1580242A (en) * | 2004-05-18 | 2005-02-16 | 北京生太平生物工程技术有限公司 | Laterosporo short bacillus, and its fermentating process and use |
Non-Patent Citations (1)
| Title |
|---|
| ADRIANA M. ALIPPI等: "Differentiation of Paenibacillus larvae subsp. larvae, the Cause of American Foulbrood of Honeybees, by Using PCR and Restriction Fragment Analysis of Genes Encoding 16S rRNA", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016135766A1 (en) | 2015-02-26 | 2016-09-01 | Università Degli Studi Di Sassari | Markers for the detection of brevibacillus laterosporus and related methods and kits |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101956018B (en) | 2015-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Detailed comparison of bacterial communities during seasonal sludge bulking in a municipal wastewater treatment plant | |
| Awasthi et al. | Effects of biochar amendment on bacterial and fungal diversity for co-composting of gelatin industry sludge mixed with organic fraction of municipal solid waste | |
| Calheiros et al. | Substrate effect on bacterial communities from constructed wetlands planted with Typha latifolia treating industrial wastewater | |
| Takaku et al. | Microbial communities in the garbage composting with rice hull as an amendment revealed by culture-dependent and-independent approaches | |
| Muyzer | DGGE/TGGE a method for identifying genes from natural ecosystems | |
| Zhang et al. | Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong | |
| Chang et al. | Aerobic degradation of tetrabromobisphenol-A by microbes in river sediment | |
| Barbosa Lima et al. | Amazonian Dark Earth and plant species from the Amazon region contribute to shape rhizosphere bacterial communities | |
| Yarwood et al. | Soil microbe active community composition and capability of responding to litter addition after 12 years of no inputs | |
| Guidi et al. | Dynamics of Bacillus thuringiensis var. israelensis and Lysinibacillus sphaericus spores in urban catch basins after simultaneous application against mosquito larvae | |
| Li et al. | Evolution of microbial communities during electrokinetic treatment of antibiotic-polluted soil | |
| Guidi et al. | A real‐time PCR method to quantify spores carrying the Bacillus thuringiensis var. israelensis cry4Aa and cry4Ba genes in soil | |
| Rintala | Actinobacteria in indoor environments: exposures and respiratory health effects | |
| Bourrain et al. | In‐depth prospection of Avène Thermal Spring Water reveals an uncommon and stable microbial community | |
| CN101956018A (en) | Method for identifying brevibacillus laterosporus in microbial fertilizer | |
| Youenou et al. | Impact of untreated urban waste on the prevalence and antibiotic resistance profiles of human opportunistic pathogens in agricultural soils from Burkina Faso | |
| Subramanian et al. | Use of 16S rRNA probes for characterization of gut microflora of silkworm (Bombyx mori L.) breeds | |
| Zhang et al. | Impact of nutrient addition on diversity and fate of fecal bacteria | |
| Doğruöz-Güngör et al. | ENZYME PROFILES AND ANTIMICROBIAL ACTIVITIES OF BACTERIA ISOLATED FROM THE KADIINI CAVE, ALANYA, TURKEY. | |
| Subasinghe et al. | Bacterial and fungal composition profiling of microbial based cleaning products | |
| McEwan et al. | Evaluation of the microbiome of decaying alder nodules by next generation sequencing. | |
| Morohoshi et al. | Secreted protease mediates interspecies interaction and promotes cell aggregation of the photosynthetic bacterium Chloroflexus aggregans | |
| Fan et al. | Combination of amplified rDNA restriction analysis and high-throughput sequencing revealed the negative effect of colistin sulfate on the diversity of soil microorganisms | |
| Tigini et al. | Wastewater-Agar as a selection environment: A first step towards a fungal in-situ bioaugmentation strategy | |
| van Leuven et al. | High resolution ITS amplicon melting analysis as a tool to analyse microbial communities of household biofilms in ex-situ models |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| DD01 | Delivery of document by public notice |
Addressee: Cao Fengming Document name: Notification of Passing Preliminary Examination of the Application for Invention |
|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150107 Termination date: 20211028 |