CN110261267A - A kind of detection method of fishing photosynthetic bacteria preparation product - Google Patents
A kind of detection method of fishing photosynthetic bacteria preparation product Download PDFInfo
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- CN110261267A CN110261267A CN201910464774.2A CN201910464774A CN110261267A CN 110261267 A CN110261267 A CN 110261267A CN 201910464774 A CN201910464774 A CN 201910464774A CN 110261267 A CN110261267 A CN 110261267A
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- photosynthetic bacteria
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- bacteria preparation
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- 241000894006 Bacteria Species 0.000 title claims abstract description 297
- 230000000243 photosynthetic effect Effects 0.000 title claims abstract description 207
- 238000002360 preparation method Methods 0.000 title claims abstract description 136
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 112
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 56
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 51
- 238000000034 method Methods 0.000 claims abstract description 50
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 44
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 43
- 230000000694 effects Effects 0.000 claims abstract description 30
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical group [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 claims abstract description 17
- 230000001419 dependent effect Effects 0.000 claims abstract description 13
- 230000003993 interaction Effects 0.000 claims abstract description 13
- 238000005259 measurement Methods 0.000 claims abstract description 10
- 230000009466 transformation Effects 0.000 claims abstract description 9
- 238000005516 engineering process Methods 0.000 claims abstract description 8
- 238000005286 illumination Methods 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 15
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims description 13
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 12
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 claims description 11
- 230000015556 catabolic process Effects 0.000 claims description 11
- 238000006731 degradation reaction Methods 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 10
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- 238000009395 breeding Methods 0.000 claims description 10
- 230000001488 breeding effect Effects 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 10
- 239000012895 dilution Substances 0.000 claims description 10
- 101150027124 nirS gene Proteins 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 108090000854 Oxidoreductases Proteins 0.000 claims description 7
- 102000004316 Oxidoreductases Human genes 0.000 claims description 7
- 238000009472 formulation Methods 0.000 claims description 7
- 102000008109 Mixed Function Oxygenases Human genes 0.000 claims description 6
- 108010074633 Mixed Function Oxygenases Proteins 0.000 claims description 6
- 101150083634 amoA gene Proteins 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 108010025915 Nitrite Reductases Proteins 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 4
- 238000005314 correlation function Methods 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 235000013399 edible fruits Nutrition 0.000 claims description 3
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- 238000003753 real-time PCR Methods 0.000 claims description 2
- 238000012163 sequencing technique Methods 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 7
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 abstract description 6
- 229910052698 phosphorus Inorganic materials 0.000 abstract description 6
- 239000011574 phosphorus Substances 0.000 abstract description 6
- 238000012165 high-throughput sequencing Methods 0.000 abstract description 3
- 239000000047 product Substances 0.000 description 80
- 238000000137 annealing Methods 0.000 description 24
- 239000000243 solution Substances 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 8
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- 239000013642 negative control Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 229910019142 PO4 Inorganic materials 0.000 description 6
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- 238000007689 inspection Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 6
- 239000010452 phosphate Substances 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 4
- 241000190932 Rhodopseudomonas Species 0.000 description 4
- 241000190950 Rhodopseudomonas palustris Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- -1 nxrA Proteins 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 101100490994 Aeromonas hydrophila amoA gene Proteins 0.000 description 3
- 101100490996 Nitrosomonas europaea (strain ATCC 19718 / CIP 103999 / KCTC 2705 / NBRC 14298) amoA2 gene Proteins 0.000 description 3
- 241000190967 Rhodospirillum Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
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- LMSDCGXQALIMLM-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;iron Chemical compound [Fe].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O LMSDCGXQALIMLM-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
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- 241000190986 Ectothiorhodospira Species 0.000 description 2
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- 241000589516 Pseudomonas Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 150000003863 ammonium salts Chemical class 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
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- 238000000691 measurement method Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000029553 photosynthesis Effects 0.000 description 2
- 238000010672 photosynthesis Methods 0.000 description 2
- 238000012372 quality testing Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 1
- 244000170916 Paeonia officinalis Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 241000191035 Rhodomicrobium Species 0.000 description 1
- 241000190946 Rhodopseudomonas sp. Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 241000605008 Spirillum Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000035568 catharsis Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000852 hydrogen donor Substances 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 208000018875 hypoxemia Diseases 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000001546 nitrifying effect Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- GQPLMRYTRLFLPF-UHFFFAOYSA-N nitrous oxide Inorganic materials [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
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- 238000004806 packaging method and process Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005464 sample preparation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001228 trophic effect Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C12Q1/6869—Methods for sequencing
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- G01N15/10—Investigating individual particles
- G01N15/1023—Microstructural devices for non-optical measurement
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/56911—Bacteria
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Abstract
The invention discloses a kind of fishing detection methods of photosynthetic bacteria preparation product, this method combines the multiple technologies such as double-layer plate cultivation, microscopic counting and high throughput sequencing technologies, the integrated measuring method for establishing bacterium amount and purity for fishing photosynthetic bacteria preparation product；Meanwhile according to photosynthetic bacteria amphimicrobian and to physiological characteristics such as illumination requirements, and its using the functional characteristic of the inorganic nitrogen and phosphorus of water body, further research establishes fishing photosynthetic bacteria preparation product to the measuring method of water body nitrogen phosphorus function and effect；And the nitrogen transformation function potentiality of microbial inoculum are determined using the method for measurement nitrogen dependent interaction functional gene.
Description
Technical field
The invention belongs to photosynthetic bacteria preparation technical fields, and in particular to a kind of detection of fishing photosynthetic bacteria preparation product
Method.
Background technique
Photosynthetic bacteria (Photosynthetic bacteria) is that one kind can carry out the photosynthetic work of non-oxygen-production under anaerobic
With the general name (Yang Suping etc., 2008) of bacterium, can using light as the energy, using organic matter, ammonia, hydrogen sulfide etc. in nature as
Hydrogen donor carries out photosynthesis.Photosynthetic bacteria is Gram-negative bacteria, and thalli morphology multiplicity has spherical, rod-shaped, helical form, half
Annular etc., at 10-45 DEG C can growth and breeding, optimum growth temperature be 20-30 DEG C, optimal pH be 7.0-8.5 (Xu Liangmei
Deng 2005).In addition, such bacterium has eurysalinity (Li Zhuojia etc., 2007), intensity of illumination is to influence photosynthetic bacterium growth
Key factor (Xu Liangmei etc., 2005).Photosynthetic bacteria has diversified metabolic way.Photosynthetic bacteria under anaerobic can be into
Row photosynthesis, under the conditions of dark micro- aerobic and aerobic progress oxidative metabolism (historian's beam etc., 1995；Qian great Yi etc., 2005).
In terms of nitrogen metabolism, it is inorganic nitrogen-sourced that it can assimilate ammonia nitrogen, nitrite nitrogen, nitrate nitrogen etc., can also be by nitrogen fixation by nitrogen
Gas is changed into ammonium salt as nitrogen source (Xu Huifang, 2015)；In terms of phosphorus metabolism, certain photosynthetic bacterias take the photograph phosphorus energy with stronger
Power, but the influence (Yuan Yingbo etc., 2010) of the environmental conditions such as intensity and pH is illuminated by the light to the absorbability of phosphorus.There is research to think light
Close bacterium have a variety of nitrogen metabolism modes such as nitrification, denitrification (Yang, et al., 2017；Kim, et al., 1999), wherein
Participate in nitrifying process key enzyme be ammona monooxygenase and nitrite oxidoreductase (Liu Hua, 2012；Li Jingyuan etc.,
2012) key enzyme for, participating in denitrification process is nitrite reductase (Wang Liying, 2005).Encode the gene of above 3 kinds of enzymes
Respectively amoA, nxrA, nirS (Li Jingyuan etc., 2012).Nitrogen conversion correlation function gene technology is applied at present photosynthetic thin
The report of bacteria preparation quality testing and evaluation is also relatively rare.
Photosynthetic bacteria it is many kinds of but only sub-fraction carried out in fish production using (Brandl et al.,
1989；Yang Ying warbler etc., 2009).The common bacteria type for being currently used in fishing photosynthetic bacteria preparation specifically includes that red pseudomonas
Belong to (Rhodopseudomonas) (Jian et al., 2013；Zhou et al., 2007), Rhodospirillum
(Rhodospirillum) (Qi et al., 2009), Ectothiorhodospira (Ectothiorhodospira) etc. (woods, which is opened, deposits,
2017), wherein using the most universal for Rhodopseudomonas palustris (Rhodopseudomonas palustris) (Zhang et
al.,2014；Lazaro et al.,2017).Lei Aiying etc. (2005) is separated from the bed mud of high yield prawn culturing pond and is acquired
One plant of Rhodopseudomonas palustris, research find that it can effectively remove ammonia nitrogen, nitrite nitrogen in breeding water body.In addition, also
Research thinks that the main bacteria seed applied in fishing is produced with photosynthetic bacteria has Rhodospirillum, Rhodopseudomonas and Rhodomicrobium
(201710561969.X)。
It is raw material, active photosynthetic bacteria for strain that photosynthetic bacteria preparation, which is using culture medium, by modern biotechnology
The microorganism formulation developed.Because it has the function of to remove harmful substances such as Ammonia Nitrogen in Aquatic Environment, nitrite nitrogen etc., from 20
Gradually it is widely used in culture fishery from the eighties in century.With the fast development of aquatic products industry, photosynthetic bacteria preparation at
For one of research hotspot.Currently, common photosynthetic bacteria preparation product is mostly the peony bacterium solution of 5L~20L in the market.Separately have
A small amount of photosynthetic bacteria freeze-dried powder product, but be not for bacillus according to the main advantage bacterium for testing and analyzing such freeze-dried powder product
Photosynthetic bacteria.Therefore, it is highly desirable to detect fishing with photosynthetic bacteria microbial inoculum product.But fishing is with photosynthetic thin on Vehicles Collected from Market
Bacteria preparation product quality is very different, and function and effect are difficult to ensure, the use environment of photosynthetic bacteria preparation is complicated in addition, strain class
Not various (Kim et al., 2000；Shapleigh et al., 2009), photosynthetic bacteria preparation product is detected in actual production
There are certain difficulty (Yang Guane etc., 2010) for quantity, purity and application effect in.China also there is no special needle at present
To the fishing evaluation method of probiotics product.
Photosynthetic bacteria preparation does not have the standard of technicality, only 2 universal standards, i.e. photosynthetic bacteria system in marine industry
Rhodopseudomonas palustris method of counting (SN/T3542-2013) in agent standard (NY 527-2002) and photosynthetic bacteria preparation.The former
Defined is classification, technical requirements, the method for inspection, inspection rule, mark, packaging, transport and storage of photosynthetic bacteria preparation etc.,
The latter is the reference of photosynthetic bacteria representative species quantitative measurement method.And the evaluation and measuring method of photosynthetic bacteria preparation function and effect
Also it is temporarily rarely reported.
Summary of the invention
The purpose of the present invention is to provide a kind of fishing detection method of photosynthetic bacteria preparation product, this method both can be accurate
Measure fishing in photosynthetic bacteria preparation product whether viable bacteria, photosynthetic bacteria bacterium amount and purity, can also measure fishing photosynthetic bacteria system
Function and effect and fishing in photosynthetic bacteria preparation product nitrogen dependent interaction functional gene content of the agent product to water body nitrogen phosphorus.
Above-mentioned purpose of the invention can be realized by following technical solution: a kind of fishing photosynthetic bacteria preparation product
Detection method, this method combine double-layer plate cultivation, microscope count method and high-flux sequence method, determine fishing with photosynthetic
In bacteria preparation product whether viable bacteria, bacterium amount and purity；Containing after photosynthetic bacteria in formulation products to be determined, further it is integrated with
Fishing is with photosynthetic bacteria preparation product to nitrogen dependent interaction in water body nitrogen phosphorus function and effect and fishing photosynthetic bacteria preparation product
The measuring method of functional gene, specifically includes the following steps:
(1) using the measurement fishing of double-layer plate cultivation with whether there is viable bacteria in photosynthetic bacteria preparation product, use is micro-
Mirror counting method measures the total bacteria count of fishing photosynthetic bacteria preparation product, measures fishing photosynthetic bacteria preparation using high-flux sequence method
The purity of product；
(2) as contained photosynthetic bacteria in formulation products, then fishing is on the one hand measured with photosynthetic bacteria preparation product to water body nitrogen
The function and effect of phosphorus；On the other hand, fishing is measured with nitrogen conversion correlation function gene in photosynthetic bacteria preparation product,
Determine if there is nitrogen transformation potentiality；If do not contained photosynthetic bacteria in formulation products, then illustrate that said preparation product is real
Border is not photosynthetic bacteria microbial inoculum product, no longer needs to carry out the step.
In detection method of the above-mentioned fishing with photosynthetic bacteria preparation product:
Preferably, step (1) specifically includes the following steps:
(1.1) fishing photosynthetic bacteria preparation sample is chosen, determines whether that there are viable bacterias using double-layer plate cultivation；
(1.2) using the total bacteria count in microscope count method measurement microbial inoculum；
(1.3) total DNA for extracting fishing photosynthetic bacteria preparation sample, is expanded the variable region 16S rRNA, is measured using high pass
Sequence technology measures the bacterial community composition of fishing photosynthetic bacteria preparation sample, determines the relative abundance of photosynthetic bacteria, and calculate
The bacterium amount and purity of photosynthetic bacteria in detected photosynthetic bacteria preparation sample.
Preferably, determine whether that there are when viable bacteria using double-layer plate cultivation in step (1.1), comprising the following steps:
Fishing photosynthetic bacteria preparation sample is chosen, dilution is prepared, is inoculated with photosynthetic bacteria under 30 DEG C ± 1 DEG C, the micro- aerobic condition of illumination
Agar double-layer plate is cultivated, 5~6d is cultivated, whether have bacterium growth, there is bacterium growth if observing on double-layer plate, that is, illustrates that the microbial inoculum produces
There is viable bacteria in product；There is no bacterium colony growth, then illustrates there is no viable bacteria in the microbial inoculum product.
Preferably, it is extracted in step (1.3) using Water DNA Kit (water body DNA extraction kit, commercially available, Omega)
The bacteria total DNA of fishing photosynthetic bacteria preparation sample expands the variable region 16S rRNA, measures microbial inoculum using high throughput sequencing technologies
Bacterial community composition, determine whether there is photosynthetic bacteria and photosynthetic bacteria in testing result according to primary Jie Shi Bacteria Identification system
Relative abundance, further according to the relative abundance of total bacteria count and photosynthetic bacteria obtain photosynthetic bacteria preparation sample in photosynthetic bacteria bacterium
Amount and purity.
Preferably, step (2) specifically includes the following steps:
(2.1) fishing is detected and is evaluated with the function and effect of photosynthetic bacteria preparation sample purification breeding water body nitrogen phosphorus；
(2.2) nitrogen transformation of fishing photosynthetic bacteria preparation sample is determined by measuring nitrogen dependent interaction functional gene
Function potentiality.
Preferably, it is imitated in step (2.1) by the effect to fishing photosynthetic bacteria preparation sample purification breeding water body nitrogen phosphorus
Fruit carry out detection and evaluation include: selection breeding water body, be added fishing photosynthetic bacteria preparation sample, test the fishing with it is photosynthetic carefully
Bacteria preparation product is to ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and phosphatic degradation effect in breeding water body.
Preferably, step (2.2) the nitrogen dependent interaction functional gene includes the amoA base for encoding ammona monooxygenase
The nirS gene of cause, the nxrA gene of coding nitrite oxidoreductase and coding nitrite reductase.
Preferably, using fluorescence quantitative PCR method (qPCR) detection nitrogen dependent interaction functional gene in step (2.2)
Content judges fishing photosynthetic bacteria preparation sample with the presence or absence of the gene of key enzyme required for nitrogen conversion process.
Compared with prior art, the invention has the following advantages that
(1) the method for the present invention combines the multiple technologies such as double-layer plate cultivation, microscopic counting and high-flux sequence, collection
At the system measurement method established for fishing photosynthetic bacteria preparation product bacterium amount and purity；Meanwhile it is simultaneous according to photosynthetic bacteria
Property anaerobism and physiological characteristics are required etc. to illumination, it is integrated to establish fishing and its using the functional characteristic of the inorganic nitrogen and phosphorus of water body
With photosynthetic bacteria preparation product to the measuring method of water body nitrogen phosphorus function and effect and its nitrogen transformation function potentiality；
(2) the method for the present invention both can in Accurate Determining microbial inoculum photosynthetic bacteria bacterium amount and purity, can also measure microbial inoculum product
Function and effect and nitrogen to water body nitrogen phosphorus convert correlation function gene content；
(3) by being surveyed to the common fishing being collected in the market with photosynthetic bacteria preparation product with the method for the present invention
Examination, the results show that this method can effectively detect the quality and using effect of fishing photosynthetic bacteria preparation, it not only can Accurate Determining
The bacterium amount and purity of photosynthetic bacteria in microbial inoculum product can also measure product and convert phase to the functioning efficiency and nitrogen of water body nitrogen phosphorus
Close functional gene content；
(4) the method for the present invention has good industrial application prospect, can use for subsequent further specification and initiative China's fishing
The quality testing of photosynthetic bacteria preparation and effect assessment standard provide effective technical support.
Detailed description of the invention
Fig. 1 is the electrophoresis detection of amoA gene qPCR in embodiment 2 as a result, wherein 1.Marker, 2. 55 DEG C of annealing temperatures,
3. 60 DEG C of annealing temperature, 4. 65 DEG C of annealing temperatures, 5. negative controls；
Fig. 2 is the electrophoresis detection of nxrA gene qPCR in embodiment 2 as a result, wherein 1.Marker, 2. 55 DEG C of annealing temperatures,
3. 60 DEG C of annealing temperature, 4. 65 DEG C of annealing temperatures, 5. negative controls；
Fig. 3 is the electrophoresis detection of nirS gene qPCR in embodiment 2 as a result, 1.Marker, 2. 55 DEG C of annealing temperatures, 3. move back
Fiery temperature 60 C, 4. 65 DEG C of annealing temperatures, 5. negative controls；
Fig. 4 is the double-layer plate that photosynthetic bacteria preparation IPB1 is detected in embodiment 2；
Fig. 5 is photosynthetic bacteria preparation IPB1 stoste microscopic photographs (400 ×) in embodiment 2；
Fig. 6 is that photosynthetic bacteria preparation IPB1 high throughput analysis measures its bacterial community composition in embodiment 2；
Fig. 7 is the double-layer plate that photosynthetic bacteria preparation IPB2 is detected in embodiment 3；
Fig. 8 is that photosynthetic bacteria preparation IPB2 dilutes 100 power microscopes inspection photo (400 ×) in embodiment 3；
Fig. 9 is that photosynthetic bacteria preparation IPB2 high throughput analysis measures its bacterial community composition in embodiment 3.
Specific embodiment
Application method of the invention is further illustrated combined with specific embodiments below.Following embodiment and attached drawing are only used for showing
Example property explanation, is not considered as limiting the invention.Unless stated otherwise, reagent raw material used in following embodiments is normal
The life reagent raw material that commercially available or commercial sources obtain is advised, unless stated otherwise, method and apparatus used in following embodiments is
Method and apparatus commonly used in the art.
Embodiment 1
The detection method of fishing provided in this embodiment photosynthetic bacteria preparation product, including Accurate Determining photosynthetic bacteria preparation
Whether the photosynthetic bacteria in product survive, photosynthetic bacteria bacterium amount and purity, and according to whether contains photosynthetic bacteria and determine whether
Detect its function and effect and nitrogen dependent interaction functional gene content to water body nitrogen phosphorus.Specifically includes the following steps:
One, the detection of fishing photosynthetic bacteria preparation product bacterium amount and purity
1. the fishing culture of photosynthetic bacteria in photosynthetic bacteria preparation product
1.1 used mediums and reagent
1.1.1 photosynthetic bacteria cultivates agar
(1) basal medium
1. ingredient
CH3COONa·3H2O3.0g, NaHCO31.0g, yeast extract 2.0g, K2HPO4·3H2O0.50g, NaCl 1.0g,
Fe-EDTA solution 1.0mL, agar powder 15g, distilled water 1L.
2. preparation method
In addition to agar, remaining ingredient is dissolved in distilled water, pH 7.0, agar is added, dissolved by heating, quantitative separating is suitable
Photosynthetic bacteria culture agar is made in suitable container, 121 DEG C of high pressure sterilization 15min.
(2) Fe-EDTA solution
FeSO4·7H2O 557mg, Na2- EDTA 745mg, distilled water 100mL.
1.1.2 phosphate-buffered dilution
(1) liquid is stored
1. ingredient
Potassium dihydrogen phosphate 34.0g, distilled water 500mL.
2. preparation method
PH to 7.2 is adjusted with the 1mol/L sodium hydroxide solution of about 175mL, is stored after being diluted to 1000mL with distilled water
In refrigerator.
(2) dilution
With distilled water dilution 1.25mL storage liquid (being 2. made above) to 1000mL, it is sub-packed in appropriate vessel, 121 DEG C of high pressures
Sterilize 15min, obtains hydrochlorate buffer diluent.
1.2 sample preparation
Draw that 25mL (g) is commercially available to be indicated that fishing is placed in photosynthetic bacteria preparation product and fill with aseptic straw or pipettor
In the sterile conical flask of 225mL sterile phosphate buffer diluent, 30min is vibrated, is mixed well, the sample that 1:10 is made is even
Liquid.
1:10 dilution 1mL is drawn with micropipettor, the test tube equipped with 9mL sterile diluent is slowly injected along tube wall
In, it is uniformly mixed, the even liquid of sample of 1:100 is made.
By aforesaid operations, 10 times of even liquid of ascending series dilute sample are successively made.
1.3 inoculated and cultured
Using double-layer plate cultivation.The 3 suitable dilutions prepared in selection 1.2, take the even liquid of the sample of 0.1mL equal
It is even to be coated on photosynthetic bacteria culture agar, 15min is hung, then 45 DEG C~50 DEG C of photosynthetic bacteria culture agar will be cooled to about
15mL is poured into each plate, and plate is overturn after agar solidification, is sealed plate mouth with sealed membrane, is provided one for thallus
The environment of hypoxemia.Plate is placed in about 3000Lx, 30 DEG C of illumination cultivation 5d-6d observe the growing state of flat-plate bacterial colony.It is each dilute
Degree of releasing is inoculated with 3 sterilized petri dishes.
1.4 record results
It records and takes pictures, whether there is bacterium growth on the double-deck plate.There is bacterium growth, that is, illustrates there is viable bacteria in the microbial inoculum product；
There is no bacterium colony growth, then illustrates there is no viable bacteria in the microbial inoculum product.
2. the measurement of total bacteria count in fishing photosynthetic bacteria preparation
2.1 sampling
Clean blood counting chamber is taken, clean dedicated coverslip is placed on two ridges of blood counting chamber.
Suitable dilution is selected, the dilution bacterium solution shaken up a little is drawn with sterile dropper and (dilutes bacterium in 1.2 sample preparations
Liquid), a droplet is instilled from the edge of coverslip, bacterium solution is made voluntarily to penetrate into counting chamber.Must not pay attention to when adding bacterium solution has bubble, is added dropwise
After bacterium solution, about 5min is stood.After finding grid under low power lens, conversion high power sem observation is counted.
2.2 method of counting
(1) method of counting of the tally of different size slightly has difference.The tally of 16 × 25 specifications, needs by diagonal line
Orientation, calculate upper left, lower-left, the 4 big lattice (totally 100 small lattice) of upper right and bottom right bacterium number.If the tally of 25 × 16 specifications,
It is above-mentioned 4 big especially except counting, the bacterium number of a central big lattice (totally 80 small lattice) must also be counted.
(2) each sample repeat count 3 times (each numerical value should not have big difference, and otherwise re-operate), its average value is taken.
(3) go out bacterium number contained by every milliliter of bacterium solution according to the following formula.
1. the tally of 16 × 25 specifications:
Total bacteria count/mL=(bacterium number/100 in 100 small lattice) × 400 × 10000 × bacterium solution extension rate.
2. the tally of 25 × 16 specifications:
Total bacteria count/mL=(bacterium number/80 in 80 small lattice) × 400 × 10000 × bacterium solution extension rate.
3. the fishing measurement of photosynthetic bacteria preparation product purity
Miillpore filter in a manner of vacuum filter, by 100mL photosynthetic bacteria preparation sample filtering at 0.2 μm
(Millipore) on, filter membrane is shredded and is put into mentioning for Water DNA Kit (water body DNA extraction kit) (commercially available, Omega)
It takes in pipe, the DNA of bacteria of microbial inoculum sample is extracted according to the operating method that kit provides.Then gained DNA sample is sent supreme
Flux sequencing company, with primer 515F (5 '-GTGCCAGCMGCCGCGGTAA-3 ') and 806R (5 '-
GGACTACHVGGGTWTCTAA T-3 '), the variable region 16S V4 is expanded, analyzes sample Bacterial community with high throughput sequencing technologies,
Relative abundance (the i.e. light for whether having photosynthetic bacteria and photosynthetic bacteria in testing result is determined according to primary Jie Shi Bacteria Identification system
Close bacteria preparation product purity).
4. the calculating of photosynthetic bacteria bacterium amount in fishing photosynthetic bacteria preparation
Photosynthetic bacteria bacterium amount=total bacteria count × photosynthetic bacteria relative abundance in photosynthetic bacteria preparation.
If commercially available indicate in fishing photosynthetic bacteria preparation product contains photosynthetic bacteria, then following steps two are carried out；Such as system
Photosynthetic bacteria is not contained in agent product, then illustrates the practical not photosynthetic bacteria microbial inoculum of said preparation product, no longer need to carry out step 2.
Two, the detection of fishing photosynthetic bacteria preparation product function and effect
1. the function and effect of fishing photosynthetic bacteria preparation measure
The preparation of 1.1 feed leachates
It by mixed feed drying, crushes, the feed for taking 100g to crush, is added 25 DEG C of 1L pure water and impregnates for 24 hours, take supernatant,
Feed leachate stoste is made.
The preparation of 1.2 experiment water bodys
It takes appropriate feed leachate stoste to be added in cultivating pool water, according to the difference that initial nitrogen phosphorus condition is arranged, is added
Appropriate ammonium salt, nitrite etc., adjust the initial of experiment water body Total inorganic nitrogrn (including ammonia nitrogen, nitrite nitrogen and nitrate nitrogen)
Concentration.Prepared experiment water body is sub-packed in the triangular flask of 1L, 121 DEG C of sterilizing 15-20min with 600mL/ bottles of amount.
The setting of 1.3 action conditions
Microbial inoculum group and control group be set, and microbial inoculum group is according to applying bacterium amount 103-106Cfu/mL addition is commercially available to indicate fishing with photosynthetic
Bacterium is not added in bacteria preparation product, control group, and the sterile saline of same volume is added, and every group setting three parallel.It sets respectively
(table 1) is tested under low nitrogen dim light, high nitrogen dim light, high nitrogen intense light conditions.Each group is put in 28 DEG C to cultivate 7 days, in experiment
The 0th day, the 3rd day, the 5th day, the 7th day be measured by sampling water body ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and reactive phosphate contain
It measures (GB17378.4-2007), calculates separately degradation rate.
The setting of 1 action condition of table
| Microbial inoculum group | Low nitrogen dim light group | High nitrogen dim light group | The strong light group of high nitrogen |
| Total inorganic nitrogrn concentration (mg/L) | 1-3mg/L | 8-13mg/L | 8-13mg/L |
| Intensity of illumination | 100Lx | 100Lx | 3000-50000Lx |
| Light To Dark Ratio | 10-12:12-14 | 10-12:12-14 | 24:0 |
| Whether vibrate | It is | It is | It is no |
The calculating of 1.4 degradation rates
Degradation rate=(the initial nutrition salt figure of experiment water body-experiment water body of culture n days nutrition salt figure)/experiment water body
Initial nutrition salt figure (n=3,5,7)
2 fishing photosynthetic bacteria preparations measure the functional gene of nitrogen dependent interaction
With key enzyme ammona monooxygenase, the nitrite oxidoreductase, nitrous in ammoxidation, nitrification, denitrification step
The gene (amoA gene, nxrA gene, nirS gene) of three kinds of enzymes of hydrochlorate reductase is target, passes through quantitative fluorescent PCR
(qPCR) method detects the content of three of the above gene in microbial inoculum, judges microbial inoculum with the presence or absence of key enzyme required for nitrogen conversion process
Gene.
The optimization of 2.1qPCR condition
Different annealing temperatures is set, qPCR reaction condition is optimized.Specific amplimer and response procedures are as shown in table 2.
Reaction system are as follows: qPCR Mix 15 μ L, Mg2+(25mmoL/L) 2 μ L, target gene primer (10 μm of ol/L) each 0.5 μ L, DNA mould
2 μ L of plate, adds dd H2O complements to 30 μ L.
The amplimer and reaction condition of 2 quantitative fluorescent PCR of table are arranged
The electrophoresis detection result of qPCR is as shown in Figure 1, wherein Marker, 2. annealing are warm under amoA gene different annealing temperature
55 DEG C of degree, 3. 60 DEG C of annealing temperatures, 4. 65 DEG C of annealing temperatures, 5. negative controls.
The electrophoresis detection result of qPCR is as shown in Fig. 2, wherein 1.Marker, 2. annealing under nxrA gene different annealing temperature
Temperature 60 C, 3. 55 DEG C of annealing temperatures, 4. 65 DEG C of annealing temperatures, 5. negative controls.
The electrophoresis detection result of qPCR is as shown in figure 3, wherein 1.Marker, 2. annealing under nirS gene different annealing temperature
55 DEG C of temperature, 3. 60 DEG C of annealing temperatures, 4. 65 DEG C of annealing temperatures, 5. negative controls.
The expanding effect (Fig. 1-Fig. 3) under different annealing temperature is compared with electrophoretogram after the completion of qPCR, with the band that becomes clear
The reaction condition that corresponding annealing temperature is detected as subsequent quantitation.AmoA gene, the nxrA gene, nirS base determined after optimization
The annealing temperature of cause is respectively 65 DEG C, 60 DEG C, 55 DEG C.
The production of 2.2 standard curves
Standard curve is standard gene using the plasmid containing qPCR target fragment, the specific steps are as follows:
(1) Escherichia coli in clone library containing target gene plasmid is selected to carry out 37 DEG C of shaking flask cultures；
(2) plasmid is extracted using Axygen plasmid miniprep Kit (Axygen) kit；
(3) plasmid concentration is measured using Qubit 3.0 (Life Biotech)；
(4) calculating of plasmid copy number；
(5) Plasmid DNA is expanded by 10 times of gradient dilution production standard samples, obtains standard curve.
The functional gene of 2.3 mesh detects
Using qPCR testing goal functional gene content, using the microbial inoculum DNA of bacteria of extraction as PCR amplification template, primer
(Feng et al.,2016；Faulwetter et al., 2011) and reaction condition is referring to the following table 3.QPCR system is 30 μ L,
Wherein, 15 μ L of qPCR Mix, 0.3 μ L of dyestuff (eva), forward and reverse primer (10 μm of ol/L) each 0.3 μ L, Mg of target gene2+
(25mmoL/L) 2.5 μ L, 2 μ L of DNA profiling, with dd H2O complements to 30 μ L.Experimental setup negative control, and with press 10 times of gradients
5 standard samples diluted carry out quantitative amplification detection.Purpose functional gene content is calculated according to standard curve.
QPCR reaction condition after the optimization of table 3
Embodiment 2
Common fishing photosynthetic bacteria preparation product I PB1 is acquired from the market of Haikou City, Hainan Province aquaculture main producing region
(its label main component is red spirillum, microelement, organized enzyme and somatomedin；The product nominally has ammonia in conversion water
Nitrogen, nitrite and other effects), in Haizhu District of Guangzhou city, Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst utilizes embodiment 1
In method carried out the detection of photosynthetic bacteria bacterium amount, purity in photosynthetic bacteria preparation product, and its to ammonia nitrogen, nitrite
The main function and effect of water quality factor such as nitrogen, nitrate nitrogen and reactive phosphate and the measurement of nitrogen transformation function gene.
1, the photosynthetic bacteria bacterium amount and purity testing of photosynthetic bacteria preparation product I PB1
Fishing photosynthetic bacteria preparation product I PB1 is sampled, using the double-layer plate cultivation in embodiment 1, is detected photosynthetic thin
It whether there is viable bacteria in bacteria preparation product；Then, according to above-mentioned microscopic counting, the total bacteria count in microbial inoculum product is measured；Again
The total DNA of microbial inoculum sample is extracted, the variable region specific amplification 16S V4 analyzes sample Bacterial community, determines the phase of photosynthetic bacteria
To abundance.Finally it is calculated from the formula photosynthetic bacteria bacterium amount and purity in photosynthetic bacteria preparation.
The double-layer plate of photosynthetic bacteria preparation IPB1 detection is as shown in Figure 4.
Photosynthetic bacteria preparation IPB1 stoste microscopic photographs (400 ×) are as shown in Figure 5.
It is as shown in Figure 6 that photosynthetic bacteria preparation IPB1 high throughput analysis measures its bacterial community composition.
There is viable bacteria detection in photosynthetic bacteria preparation product I PB1 it can be seen from Fig. 4-6；Microscope inspection is the result shows that it is thin
Bacterium sum is 3.25 × 108Cells/mL, according to high-flux sequence as a result, the main advantage bacterium of photosynthetic bacteria preparation product I PB1
Belong to Rhodopseudomonas (Rhodopseudomonas sp.), relative abundance (purity) is 50.58%, thus calculates and obtains
The photosynthetic bacteria bacterium amount of photosynthetic bacteria is 1.64 × 10 in microbial inoculum IPB18cells/mL。
Due to having photosynthetic bacteria detection in photosynthetic bacteria preparation product I PB1,2 are followed the steps below.
2, fishing is measured with the breeding water body clean-up effect of photosynthetic bacteria preparation product I PB1
Based on the cultivating pool water that sterilizes, suitable aseptic feed leachate stoste is added, it is common to reach cultivating pool
Trophic level.Microbial inoculum group and control group be set, and microbial inoculum group is according to applying bacterium amount 103-106Cfu/mL adds microbial inoculum product I PB1, control
Group is not added microbial inoculum IPB1 but the sterile saline of same volume is added, and every group is respectively provided with three in parallel.They are set respectively
It is cultivated 7 days in low nitrogen dim light, high nitrogen dim light, lower 28 DEG C of high nitrogen intense light conditions, in the 0th day, the 3rd day, the 5th day, the 7th day of experiment
Ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and the phosphate content (GB17378.4-2007) of water body is measured by sampling, calculates separately drop
Solution rate.
The results show that testing the phosphate of water body, the maximum of nitrate nitrogen, nitrite nitrogen under low nitrogen low light condition
Degradation rate is respectively 40.98%, 28.28%, 20.12%；To ammonia nitrogen, nitrite nitrogen, nitrate under high nitrogen low light condition
Nitrogen and phosphatic most degradation rate are respectively 33.18%, 26.77%, 24.16% and 45.24%；Under high nitrogen intense light conditions
It is respectively 18.13%, 48.07%, 62.97% and to ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and phosphatic most degradation rate
8.78% (table 4).
4 photosynthetic bacteria preparation IPB1 of table is at different conditions to the most degradation rate of inorganic nitrogen phosphorus
| Ammonia nitrogen | Nitrite nitrogen | Nitrate nitrogen | Phosphate | |
| Low nitrogen dim light | / | 20.12% | 28.28% | 40.98% |
| High nitrogen dim light | 33.18% | 26.77% | 24.16% | 45.24% |
| The strong light of high nitrogen | 18.13% | 48.07% | 62.97% | 8.78% |
3, the measurement of the fishing nitrogen transformation function gene of photosynthetic bacteria preparation product I PB1
Using qPCR method testing goal functional gene content, primer and reaction condition are referring to table 1.By the microbial inoculum bacterium of extraction
Total DNA is as PCR amplification template.Fluorescent quantitation amplification system is 30 μ L, wherein 15 μ L of qPCR Mix, 0.3 μ of dyestuff (eva)
L, forward and reverse primer (10 μm of ol/L) each 0.3 μ L, Mg of target gene2+(25mmoL/L) 2.5 μ L, 2 μ L of DNA profiling, uses dd
H2O complements to 30 μ L.According to standard curve, the content of functional gene is calculated.
The results show that the nirS gene copy number for encoding nitrite reductase in photosynthetic bacteria preparation product I PB1 is
5.33×103Copies/ μ L encodes the amoA gene of ammona monooxygenase and the nxrA gene of coding nitrite oxidoreductase
Detected value is very low, can not detect (table 5) with qPCR.
3 kinds of gene (amoA, nxrA, nirS) qPCR detected values in 5 photosynthetic bacteria preparation IPB1 of table
The above result shows that microbial inoculum IPB1 is the composite bacteria agent comprising multi-cultur es, dominant bacteria is Rhodopseudomonas,
Relative abundance (purity) is 50.58%, and total bacterium amount is 3.25 × 10 in microbial inoculum8Cells/mL, and photosynthetic bacteria bacterium amount is 1.64
×108cells/mL.The microbial inoculum product has the function of inorganic nitrogen phosphorus of degrading, and to nitrite under high nitrogen intense light conditions
Nitrogen, the degradation effect of nitrate nitrogen are good.In addition, the microbial inoculum product contains the nirS gene of coding nitrite reductase,
Copy number is 5.33 × 103Copies/ μ L encodes the amoA gene of ammona monooxygenase and encodes nitrite oxidoreductase
NxrA genetic test value is low.
The result shows that the present embodiment method can accurately detect photosynthetic bacteria in fishing photosynthetic bacteria preparation product I PB1
Bacterium amount, purity also scientific can measure its catharsis effect and nitrogen dependent interaction functional gene content to water body nitrogen phosphorus.
Embodiment 3
Common fishing photosynthetic bacteria preparation product I PB2 is acquired from the market of Nanjing aquaculture main producing region
(its label main component is photosynthetic bacteria and its metabolite etc.；The product nominally has ammonia nitrogen, nitrous acid in fast degradation water
The effect of harmful substances such as salt), in Haizhu District of Guangzhou city, Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst utilizes embodiment 1
In method carried out the photosynthetic bacteria bacterium amount of photosynthetic bacteria preparation product, purity testing.
1, the photosynthetic bacteria bacterium amount and purity testing of photosynthetic bacteria preparation product I PB2
Fishing photosynthetic bacteria preparation product I PB2 is sampled, using the double-layer plate cultivation in embodiment 1, is detected photosynthetic thin
It whether there is viable bacteria in bacteria preparation product；Then, according to above-mentioned microscopic counting, the total bacteria count in microbial inoculum product is measured；Again
The bacteria total DNA of microbial inoculum sample is extracted, the variable region specific amplification 16SV4 analyzes sample Bacterial community, determines photosynthetic bacteria
Relative abundance (purity).Finally it is calculated from the formula photosynthetic bacteria bacterium amount in photosynthetic bacteria preparation.
The double-layer plate of photosynthetic bacteria preparation IPB2 detection is as shown in Figure 7.
It is as shown in Figure 8 that photosynthetic bacteria preparation IPB2 dilutes 100 power microscopes inspection photo (400 ×).
It is as shown in Figure 9 that photosynthetic bacteria preparation IPB2 high throughput analysis measures its bacterial community composition.
There is viable bacteria detection in photosynthetic bacteria microbial inoculum IPB2 it can be seen from Fig. 7-9；Microscope inspection is the result shows that its bacterium is total
Number is 7.25 × 109cells/mL；According to high-flux sequence as a result, the main advantage Pseudomonas of microbial inoculum IPB2 is in bacillus
(Bacillus sp.), relative abundance 97.00%, without photosynthetic Detection of pathogenic bacteria.Therefore, in microbial inoculum IPB2 photosynthetic bacteria bacterium
Amount is 0.
The above results show that the bacterium amount of photosynthetic bacteria is 0 in microbial inoculum product I PB2, and practical non-photosynthetic bacteria preparation produces
Product.It is subsequent no longer to detect its effect effect to the main water quality factor such as ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and reactive phosphate
Fruit and nitrogen transformation function gene content.
The result shows that the present embodiment method can in accurate testing product IPB2 photosynthetic bacteria bacterium amount, purity, really sentence
Whether with the presence of photosynthetic bacteria in other product.
The present invention is not limited within the scope of above-mentioned specific embodiment, and the embodiment above is used for the purpose of can be to this
The use process of invention is described in detail, and has the production method of equal function and technical detail to also belong in the present invention
A part of appearance.In fact, those skilled in the art are according to description above, it will be able to according to respectively needing to find different tune
Perfect square case, these adjustment all should be in the scope of the claims by the appended claims herein.
Claims (8)
1. a kind of fishing detection method of photosynthetic bacteria preparation product, it is characterized in that: this method combine double-layer plate cultivation,
Microscope count method and high-flux sequence method, whether determining viable bacteria in fishing photosynthetic bacteria preparation product, bacterium amount and purity；To
It determines after containing photosynthetic bacteria in formulation products, is further integrated with fishing photosynthetic bacteria preparation product and effect is acted on to water body nitrogen phosphorus
The measuring method of fruit and fishing nitrogen dependent interaction functional gene in photosynthetic bacteria preparation product, specifically includes the following steps:
(1) using the measurement fishing of double-layer plate cultivation with whether there is viable bacteria in photosynthetic bacteria preparation product, using microscope meter
The total bacteria count of number method measurement fishing photosynthetic bacteria preparation product, measures fishing photosynthetic bacteria preparation product using high-flux sequence method
Purity；
(2) as contained photosynthetic bacteria in formulation products, then fishing is on the one hand measured with photosynthetic bacteria preparation product to water body nitrogen phosphorus
Function and effect；On the other hand, fishing is measured with nitrogen conversion correlation function gene in photosynthetic bacteria preparation product, is determined
Whether it has the function of nitrogen transformation potentiality；If do not contained photosynthetic bacteria in formulation products, then illustrate that said preparation product is practical simultaneously
Non-photosynthetic bacterium microbial inoculum product no longer needs to carry out the step.
2. the fishing according to claim 1 detection method of photosynthetic bacteria preparation product, it is characterized in that: step (1) is specific
The following steps are included:
(1.1) fishing photosynthetic bacteria preparation sample is chosen, determines whether that there are viable bacterias using double-layer plate cultivation；
(1.2) using the total bacteria count in microscope count method measurement microbial inoculum；
(1.3) total DNA for extracting fishing photosynthetic bacteria preparation sample, expands the variable region 16S rRNA, using high-flux sequence skill
Art measures the bacterial community composition of fishing photosynthetic bacteria preparation sample, determines the relative abundance of photosynthetic bacteria, and calculate and examined
Survey the bacterium amount and purity of photosynthetic bacteria in photosynthetic bacteria preparation sample.
3. the fishing according to claim 2 detection method of photosynthetic bacteria preparation product, it is characterized in that: in step (1.1)
Determine whether that there are when viable bacteria using double-layer plate cultivation, comprising the following steps: choose fishing photosynthetic bacteria preparation sample, system
Standby dilution is inoculated with photosynthetic bacteria under 30 DEG C ± 1 DEG C, the micro- aerobic condition of illumination and cultivates agar double-layer plate, cultivates 5~6d,
Whether there is bacterium growth on observation double-layer plate, has bacterium growth, that is, illustrate there is viable bacteria in the microbial inoculum product；There is no bacterium colony growth, then
Illustrate there is no viable bacteria in the microbial inoculum product.
4. the fishing according to claim 2 detection method of photosynthetic bacteria preparation product, it is characterized in that: in step (1.3)
The bacteria total DNA of fishing photosynthetic bacteria preparation sample is extracted using WaterDNAKit, the variable region 16S rRNA is expanded, using height
Flux sequencing technologies measure the bacterial community composition of microbial inoculum, determine in testing result whether have according to primary Jie Shi Bacteria Identification system
The relative abundance of photosynthetic bacteria and photosynthetic bacteria obtains photosynthetic bacteria system further according to the relative abundance of total bacteria count and photosynthetic bacteria
The bacterium amount and purity of photosynthetic bacteria in agent sample.
5. the fishing according to claim 1 detection method of photosynthetic bacteria preparation product, it is characterized in that: step (2) is specific
The following steps are included:
(2.1) fishing is detected and is evaluated with the function and effect of photosynthetic bacteria preparation sample purification breeding water body nitrogen phosphorus；
(2.2) the nitrogen transformation function of fishing photosynthetic bacteria preparation sample is determined by measuring nitrogen dependent interaction functional gene
Potentiality.
6. the fishing according to claim 5 detection method of photosynthetic bacteria preparation product, it is characterized in that: in step (2.1)
By to fishing with the function and effect of photosynthetic bacteria preparation sample purification breeding water body nitrogen phosphorus carry out detection and evaluation include: selection support
Grow water body, fishing photosynthetic bacteria preparation sample be added, test the fishing photosynthetic bacteria preparation product to ammonia nitrogen in breeding water body,
Nitrite nitrogen, nitrate nitrogen and phosphatic degradation effect.
7. the fishing according to claim 5 detection method of photosynthetic bacteria preparation product, it is characterized in that: step (2.2) institute
Stating nitrogen dependent interaction functional gene includes the amoA gene for encoding ammona monooxygenase, coding nitrite oxidoreductase
The nirS gene of nxrA gene and coding nitrite reductase.
8. the fishing according to claim 5 detection method of photosynthetic bacteria preparation product, it is characterized in that: in step (2.2)
Using the content of fluorescence quantitative PCR method detection nitrogen dependent interaction functional gene, judge that fishing is with photosynthetic bacteria preparation sample
The gene of key enzyme required for the no conversion process there are nitrogen.
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