CN107287132B - Luminescent bacterium, separation and identification method and application thereof - Google Patents
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Abstract
The invention discloses a luminous bacterium, a separation and identification method and application thereof, and belongs to the technical field of microorganisms. The bacterium is a luminous bacterium Fc-11-1, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation date of 2016, 12 and 05 months, has the preservation number of CGMCC No.13424, and is named in classification as: vibrio toranziae. The invention also discloses a separation and identification method of the luminous bacteria and application thereof. The luminous bacteria Fc-11-1 enriches luminous bacteria species groups, the separation method solves the problem of difficult separation and screening of luminous bacteria in the prior art, and the identification method introduces modern microorganism identification instrument and equipment and combines with a modern molecular biology method.
Description
Technical Field
The invention relates to a luminous bacterium, a separation and identification method and application thereof, belonging to the technical field of microorganisms.
Background
The luminous bacteria are widely parasitic, symbiotic or saprophytic in marine organisms or plankton, and the metabolic process of the luminous bacteria can emit light visible to naked eyes in dark environment. Researches find that a plurality of toxic substances can inhibit the luminescence of luminescent bacteria, and the toxic substances can be detected or food and environment safety evaluation can be carried out by utilizing the toxic effect of the toxic substances on the luminescent bacteria. For example, the method can be used for detecting pesticide residues, veterinary drug residues, heavy metal pollution and the like by utilizing luminous bacteria, and also can be used for evaluating the comprehensive toxicity of foods, water samples and the like. The currently isolated luminescent bacteria include Vibrio (Vibrio), Photobacterium (Photobacterium), Shewanella (Shewanella), and Brevibacterium (Xenorhabdus), and about 11 kinds of luminescent bacteria have been found and named, which are very small compared with the abundant biological resources of nature. This also makes the application of photobacterium to be still greatly limited.
The applicant of the invention separates and screens the luminous bacteria in seawater and marine organisms to obtain more luminous bacteria resources and lay a foundation for the application of the luminous bacteria resources in the detection of harmful substances and environmental monitoring in multiple fields.
Disclosure of Invention
One of the purposes of the invention is to provide a luminous bacterium. The luminous bacterium Fc-11-1 enriches the species resource of luminous bacteria, is a novel luminous strain different from 11 kinds of luminous bacteria reported in the prior art, and is expected to be used for quickly detecting harmful substances in food.
The technical scheme for solving the technical problems is as follows: a strain of luminous bacteria Fc-11-1 is preserved in the general microbiological culture collection center (CGMCC for short) of China Committee for culture Collection of microorganisms of the institute of Chinese academy of sciences in the great Tunny of the sunward area, Beijing, 12 months and 05 days in 2016, the preservation number is CGMCC No.13424, and the strain is classified and named Vibrio tornazoniae.
Another object of the present invention is to provide a method for isolating the above-mentioned luminescent bacteria. The separation method of the luminous bacteria solves the problem that the luminous bacteria in the prior art are difficult to separate and screen. The method comprises the steps of firstly coating a seawater 2216E agar plate with sterile solution and suspension of the surface and internal tissues of marine organisms, and then transferring a solid luminous culture medium to observe the technology of luminescence, so that the growth of the non-salt-tolerant saprophytic bacteria can be effectively inhibited, and the growth of the marine luminous bacteria is indirectly promoted.
The technical scheme for solving the technical problems is as follows: the separation method of the luminous bacteria comprises the following steps:
(1) preparation of the Medium
Solid culture medium for luminescent bacteria (isolation medium): 3mL of glycerol, 5g of yeast powder, 5g of tryptone, 1g of calcium carbonate, 20g of agar and 1000mL of aged seawater, wherein the pH value is 7.8-8.0, and the raw materials are sterilized for 20 minutes at 121 ℃;
luminous bacteria liquid culture medium: 5g of yeast powder, 5g of tryptone, 30g of sodium chloride, 5g of disodium hydrogen phosphate, 1g of monopotassium phosphate and 3mL of glycerol, wherein the pH value is 7.6, and the yeast powder is sterilized for 20 minutes at 121 ℃;
seawater 2216E agar medium: 5g of peptone, 1g of yeast powder, 0.01g of iron phosphate, 20g of agar and 1000mL of aged seawater, wherein the pH value is 7.6, and the sterilization is carried out for 20 minutes at 121 ℃;
(2) washing impurities on the surface of a fish body of a freshly captured marine fish by using a sterile 3% NaCl solution in a sterile environment, respectively collecting 5-6 fish scales, gills and 80% intestines of the marine fish, respectively putting the fish scales, gills and intestines of the marine fish into 45mL of sterile 3% NaCl solution containing glass beads, placing the fish scales, gills and intestines of the marine fish on a vortex oscillator, oscillating the fish scales, gills and intestines of the marine fish for 2-3 minutes, fully and uniformly mixing, and placing the fish on a sterile super clean bench;
(3) respectively sucking 200 μ L of the mixed fish scale, fish gill and fish intestine suspension, injecting into 2216E seawater agar culture medium plate, coating with coating rod, standing for 1min, placing into 25 deg.C incubator, and performing inverted culture;
(4) picking the growing bacterial colony with an inoculating needle, streaking and inoculating the bacterial colony in a luminous bacteria solid culture medium plate, putting the luminous bacteria solid culture medium plate into an incubator at 25 ℃, carrying out inverted culture until bacterial colony is generated, and taking out; observing in a dark room, marking the colonies which emit fluorescence, and numbering;
(5) and (4) picking the marked luminous colonies by using an inoculating needle, and carrying out continuous streak inoculation until pure colonies which can emit fluorescence in a dark room are obtained.
The stale seawater is seawater obtained by storing seawater which is not polluted by land dirt or is rarely mixed with fresh water in a glass bottle for several weeks.
It is a further object of the present invention to provide a method for identifying the above luminescent bacterium. The invention provides a method for identifying luminous bacteria by combining traditional microbiology and modern molecular biology, and utilizes a biologics microorganism identification instrument which is a modern physiological biochemical identification instrument to identify the separated luminous bacteria, thereby avoiding the defects of time and labor waste and inaccurate result of manual operation methods.
The technical scheme for solving the technical problems is as follows: the identification method of the luminous bacteria comprises the following steps:
(1) observing the morphological size of plate colonies, and observing the cell morphology by gram staining;
(2) biolog identification
According to an operation manual provided by a Biolog microorganism automatic identifier, the turbidity of the inoculated strain suspension is adjusted, the inoculated strain suspension is inoculated into a 96-hole GN2 micropore identification plate, the plate is cultured for 24 hours, 48 hours and 72 hours at the temperature of 28 ℃, and the plate reading modes of manual operation and automatic operation are combined to respectively measure for 3 times, so that the identification result is obtained.
(3) Preparation of PCR template
The overnight cultured suspension of the photobacteria was formulated to 108CFU/mL (OD600 ═ 0.1), 3. mu.L of the suspension was transferred to a 1.5mL centrifuge tube containing 100. mu.L of sterile double distilled water, and the suspension was subjected to water bath at 99 ℃ for 10min, centrifugation at 10000g for 10min, and after cooling, the supernatant was used as a template for PCR reaction. The extracted DNA template can be stored in a refrigerator at-20 ℃.
(4)16S rDNA PCR amplification
The primers used for the PCR amplification of the 16S rDNA of the luminous bacteria are bacterial 16S rDNA universal primers, and the upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; a downstream primer 1492R: 5'-TACGGTTACCTTGTTACGACTT-3' are provided.
PCR amplified system (25. mu.L): GoTaq Green Master Mix 12.5. mu.L, upstream primer 27F 1. mu.L, downstream primer 1492R 1. mu.L, template DNA 2.5. mu.L, ddH2O8. mu.L. The procedure for PCR amplification was: pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min, 30 cycles, and extension at 72 ℃ for 10 min.
The PCR product was detected by electrophoresis on a 1.5% agarose gel.
(5)16S rDNA PCR product sequencing and phylogenetic tree construction
The 16S rDNA PCR product is purified and then sent to the Boehringer Bioengineer (Dalian) Co., Ltd for sequencing. The gene sequence obtained by sequencing is submitted to NCBI website (http:// Blast. NCBI. nlm. nih. gov) for Blast alignment. The construction of the phylogenetic tree was carried out 1000 times by self-development with a Neighbor-join statistical method using MEGA 5.05 software.
The fourth purpose of the present invention is to provide the application of the above mentioned luminescent bacteria in the detection of antibiotic and its metabolite sensitivity characteristics. The luminous bacteria Fc-11-1 of the invention is sensitive to various toxic substances including furazolidone and metabolites thereof, and can be used for detecting drug residues in products such as aquaculture and the like.
The technical scheme for solving the technical problems is as follows: the luminescent bacteria can be used for detecting the sensitivity characteristics of antibiotics and metabolites thereof.
The detection method of the sensitivity characteristics of the luminous bacteria to antibiotics and metabolites thereof comprises the following steps:
(1) cup and dish method for detecting sensitivity of luminous bacteria to antibiotics and metabolites thereof
(step 1.1) preparing a solution of 0.5mg/mL of antibiotic or antibiotic metabolite;
(step 1.2) coating the bacterial suspension of the luminous bacteria Fc-11-1 cultured overnight on a 2216E plate, placing an oxford cup on the plate, and respectively adding 100 mu L of the solution of the antibiotics or the antibiotic metabolites prepared in the step (1.1) into the oxford cup;
(step 1.3) observing the size of a bacteriostatic zone after culturing at 25 ℃, wherein the growth has good sensitivity to antibiotics if the bacteriostatic zone is large;
(2) luminescence inhibition rate of antibiotic and metabolite thereof on luminous bacteria
Preparing solutions of antibiotics or antibiotic metabolites with gradient concentrations, respectively reacting with the bacterial suspension of the luminous bacteria Fc-11-1 cultured overnight, and determining the luminous inhibition rate of the luminous bacteria Fc-11-1, wherein the inhibition rate is high, and the sensitivity of luminescence to antibiotics is good;
(3) and (3) determining the growth and luminescence sensitivity characteristics of the photobacteria Fc-11-1 to antibiotics and metabolites thereof by combining the results of the steps (1) and (2).
On the basis of the technical scheme, the invention can be further improved as follows.
Further, in the step (1), the antibiotic is one of furazolidone, chloramphenicol, penicillin and erythromycin, and the antibiotic metabolite is a furazolidone metabolite.
Still further, the furazolidone metabolite is 3-amino-2-oxazolidinone.
Further, the concentration gradient in the step (2) is 10 mug/L, 50 mug/L and 100 mug/L.
The invention has the beneficial effects that:
1. the invention separates out a luminous bacterium. The luminous bacteria Fc-11-1 enriches the species resource of the luminous bacteria, and is a novel luminous strain different from 11 kinds of luminous bacteria reported in the prior art.
2. The invention provides a method for separating luminous bacteria, which solves the problem of difficult separation and screening of luminous bacteria in the prior art. The separation method adopts the sterile solution and the suspension of the surface and internal tissues of the marine organism to coat the seawater 2216E agar plate, can effectively inhibit the growth of the non-salt-tolerant saprophytic bacteria, and indirectly promotes the growth of the marine luminous bacteria.
3. The invention provides a method for identifying luminous bacteria by combining traditional microbiology and modern molecular biology, and utilizes a biologics identification system of modern physiological and biochemical identification instruments to identify the separated luminous bacteria, thereby avoiding the defects of time and labor waste and inaccurate result of manual operation methods.
4. The luminous bacteria Fc-11-1 disclosed by the invention are sensitive to antibiotics and metabolites thereof, and can be used for detecting residues of the antibiotics and the metabolites thereof.
Drawings
FIG. 1 is a colony observation image of a luminescent bacterium Fc-11-1.
FIG. 2 is a cell morphology observation chart of a luminescent bacterium Fc-11-1.
FIG. 3 is a graph showing the growth and luminescence intensity of a luminescent bacterium Fc-11-1.
FIG. 4 is a phylogenetic diagram of a luminescent bacterium Fc-11-1.
FIG. 5 luminescence inhibition efficiency of antibiotics and their metabolites on the luminescent bacterium Fc-11-1.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
Example 1 isolation and identification of Photobacterium Fc-11-1
1 strain of luminous bacteria is obtained after separation, purification and culture, and the luminous bacteria can emit bright blue-green visible light in a dark room and have the luminous characteristic; the gram-stained thalli is red, so that the gram-negative bacteria can be judged; the colony on the luminous solid culture medium is round and similar to a steamed bread, the surface is smooth and moist, the edge is neat, and the periphery of the colony is in a sawtooth shape after more than 24 hours; observed under an oil lens, the shape of the rod or the arc is shown in figures 1 and 2.
Example 2Biolog identification System analysis
The Biolog identification system analysis result of the luminous bacteria Fc-11-1 has low similarity with the standard strains in the database, and the species cannot be identified. However, the similarity (SIM value 0.128) and the distance (DIST value 8.955) obtained by the statistics of the Biolog identification system show that the strain has high homology with the Vibrio, and the physiological and biochemical characteristics of the strain are highly consistent with those of Vibrio torazoniae described by Aide Lasa et al[1]。
TABLE 1 Biolog identification of Photobacterium Fc-11-1
Note: "+" indicates positive reaction, and "-" indicates negative reaction.
EXAMPLE 3 Effect of incubation time on the growth and luminescence intensity of luminescent bacteria
Effect of culture time on growth and luminescence intensity of the photogenic bacteria Fc-11-1 in the luminescence medium under shaking culture conditions of 150rpm/min at 25 ℃ (FIG. 3). Selecting a single colony of luminescent bacteria, inoculating the single colony of the luminescent bacteria into 10mL of luminescent liquid culture medium, and carrying out shaking culture at 25 ℃ and 150r/min for 20h to obtain seed liquid; inoculating 1mL of the seed solution into 150mL of liquid luminescence culture medium, culturing at 25 deg.C and 150r/min with shaking, and measuring luminescence intensity (photon count per second) and OD600nm absorbance every 4h with weak luminometer. And measuring the luminous intensity by taking 1mL of the bacterial liquid. Centrifuging 1mL of bacterial solution at 5000r/min for 5min, collecting cell precipitate, dissolving in 1mL of physiological saline, and measuring with spectrophotometer at wavelength of 600 nm.
As can be seen from fig. 3, the luminescence and growth of the luminescent bacterium Fc-11-1 were not synchronized, and the luminescence began when the bacterial concentration reached OD600nm ═ 0.1. When the luminous bacteria grow into the logarithmic growth phase and the bacterial density reaches about OD600 nm-0.7, the luminous bacteria can continuously and stably emit light for 12 h. In the actual detection, the strain with short initial luminescence time and long continuous stable luminescence time is easy to operate and control, and in this point, the luminous bacterium Fc-11-1 is suitable for the detection of toxic substances.
Example 416S rDNA sequencing and phylogenetic analysis
The 16S rDNA sequence of the photobacteria Fc-11-1, with a length of 1471bp, was sequenced and the results were submitted to the NCBI for alignment, as shown in Table 2. The morphological identification result and the highest similarity of the experimental strain 16S rDNA sequence are combined to obtain the separated luminescent bacteria, and the similarity of the separated luminescent bacteria and Vibrio torazoniae is the highest and reaches 100 percent.
TABLE 2 16S rDNA sequence similarity of Photobacterium Fc-11-1
The isolated photobacteria Fc-11-1 were aligned and phylogenetic tree analyzed as shown in FIG. 4.
The results of physiological and biochemical identification and molecular biological identification prove that the luminous bacteria Fc-11-1 obtained by separation is Vibrio torazoniae, and the luminous bacteria are not reported at home.
Example 5 method for detecting the sensitivity characteristics of luminescent bacteria to antibiotics and their metabolites, comprising the following steps:
(1) cup and dish method for detecting sensitivity of luminous bacteria to antibiotics and metabolites thereof
(step 1.1) preparing 0.5mg/mL furazolidone, a furazolidone metabolite, chloramphenicol, penicillin, and erythromycin solution;
(step 1.2) coating the bacterial suspension of the luminous bacteria Fc-11-1 cultured overnight on a 2216E plate, placing an oxford cup on the plate, and respectively adding 100 mu L of the solution of the antibiotics or the antibiotic metabolites prepared in the step (1.1) into the oxford cup;
(step 1.3) observing the size of an inhibition zone after 25 ℃ culture, and judging the sensitivity of the strain to antibiotics by referring to a CLSI (CLSI) inhibition zone diameter judgment standard, wherein if the inhibition zone is large, the sensitivity of the growth to antibiotics is good (see table 3);
TABLE 3 growth inhibitory Activity of different toxic substances on the luminescent bacteria Fc-11-1
| Solutions of | Furazolidones | Furazolidone metabolites | Chloromycetin | Penicillin | Erythromycin |
| Diameter of zone of inhibition | 29mm | 31mm | 27mm | 20mm | 23mm |
Table 3 shows that the luminous bacteria Fc-11-1 is sensitive to various toxic substances, wherein the luminous bacteria Fc-11-1 is particularly obvious to furazolidone and metabolites thereof, the diameter of a inhibition zone is about 30mm, the luminous bacteria Fc-11-1 is also sensitive to chloramphenicol, and the diameter of the inhibition zone reaches 27 mm. As for penicillin and erythromycin, although the sensitivity is slightly lower, the diameter of the inhibition zone reaches more than 20mm, and the luminescent bacterium Fc-11-1 also reaches at least a moderate sensitivity level to penicillin and erythromycin by referring to the experimental standard for antibiotic sensitivity in CLSI (American society for clinical laboratory standardization). The results confirmed that the photobacteria Fc-11-1 has better sensitivity to several toxic substances.
(2) Luminescence inhibition rate of antibiotic and metabolite thereof on luminous bacteria
Inoculating the separated single colony of the luminous bacteria Fc-11-1 into 10mL of liquid culture medium, and carrying out shake culture at 25 ℃ and 150r/min for 20h to obtain seed liquid; inoculating 1mL of seed solution into 150mL of liquid luminous culture medium, and performing shaking culture at 25 ℃ at 150r/min for 20 h;
preparing furazolidone, furazolidone metabolite, chloramphenicol, penicillin and erythromycin solutions with the concentrations of 10 mug/L, 50 mug/L and 100 mug/L respectively; respectively reacting with the bacterial suspension of the luminous bacteria Fc-11-1 cultured overnight, and determining the luminous inhibition rate of the luminous bacteria Fc-11-1, wherein the inhibition rate is high, and the sensitivity of the luminescence to antibiotics is good;
the luminescence inhibition efficiency of the antibiotic against the luminescent bacteria was calculated according to the following formula:
in the formula:
x%: a luminescence suppression efficiency;
Kc: the luminous intensity of luminous bacteria in the solution without toxic substances is increased;
Kt: and (3) the luminous intensity of the luminous bacteria in the solution to be detected.
As can be seen from FIG. 5, the luminescence intensity of the luminescent bacteria Fc-11-1 is obviously inhibited by several solutions, wherein the luminescence inhibition rates of furazolidone and its metabolites and chloramphenicol to the luminescent bacteria Fc-11-1 are higher than those of erythromycin and penicillin, when the concentration reaches 100 μ g/L, the luminescence inhibition rate of furazolidone and its metabolites can reach 55%, the luminescence inhibition rate of chloramphenicol to its metabolites exceeds 45%, but the luminescence inhibition efficiency of 10 μ g/L chloramphenicol is obviously higher than that of other antibiotics, with the increase of the concentration, the inhibition rates are gradually increased, but the increase range is lower than that of furazolidone and its metabolites, and when the concentration of antibiotics is increased to 100 μ g/L, the luminescence inhibition rates of the three antibiotics are basically equal. The inhibition rate of the erythromycin and the penicillin is over 20 percent. Even erythromycin and penicillin with the concentration of 10 mug/L can inhibit the luminous efficiency by nearly 10 percent, and show better sensitivity. Therefore, the luminous bacteria Fc-11-1 is suitable for the detection of antibiotics and metabolites thereof.
Reference to the literature
[1].Aide Lasa,Ana L.Dieguez,Jesus L.Romalde，Vibrio toranzoniaesp.nov.,a new member of the Splendidus clade in the genus Vibrio[J].Systematic and Applied Microbiology,2013,(36):96-100.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
Inspection and quarantine technology center of < 110 > cigarette table entry and exit inspection and quarantine bureau
Luminous bacterium strain of < 120 >, separation and identification method and application thereof
〈160〉1
〈170〉Patent In version 3.5
〈210〉1
〈211〉1471
〈212〉DNA
〈213〉Vibrio toranzoniae
〈400〉1
CTAACACATGCAAGTCGAGCGGTAACGACACTAACAATCCTTCGGGTGCGTTAATGGGCGTCGAGCGGCGGACGGGTGAGTAATGCCTAGGAAATTGCCTTGATGTGGGGGATAACCATTGGAAACGATGGCTAATACCGCATGATGCCTACGGGCCAAAGAGGGGGACCTTCGGGCCTCTCGCGTCAAGATATGCCTAGGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGTTGTGAGGAAGGGGGTAACGTTAATAGCGCTATCTCTTGACGTTAGCAACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCATGCAGGTGGTTCATTAAGTCAGATGTGAAAGCCCGGGGCTCAACCTCGGAACTGCATTTGAAACTGGTGAACTAGAGTACTGTAGAGGGGGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCAGTGGCGAAGGCGGCCCCCTGGACAGATACTGACACTCAGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTTGGAGGTTGTGGCCTTGAGCCGTGGCTTTCGGAGCTAACGCGTTAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAATCCAGCGGAGACGCAGGAGTGCCTTCGGGAGCTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTGTTTGCCAGCGAGTCATGTCGGGAACTCCAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAGAGGGCAGCGAGCCAGCGATGGTAAGCGAATCCCAAAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCAAAAGAAGTAGGTAGTCTAACCTTCGGGAGGACGCTTACCACTTTGTGGTTCATGACTGGGGTGAAGTCGTAACAAGGTA 1471
Claims (1)
1. An application of luminous bacteria in detecting sensitivity characteristics of antibiotics and metabolites thereof is disclosed, wherein the luminous bacteria is luminous bacteria Fc-11-1, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.13424, and is classified and named as: vibriotorazoniae; the antibiotic is one of furazolidone, chloramphenicol, penicillin and erythromycin, and the antibiotic metabolite is furazolidone metabolite.
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| CN102071159A (en) * | 2009-11-20 | 2011-05-25 | 中国海洋大学 | Detection of environmental pollutants through photobacterium leiognathi |
| CN101560491B (en) * | 2008-04-15 | 2012-01-04 | 中国科学院上海生命科学研究院 | Luminous bacteria and application thereof in detecting general biological toxicity in food or water sample |
| CN105733982A (en) * | 2016-02-24 | 2016-07-06 | 青岛农业大学 | Bacillus amyloliquefaciens used for preventing blueberry lasiodiplodia theobromae branch withering and inoculant and preparation method thereof |
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| CN101560491B (en) * | 2008-04-15 | 2012-01-04 | 中国科学院上海生命科学研究院 | Luminous bacteria and application thereof in detecting general biological toxicity in food or water sample |
| CN102071159A (en) * | 2009-11-20 | 2011-05-25 | 中国海洋大学 | Detection of environmental pollutants through photobacterium leiognathi |
| CN105733982A (en) * | 2016-02-24 | 2016-07-06 | 青岛农业大学 | Bacillus amyloliquefaciens used for preventing blueberry lasiodiplodia theobromae branch withering and inoculant and preparation method thereof |
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