CN101067600A - Aquatic pathogenic bacterium colony induction signaling molecule detecting method and special reagent kit thereof - Google Patents
Aquatic pathogenic bacterium colony induction signaling molecule detecting method and special reagent kit thereof Download PDFInfo
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Abstract
The invention discloses an examination method of aquatic product disease germ community induce signal member and its special-purpose reagent box. The method including carrying on the following experiment of the received test fungus: 1) adds X-Gal or ONPG on the plate with the root cancer farming bacillus together streak cultivation, observes whether demonstrates the blue color; 2) with the purple bacillus CV026 on the identical plate streak cultivation, observes whether demonstrates the purple color; 3) puts the received test fungus score inoculate on the LB plate which contains 500nM N-acylation homoserine lactone cultivated, observes whether demonstrated the purple discoloration will be shallow or removes; 4) draws a line together with Esc.coli pSB403 on the plate cultivated, observes whether shines; in above experiment, if at least an experiment demonstrates the masculine gender, then the received test fungus produces the N- acylation homoserine lactone. The invention method has high accurate rate, have no false negative phenomenon, easy to qualitative and quantitative analysis, operation simple, cost low, rapid to obtain the result, suits the large-scale high efficiency examination.
Description
Technical field
The present invention relates to a kind of method for quick and dedicated kit thereof of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule.
Background technology
China every year by aquaculture economic loss that bacterial disease caused up to 50,000,000,000 yuan, aquatic pathogenic bacterium is mainly Gram-negative bacteria (Jiang Lan, Deng. Chinese aquatic science .9 (2): 152-156,2002), the common Vibrio anguillarum that comprises, Aeromonas salmonicida, Aeromonas hydrophila, Yersiniaruckeri, Vibrio salmonicida, Vibrio vulnificus, (Jesper B.B.et al.Dis Aquat Org.65:43-52 such as Aeromonas salmonicida and Vibrio splendidus, 2005), these pathogens are all relevant with water environment, should distinguish (the Gao Yijing that comes with the vegetalitas pathogen that present research is more, Agricultural University Of Nanjing's master thesis, in give birth to the influence of the quorum sensing system of magnificent certain herbaceous plants with big flowers rhizobium slowly, 2006 to its growth and symbiosis dross).Conventional chemical medicament and antibiotic use press for exploitation new controlling way and medicine owing to have environmental pollution, food security hidden danger, inducible resistance bacterial strain equivalent risk in the aquaculture practices.
1994, Fuqua etc. propose " quorum sensing (quorum sensing; write a Chinese character in simplified form QS) " speech first and describe the molecule communication of bacterial cell particular form, be that bacterium produces signaling molecule and is discharged in the environment and goes, the expression of the specific gene that induction cell densities is trusted after the signaling molecule in the environment reaches certain concentration, thereby represent the behavioural characteristic (Fuqua W.G.et al., Journal of Bacteriology.176:269-275,1994) that makes new advances.Many functions by the cell-intercellular signal scalable bacterium of signaling molecule mediation, as antibiotic biosynthesizing, the generation of virulence factor, plasmid in conjunction with shift to wait (Chen Feng. Shanghai Agricultural journal .21 (1): 98-102,2005), therefore, the quorum sensing approach becomes the new breakthrough mouth of biological control and treatment bacterial disease, provide new target spot (Lars R.et al.Journalof Microbiological Methods.44:239-251 for solving the drug resistance problem that is on the rise because of the antibiotic abuse of tradition at present, 2001), also be present international hot research subject under discussion.For example: by more synthetic colony induction signaling molecule analogues; combine with corresponding receptor protein is competitive; can produce halogenation furanone (halogenated furanones) a kind of and signaling molecule (N-acidylate homoserine lactone) similar as marine red alga (Delisea pulchra); this material is cultivated the degraded that can promote LuxR (a kind of signaling molecule receptor protein) with Fei Shi vibrios V.fischeri; therefore and destroy its QS behavior (Haviv Y.S.et al.Cancer Res.62 (15): 4273-4281; 2002), thus reach the purpose of disease control.
For Gram-negative (G-) bacterium; most this class signaling molecule all belongs to N-acidylate homoserine lactone (Acylated Homoserine Lactone; AHL); its structure contains the acyl side-chain of homoserine lactone ring and 4-14 carbon; the 3rd carbon atom of side chain replaced by oxygen or hydroxyl; or be not substituted; in other words; the key distinction between them is the length of N-side chain; or the substituent difference of 3-carbon location; or have or not one or more unsaturated links (Camara M.et al.Methods in Microbiol.27:319-330,1998) in the side chain.For detection and the evaluation of AHLs, be the basis of carrying out the aquatic pathogenic bacterium Control Study by the quorum sensing approach.
Because the concentration of colony induction signaling molecule is extremely low, general means can't detect.Adopted microorganism to diffusivity signaling molecule sensitivity as the biological detection bacterium in recent years, making quick bio to signaling molecule detect becomes possibility.The bacterium that majority is used for biological detection be can't self synthetic AHL mutant strain, and after external source AHL adds, just can show wild type phenotype (Chen Feng. Shanghai Agricultural journal .21 (1): 98-102,2005).Yet, more single (the Zhan Kehang of biological reporting system that has delivered, Deng. Chinese Medical Journal .86 (37): 2655-2658,2006), on sensing range and accuracy rate, all there is certain limitation, and can occur false-negative report (Yang Menghua, etc. microorganism journal .46 (6): 1007-1010,2006).The evaluation of relevant aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule does not appear in the newspapers as yet.
Summary of the invention
In order to address the above problem, the purpose of this invention is to provide a kind of method for quick and dedicated kit thereof of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule.
The quick detection kit of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule provided by the invention comprises Agrobacterium tumefaciems (Agrobac terium tumefaciens) A136, chromabacterium biolaceum (Chromobac terlerviolaceum) CV026, N-acidylate homoserine lactone, 5-bromo-4-chloro-3-indyl-β-D-galactopyranoside (X-Gal) or ortho-nitrophenol β-D-galactoside (ONPG) and Escherichia coli (Escherichia coli) pSB403.
More accurately credible for the testing result that makes described kit, described kit also comprises positive control bacterial strain liquefied Serratia (Serratia Liquefaciens) MG1, Pseudomonas aeruginosa (Pseudomonas aeruginosa) PA01 and Fei Shi vibrios (Vibrio fischeri) MJ1.
Described N-acidylate homoserine lactone can be 3-OH-C6-HSL, 3-OH-C8-HSL, 3-OH-C10-HSL, 3-Oxo-C10-HSL, C8-HSL, 3-Oxo-C8-HSL, 3-Oxo-C12-HSL, 3-Hydroxy-7-C14-HSL, C4-HSL, 3-Hydroxy-C4-HSL and 3-OH-C12-HSL (3-carboxyl 12 carbon-N-1-homoserine lactone, OHHL) one or more combination in any in.
Gram-negative pathogen provided by the invention produces the method for quick of colony induction signaling molecule, comprises and is carried out four experiments as described below with trying bacterium:
1) tried bacterium and reporting bacterial strain Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 common line on the same solid plate that has added X-Gal or ONPG and cultivated described, observe Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 thalline and whether show blueness, if it is show that blueness is promptly positive, otherwise negative;
2) tried bacterium and reporting bacterial strain chromabacterium biolaceum (Chromobacterler violaceum) CV026 common line on same solid plate and cultivated described, observe chromabacterium biolaceum (Chromobacterler violaceum) CV026 thalline and whether show purple, if it is show that purple is promptly positive, otherwise negative;
3) tried the bacterium streak inoculation and on the LB flat board that contains 500nM N-acidylate homoserine lactone, cultivated 24-48 hour described, again reporting bacterial strain chromabacterium biolaceum (Chromobac terler violaceum) CV026 is being tried the other parallel scribing of bacterium line, observe chromabacterium biolaceum (Chromobacterler violaceum) CV026 thalline and whether show that purple shoals or takes off, if show purple shoal or take off promptly positive, otherwise negative;
4) tried bacterium and reporting bacterial strain Escherichia coli (Escherichia coli) pSB403 common line on solid plate and cultivated described, whether observe Escherichia coli (Escherichia coli) pSB403 thalline luminous, if it is luminous promptly positive, otherwise negative;
In above-mentioned four experiments; if it is positive to have at least an experiment to show; judge that then being tried bacterium produces N-acidylate homoserine lactone (AHLs) colony induction signaling molecule, otherwise, judge that then being tried bacterium does not produce N-acidylate homoserine lactone (AHLs) colony induction signaling molecule.
Described N-acidylate homoserine lactone can be 3-OH-C6-HSL, 3-OH-C8-HSL, 3-OH-C10-HSL, 3-Oxo-C10-HSL, C8-HSL, 3-Oxo-C8-HSL, 3-Oxo-C12-HSL, 3-Hydroxy-7-C14-HSL, C4-HSL, 3-Hydroxy-C4-HSL and 3-OH-C12-HSL (3-carboxyl 12 carbon-N-1-homoserine lactone, OHHL) one or more combination in any in.
In the described method, also comprise detection is shown that the bacterium of being tried that produces the AHLs colony induction signaling molecule cultivates in a large number, extract the AHLs signaling molecule, react, carry out qualitative detection in conjunction with thin-layered chromatography with this signaling molecule and the reporting bacterial strain that is tried the bacterium reacting positive.
In the described method, comprise that also the AHLs signaling molecule with described extraction utilizes the biological diffusion method of well formula to measure the AHLs activity.
Being tried the used nutrient culture media of a large amount of cultivations of bacterium is the ABT nutrient culture media, and described ABT nutrient culture media is for containing 0.4g/L (NH
4)
2SO
4, 0.6g/L Na
2HPO
4, 0.3g/L KH
2PO
4, 0.3g/L NaCl, 1mmol/L MgCl
2, 0.1mmol/LCaCl
2, 0.01mmol/L FeCl
3, 2.5mg/L thiamines, quality percentage composition be that 0.5% glucose and quality percentage composition are the nutrient culture media of 0.5% acid hydrolysis casein; The method of described extraction AHLs signaling molecule is to be used in to add ethyl acetate extraction in the supernatant that is tried bacteria culture fluid and obtain AHLs.
Described thin-layered chromatography will be tried AHLs extract and the standard items point sample on chromatographic sheet that bacterium produces, launch with methanol solution, the reporting bacterial strain that utilizes then and tried the bacterium reacting positive detects its variable color or luminescence sites, determines specifically to be tried the AHL that bacterium produces; When above-mentioned experiment 1) during reacting positive, the standard items that described thin-layered chromatography detects are 3-OH-C6-HSL, 3-OH-C8-HSL, 3-OH-C10-HSL and 3-Oxo-C10-HSL etc.; When above-mentioned experiment 2) during reacting positive, the standard items that described thin-layered chromatography detects are C4-HSL, 3-Hydroxy-C4-HSL etc.; When above-mentioned experiment 3) during reacting positive, the standard items that described thin-layered chromatography detects are C8-HSL, 3-Oxo-C8-HSL, 3-Oxo-C12-HSL, 3-Hydroxy-7-C14-HSL etc.; When above-mentioned experiment 4) during reacting positive, the standard items that described thin-layered chromatography detects are 3-carboxyl 12 carbon-N-1-homoserine lactone (OHHL).
In the described method, described to be tried bacterium be from sick fish and/or breeding water body and/or culture to separate the bed mud and obtain.
In the described method, described solid plate is a solid LB flat board; Described to be tried bacterium and the reporting bacterial strain cultured method of ruling jointly be to make to be tried bacterium and reporting bacterial strain is cultivated on same flat board, and the bacterium colony distance is below the 1cm, and described method is preferably two strain bacterium parallel scribings; The described temperature of being tried bacterium and reporting bacterial strain co-incubation is 25 ℃, and the time of cultivation is 24 hours.
The concentration of X-Gal is 30-80 μ g/ml in the described solid plate that adds X-Gal, is preferably 50.0 μ g/ml; The concentration of ortho-nitrophenol β-D-galactoside (ONPG) is 40-80 μ g/ml in the described solid plate that adds ortho-nitrophenol β-D-galactoside (ONPG), is preferably 60 μ g/ml; The concentration of having added N-acidylate homoserine lactone in the solid plate of N-acidylate homoserine lactone is 400-600nmol/L, is preferably 500nmol/L.
In the described method, also comprise and described four control experiments that experiment is carried out synchronously, in described four experiments, all rule separately and cultivate negative contrast with described reporting bacterial strain; Described 1) and 3) in, with Pseudomonas aeruginosa (P.aeruginosa) PA01 rule separately cultivate or with tried bacterium and rule jointly and cultivate positive contrast, described 2) in, with liquefied Serratia (Serratia Liquefaciens) MG1 rule separately cultivate or with tried bacterium and rule jointly and cultivate positive contrast, described 4) in, with Fei Shi vibrios (Vibrio fischeri) MJ1 rule separately cultivate or with tried bacterium and rule jointly and cultivate positive contrast.
Three strain reporting bacterial strains of the present invention:
One of reporting bacterial strain Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 contains traG:lacZ fusion and traR gene (Fuqua C.and Winans S.C.J Bacteriol.178:435-440,1996).Itself does not produce AHLs, external source AHLs can induce lacZ gene great expression, and hydrolysis X-Gal, ONPG substrates such as (ortho-nitrophenol β-D-galactoside) produces obvious color and changes (Shaw P.D.et al., ProcNatl Acad Sci USA.94:6036-6041,1997).It is wider that this report bacterial strain detects spectrum, and HSL such as most Oxo-, Hydroxy-, Unsubstituted are detected sensitivity, but to insensitive (Shaw P.D.et al.Proc Natl Acad Sci USA.94:6036-6041,1997) such as C4-HSL.
Two chromabacterium biolaceums of reporting bacterial strain (Chromobacterler violaceum) CV026, the ability of its generation purple pigment is regulated and control by AHLs, the functional gene of the synthetic AHLs of this bacterium disappearance, can not synthesize AHLs, do not produce purple pigment, and dividing the period of the day from 11 p.m. to 1 a.m as external source AHLs, its restriction enzyme CviR produces purple pigment (Throup J.P.etal.Bioluminescence and Chemi luminescence:Fundamental and Appl ied Aspects.89-92,1995).This report bacterium mainly detects responsive (McClean K.H.et al., Microbiology.143:3703-3705,1997) to short chain or medium chain Alkanoyl-AHLs; Be the colour developing state and (add external source AHLs, as N-hexanoyl-L-homoserine lactone (HHL)) chromabacterium biolaceum (Chromobac terler violaceum) CV026, because some long-chain AHLs can suppress restriction enzyme CviR, so the violacein amount that is accumulated when adding long-chain AHLs in the nutrient culture media will less (Throup J.P.et al.Bioluminescence andChemiluminescence:Fundamental and Applied Aspects.89-92,1995).
Three Escherichia coli of reporting bacterial strain (Escherichia coli) pSB403, make up based on lux R, self can not secrete 3-carboxyl 12 carbon-N-1-homoserine lactone (OHHL), and under external source OHHL condition, can bring out bioluminescence (Lars R.et al.Journal of Microbiological Methods.44:239-251,2001).
Experiment showed, that method of the present invention detects aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule (AHLs) accuracy rate height, do not have false negative phenomenon, can carry out the qualitative and quantitative analysis.The method utilization of the present invention in addition cultured method of ruling jointly detects, and easy and simple to handle, cost is low, and it is rapid to obtain the result, is fit to extensive high-level efficiency and detects.Operation that the kit of the special use that bacterial strain that the method according to this invention is all and material are made is more convenient.
Embodiment
Method among the following embodiment if no special instructions, is conventional method, and all experiments are all carried out under aseptic condition.
Percentage composition described in the following embodiment if no special instructions, is the quality percentage composition.
Method of the present invention, with following three kinds of reporting bacterial strains through being combined into detection system:
Reporting bacterial strain one:
Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 (available from Oxoid, West Heidelberg, Victoria, Australia).
Reporting bacterial strain two:
Chromabacterium biolaceum (Chromobacterler violaceum) CV026 (Hach Company, Loveland, Colo.USA).
With chromabacterium biolaceum (Chromobacterler violaceum) CV026 that is colour developing state (add HHL) (HachCompany, Loveland, Colo.USA).
Reporting bacterial strain three:
Escherichia coli (Escherichia coli) pSB403 (D IFR, TUD, DK-2800Kgs Lyngby, Denmark).
The method for quick of embodiment 1, a kind of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule
1, the preparation of the quick detection kit of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule
1) cultivation of reporting bacterial strain
With LB solid medium (1% peptone of above-mentioned reporting bacterial strain Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 at interpolation 4.5 μ g/ml (final concentration) tetracyclines, 50.0 μ g/ml (final concentration) miramycins, 0.5% yeast extract, 0.5%NaCl, 1.2% agar) go up line, cultivated 1 day at 25 ℃;
Chromabacterium biolaceum (Chromobac terler violaceum) CV026 is rule on the LB solid medium that adds 20.0 μ g/ml (final concentration) kanamycins, cultivated 1 day at 25 ℃;
Escherichia coli (Escherichia coli) pSB403 is added the LB solid medium (1% peptone, 0.5% yeast extract, 0.5%NaCl, 1.2% agar) of tetracycline 20.0 μ g/ml (final concentration) and go up line, cultivated 1 day at 25 ℃.
2) cultivation of positive control bacterial strain
Positive control bacterial strain liquefied Serratia (Serratia Liquefaciens) MG1 (DIFR, TUD, DK-2800Kgs Lyngby, Denmark), Pseudomonas aeruginosa (Pseudomonas aeruginosa) PA01 (DIFR, TUD, DK-2800 Kgs Lyngby, Denmark), Fei Shi vibrios (Vibrio fischeri) MJ1 (DIFR, TUD, DK-2800Kgs Lyngby is Denmark) respectively at LB solid medium (1% peptone, 0.5% yeast extract, 0.5%NaCl, 1.2% agar) go up line, cultivated 1 day at 25 ℃.
3) preparation of the quick detection kit of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule
Reporting bacterial strain (streak plate of each bacterial strain), step 2 that step 1) is cultivated) positive control bacterial strain (streak plate of each bacterial strain), the 20mg/ml X-Gal solution cultivated, (N-hexanoyl-L-homoserine lactone, HHL) 1 μ g is combined into matching used quick detection kit for 5ml and N-hexanoyl homoserine lactone.
Preserve for bacterial strain is convenient, wherein, the also available liquid LB of each bacterial strain nutrient culture media 25 ℃ shake train to 0D value for behind the 0.2-0.6, collect bacterium liquid, adding volumn concentration is the glycerine of 20% (final concentration), makes things convenient for low temperature (80 ℃) preservation.Each reagent in the kit also can be regulated according to consumption.
2, the fast detecting of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule
1) separation of aquatic products Gram-negative pathogen and cultivation
From suffering from the river line jenoar (Lutjanus sebae) of explosive vibriosis, take from Zhanjiang City, Guangdong Province East Sea Island net cage for sea farming, the same document of separation method (Mo Zhaolan etc. microorganism journal .42 (3): 263-269,2002), the result obtains a strain Gram-negative pathogen, through morphological feature, on the growth conditions, Physiology and biochemistry, 16S rRNA sequential analysis (Mo Zhaolan etc. microorganism journal .42 (3): 263-269,2002), with the monoclonal antibody immunity fluorescent technique carry out the serotype Analysis and Identification (Yu Junhong etc. marine fishery research .23 (2): 38-44,2002), the result shows that separating the Gram-negative pathogen that obtains is that Vibrio anguillarum (Vibrio anguillarum) 01 (can be available from Oxoid, West Heidelberg, Victoria, Austral ia).
This pathogenic strains Vibrio anguillarum (Vibrio anguillarum) 01 is seeded in ABT nutrient culture media (1L ABT component content: 0.4g (NH
4)
2SO
4, 0.6g Na
2HPO
4, 0.3g KH
2PO
4, 0.3g NaCl, 1mM MgCl
2, 0.1mMCaCl
2, 0.01mM FeCl
3, the 2.5mg thiamines contains 0.5% glucose and 0.5% acid hydrolysis casein) on, cultivated 1 day at 25 ℃, standby as being tried bacterium.
In addition, we are separated to Aeromonas hydrophila (Aeromonashydrophila) C2 from the cultivating pool bed mud of Zhejiang Jiaxing area (can be available from Oxoid, West Heidelberg, Victoria, Australia), being separated to Aeromonas hydrophila (Aeromonas hydrophila) C3 in the cultivating pool grass carp intestinal of Zhejiang Jiaxing area (can be available from Oxoid, West Heidelberg, Victoria, Australia), being separated to aeromonas salmonicida (Aeromonas salmonicida) T01 from Tianjin Tilapia mossambica body surface (can be available from Oxoid, West Heidelberg, Victoria, Australia) (separating and authentication method of separation and the same Vibrio anguillarum of authentication method (Vibrio anguillarum) 01).
2) fast detecting of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule (AHLs)
Utilize the kit of step 1 preparation or directly use bacterial strain and the reagent that needs, tried pathogen with the detection of parallel scribing method and whether produce AHLs, concrete operations are as follows:
What 1. step 1) is obtained is tried bacterium (Vibrio anguillarum (Vibrio anguillarum) 01, Aeromonas hydrophila (Aeromonas hydrophila) C2, Aeromonas hydrophila (Aeromonas hydrophila) C3 or aeromonas salmonicida (Aeromonas salmonicida) T01) respectively with reporting bacterial strain chromabacterium biolaceum (Chromobacterlerviolaceum) CV026 parallel scribing on 3.5cm LB flat board, make the distance between two strain bacterium bacterium colonies be no more than 1cm, liquefied Serratia (Serra tia Liquefaciens) MG1 as the positive control bacterium separately 3.5cm LB dull and stereotyped with the reporting bacterial strain parallel scribing, chromabacterium biolaceum (Chromobacterler violaceum) CV026 separately on 3.5cm LB flat board parallel scribing as negative control the flat board of line is all cultivated 24h at 25 ℃.After the cultivation, observe the change color of thalline on the nutrient culture media.Whether record chromabacterium biolaceum (Chromobac terler violaceum) CV026 thalline shows purple, if show that purple is promptly positive, otherwise it is negative not show purple;
What 2. step 1) is obtained is tried bacterium (Vibrio anguillarum (Vibrio anguillarum) 01, Aeromonas hydrophila (Aeromonas hydrophila) C2, Aeromonas hydrophila (Aeromonas hydrophila) C3 or aeromonas salmonicida (Aeromonas salmonicida) T01) streak inoculation is in containing 500nM N-hexanoyl homoserine lactone (N-hexanoyl-L-homoserine lactone respectively, HHL) on the LB flat board, cultivate 24h for 25 ℃, again reporting bacterial strain chromabacterium biolaceum (Chromobac terler violaceum) CV026 is being tried the other parallel scribing of bacterium line respectively, make the distance between two strain bacterium bacterium colonies be no more than 1cm, continue at 25 ℃ then and cultivate 24h down.Pseudomonas aeruginosa (Pseudomonas aeruginosa) PA01 is as positive control, chromabacterium biolaceum (Chromobac terlerviolaceum) CV026 separately behind parallel scribing on the 3.5cm LB flat board 25 ℃ cultivate 48h as negative control, observe the change color of thalline on the nutrient culture media.Whether record chromabacterium biolaceum (Chromobacterler violaceum) CV026 thalline shows that purple shoals or takes off, if show purple shoal or take off promptly positive, if instead purple does not shoal or takes off negative;
3. step 1) is obtained tried bacterium (Vibrio anguillarum (Vibrio anguillarum) 01, Aeromonas hydrophila (Aeromonas hydrophila) C2, Aeromonas hydrophila (Aeromonas hydrophila) C3 or aeromonas salmonicida (Aeromonas salmonicida) T01) respectively with Escherichia coli (Escherichia coli) pSB403 parallel scribing on the LB flat board, make the distance between two strain bacterium bacterium colonies be no more than 1cm, cultivate 24h for 25 ℃.Fei Shi vibrios Vibrio fischeriMJ1 cultivates 24h for 25 ℃, as the positive control bacterium separately 3.5cm LB dull and stereotyped with the reporting bacterial strain parallel scribing.Escherichia coli (Escherichia coli) pSB403 separately behind parallel scribing on the 3.5cm LB flat board 25 ℃ cultivate 24h as negative control.Take and the record biological luminescence by photographic camera.Whether record Escherichia coli (Escherichia coli) pSB403 thalline is luminous, if luminous promptly positive, if instead not luminous negative.
4. the strain subject that step 1) is obtained (Vibrio anguillarum (Vibrio anguillarum) 01, Aeromonas hydrophila (Aeromonas hydrophila) C2, Aeromonas hydrophila (Aeromonas hydrophila) C3 or aeromonas salmonicida (Aeromonas salmonicida) T01) and Agrobacterium tumefaciems (Agrobacteriumtumefaciens) A136 parallel scribing on the LB flat board that contains 50.0 μ g/ml X-Gal, make the distance between two strain bacterium bacterium colonies be no more than 1cm, cultivate 24h for 25 ℃.Pseudomonas aeruginosa (Pseudomonas aeruginosa) PA01 cultivates 24h for 25 ℃, as the positive control bacterium separately 3.5cm LB dull and stereotyped with the reporting bacterial strain parallel scribing; Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 separately behind parallel scribing on the 3.5cm LB flat board 25 ℃ cultivate 24h as negative control.Observe the change color of thalline on the nutrient culture media, whether record Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 thalline shows blueness, if show that blueness is promptly positive, on the contrary negative.
In the above-mentioned detection; had at least one to detect the demonstration positive if try bacterium; judge that then this is tried bacterium and produces N-acidylate homoserine lactone (AHLs) colony induction signaling molecule, otherwise, judge that then being tried bacterium does not produce N-acidylate homoserine lactone (AHLs) induction signal molecule.Wherein, 3. step detects through judgement and has luminescence-producing reaction, can conclude directly that AHLs is OHHL, needs further to identify for other reaction.
Above-mentioned test result shows, above-mentioned bacterium Vibrio anguillarum (Vibrio anguillarum) 01 and the Agrobacterium tumefaciems A136 performance obvious blue tried, all the other reactions that are negative prove that there is colony induction signaling molecule AHLs in Vibrio anguillarum 01, and its characteristic reporting bacterial strain is Agrobacterium tumefaciems A136.And aquatic products gram negative pathogenic bacteria AHLs commonly used at present only relies on chromabacterium biolaceum (Chromobacterler violaceum) CV026 and chromabacterium biolaceum (Chromobacterler violaceum) the CV026 dual test that is colour developing state (adding HHL), testing result is negative like this, then misjudgment.Whole above-mentioned bacterium testing result such as following tables 1 tried, the single detection system of invisibility does not cause false negative result easily, and physical detection means need detect by high-efficient liquid phase chromatogram technology, the sample purifying requires high, when doing qualitative analysis, also need by instrument and equipments such as mass spectrum and nuclear magnetic resonance, inappropriate extensive evaluation is used (Bruhn et al.Dis Aquat Org.65:43-52,2005) with screening.
Table 1 difference is tried the water produces the pathogenic bacterium colony induction signaling molecule testing result
| Tried bacterium | Testing result | |||
| C. violaceum CV026 | C. violaceum CV026+HHL | Escherichia coli pSB403 | A. tumefaciens A136 | |
| V.anguillarum 01 | - | - | - | + |
| A.hydrophila C2 | - | + | - | - |
| A.hydrophila C3 | + | - | + | - |
| A.salmonicida T01 | - | - | + | - |
In the table ,+expression reacting positive; The no significant reaction of-expression, feminine gender.
3, AHLs extracts
With above-mentioned strain subject (Vibrio anguillarum (Vibrio anguillarum) 01, Aeromonas hydrophila (Aeromonashydrophila) C2, Aeromonas hydrophila (Aeromonas hydrophila) C3 or aeromonas salmonicida (Aeromonas salmonicida) T01) be inoculated in the ABT nutrient culture media respectively, under 25 ℃, 150rpm, cultivate 24h.The centrifugal 30s of 8000g, get supernatant 10ml respectively, add isopyknic ethyl acetate (add final concentration in addition and be 0.5% formic acid carry out acidifying), the 30s that fully vibrates, triplicate, the ethyl acetate that adds 10ml after the absorption organic phase again carries out same treatment, repeat twice, three liquid draws merged, in Rotary Evaporators evaporate to dryness and Hui Rong to 2ml in acidifying ethyl acetate, once more behind the evaporate to dryness molten respectively to 500 μ l in acidifying ethyl acetate, preserve down standby for-20 ℃.
4, the biological diffusion method of well formula is measured the AHLs activity
By measuring the AHLs activity with the reporting bacterial strain that is tried the apparent positive of bacterium co-incubation.Concrete grammar is as described below:
1) contain the flat board preparation of reporting bacterial strain:
Biological reporting bacterial strain (Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136), chromabacterium biolaceum (Chromobacterler violaceum) CV026, Escherichia coli (Escherichia coli) pSB403 are inoculated into reporting bacterial strain Agrobacterium tumefaciems A136 respectively in adding antibiotic LB nutrient solution, under 25 ℃ of aerobic conditions, cultivate 24h and obtain nutrient solution.Wherein, Agrobacterium tumefaciems A136 microbiotic is 4.5 μ g/ml (final concentration) tetracyclines and 50.0 μ g/ml (final concentration) miramycins; Chromabacterium biolaceum (Chromobacterler violaceum) CV026 microbiotic is 20.0 μ g/ml (final concentration) kanamycins; Escherichia coli (Escherichia coli) pSB403 microbiotic 20.0 μ g/ml (final concentration) tetracyclines.The nutrient solution that obtains is respectively applied for following processing:
Getting 1ml Agrobacterium tumefaciems A136 nutrient solution is inoculated in the 50ml ABT nutrient solution, under 25 ℃ of aeration conditions, cultivate 24h, then nutrient solution is all poured into to be equipped with in the triangular flask of ABT nutrient culture media (liquid) that 46 ℃ of 100ml are added with 1.2% agar and 75 μ g/ml X-gal and shaken up, will pour the flat board that obtains containing Agrobacterium tumefaciems A136 in the double dish into by the amount of 20ml/ ware rapidly again.
Get the LB nutrient solution of 2 parts of 1ml chromabacterium biolaceums (Chromobac terler violaceum) CV026, insert respectively in the 50ml LB nutrient solution, under 25 ℃ of aeration conditions, cultivate 24h, then nutrient solution is poured into respectively be equipped with 46 ℃ of 100ml add the LB nutrient culture media (liquid) of 1.2% agar or add 1.2% agar and the triangular flask of the LB nutrient culture media of 75nMN-hexanoyl-L-homoserine lactone (HHL) (liquid) in shake up, will pour the flat board that obtains containing the dull and stereotyped of chromabacterium biolaceum (Chromobacterlerviolaceum) CV026 in the double dish or contain chromabacterium biolaceum (Chromobacterler violaceum) CV026 and HHL into by the amount of 20ml/ ware rapidly again.
Get 1ml Escherichia coli (Escherichia coli) pSB403LB nutrient solution, insert in the 50ml LB nutrient solution, under 25 ℃ of aeration conditions, cultivate 24h, then nutrient solution is poured into to be equipped with in the triangular flask of LB nutrient culture media (liquid) that 46 ℃ of 100ml add 1.2% agar and shaken up, will pour the flat board that obtains containing Escherichia coli (Escherichia coli) pSB403 in the double dish into by the amount of 20ml/ ware rapidly again.
All be added with corresponding microbiotic in above-mentioned all nutrient culture media, wherein, Agrobacterium tumefaciems (Agrobac teriumtumefaciens) A136 microbiotic is 4.5 μ g/ml (final concentration) tetracyclines and 50.0 μ g/ml (final concentration) miramycins; Chromabacterium biolaceum (Chromobac terler violaceum) CV026 microbiotic is 20.0 μ g/ml (final concentration) kanamycins; Escherichia coli (Escherichia coli) pSB403 microbiotic 20.0 μ g/ml (final concentration) tetracyclines.
2) point sample is measured: with above-mentioned steps 1) flat board that obtains, sting into 6mm aperture (point sample hole), the AHLs extract (A that is respectively tried bacterium that step 3 is obtained in the center
iConcentration C
i) or synthetic HHLs (Sigma; The maximum composition AHL that selection is undertaken judging when the AHLs fast qualitative detects by step 5 is as active examination criteria thing, and the active examination criteria thing of AHLs extract that is respectively tried bacterium is specifically as shown in table 2) (A
oConcentration C
o=1500nM) (contrast) point sample is in showing in the aperture of positive flat board with its reaction respectively, the point sample amount is respectively 60 μ l.Behind the point sample, measure variable color loop diameter (R after respectively flat board being placed 25 ℃ to cultivate 48h
i(AHLs extract variable color loop diameter) and R
o(synthetic HHLs variable color loop diameter), mm).
Then: activity (concentration) computing formula of respectively being tried the AHLs extract of bacterium is C
i(nM)=C
o(nM) * R
i(mm)/R
o(mm) testing result is as shown in table 2.
Table 2. difference is tried the water produces pathogenic bacterium colony induction signaling molecule AHL composition with active
| Tried bacterium | TLC examination criteria thing (HSL) | TLC result (HSL) | Active examination criteria thing | Dull and stereotyped contained report bacterium of active and qualitative detection and auxiliary element | AHL activity (nM) |
| V. anguillar um 01 | 3-OH-C6、 3-OH-C8、 3-OH-C10、 3-Oxo-C10 | 3-OH-C6、 3-Oxo-C10 | 3-OH-C6 | A. tumefaciens A136 | 1230. 0 |
| A. hydrophil a C2 | C8， 3-Oxo-C8， 3-Oxo-C12， 3-Hydroxy-7-C 14 | 3-Oxo-C8、 3-Hydroxy-7-C 14 | 3-Hy dr oxy-7 -C14 | C.violaceum CV026+HHL | 870.2 |
| A. hydrophil a C3 | C4， 3-Hydroxy-C4， OHHL | C4， OHHL | OHHL | Escherichia coli pSB403 | 435.7 |
| A.salmoni cida T01 | OHHL | OHHL | OHHL | Escherichia coli pSB403 | 1895. 0 |
Annotate: HSL is Homoserine Lactone, homoserine lactone; OHHL is 3-carboxyl 12 carbon-N-1-homoserine lactone.
5, the AHLs fast qualitative detects
(TLC) carries out the AHLs qualitative detection by thin-layer chromatography:
The signaling molecule that produces according to the reference material Vibrio anguillarum (Vibrio anguillarum) 01 of each strain subject shown in the table 2 makes Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 variable color, and therefore selecting 3-OH-C6-HSL, 3-OH-C8-HSL, 3-OH-C10-HSL and 3-Oxo-C10-HSL (Sigma) is AHLs titer (600pmol); All the other are tried bacterium and are also selected the AHLs reference material according to this principle, and are concrete as being shown in Table 2.
The above-mentioned detection positive of 2 μ l tried bacterium (Vibrio anguillarum (Vibrio anguillarum) 01, Aeromonas hydrophila (Aeromonas hydrophila) C2, Aeromonas hydrophila (Aeromonas hydrophila) C3, aeromonas salmonicida (Aeromonas salmonicida) T01) AHLs extract or AHLs corresponding standard liquid (tried bacterium AHLs extract relevant detection reference material and see Table 2, each 600pmol) difference point sample is at C
18The inverse thin layer chromatography plate (20cm * 20cm, TLC, Darmstadt, Germany) on, each is tried bacterium AHLs extract sample or its titer respectively o'clock at a C
18On the inverse thin layer chromatography plate.Chromatosheet placed respectively in the 10ml methanol solution (60%, volumn concentration) fully launches, wait to launch the forward position near the chromatogram edges of boards along the time (about 4h), with chromatosheet taking-up, dry 10min.
Method according to the step 1) of step 4 prepares the flat board that contains reporting bacterial strain and auxiliary element shown in the table 2, and being laid on point sample respectively has the corresponding AHLs extract of bacterium or the C of AHLs corresponding standard liquid of being tried shown in the table 2
18On the inverse thin layer chromatography plate, after waiting to solidify, place closed container to cultivate 48h for 25 ℃.
The AHLs extract sample luminescence sites of respectively being tried bacterium is compared with the luminescence sites of its titer, find that Vibrio anguillarum (Vibrio anguillarum) 01 AHLs that contains is 3-OH-C6 and 3-Oxo-C10.All the other are tried the same Vibrio anguillarum of bacterium detection method (Vibrio anguillarum) 01, and test result is as shown in table 2.
Claims (10)
1, a kind of quick detection kit of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule comprises Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136, chromabacterium biolaceum (Chromobacterler violaceum) CV026, N-acidylate homoserine lactone, 5-bromo-4-chloro-3-indyl-β-D-galactopyranoside or ONPG and Escherichia coli (Escherichia coli) pSB403.
2, quick detection kit according to claim 1 is characterized in that: described kit also comprises positive control bacterial strain liquefied Serratia (Serratia Liquefaciens) MG1, Pseudomonas aeruginosa (Pseudomonasaeruginosa) PAO1 and Fei Shi vibrios (Vibrio fischeri) MJ1.
3, quick detection kit according to claim 2 is characterized in that: described N-acidylate homoserine lactone is one or more combination in any among 3-OH-C6-HSL, 3-OH-C8-HSL, 3-OH-C10-HSL, 3-Oxo-C10-HSL, C8-HSL, 3-Oxo-C8-HSL, 3-Oxo-C12-HSL, 3-Hydroxy-7-C14-HSL, C4-HSL, 3-Hydroxy-C4-HSL and the 3-OH-C12-HSL.
4, a kind of method for quick of aquatic products Gram-negative pathogenic bacterium colony induction signaling molecule comprises and is carried out four experiments as described below with trying bacterium:
1) tried bacterium and reporting bacterial strain Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 common line on the same solid plate that has added X-Gal or ONPG and cultivated described, observe Agrobacterium tumefaciems (Agrobacterium tumefaciens) A136 thalline and whether show blueness, if it is show that blueness is promptly positive, otherwise negative;
2) tried bacterium and reporting bacterial strain chromabacterium biolaceum (Chromobacterler violaceum) CV026 common line on same solid plate and cultivated described, observe chromabacterium biolaceum (Chromobacterler violaceum) CV026 thalline and whether show purple, if it is show that purple is promptly positive, otherwise negative;
3) tried the bacterium streak inoculation and on the LB flat board that contains 500nM N-acidylate homoserine lactone, cultivated 24-48 hour described, again reporting bacterial strain chromabacterium biolaceum (Chromobacterler violaceum) CV026 is being tried the other parallel scribing of bacterium line respectively, observe chromabacterium biolaceum (Chromobacterler violaceum) CV026 thalline and whether show that purple shoals or takes off, if show purple shoal or take off promptly positive, otherwise negative;
4) tried bacterium and reporting bacterial strain Escherichia coli (Escherichia coli) pSB403 common line on solid plate and cultivated described, whether observe Escherichia coli (Escherichia coli) pSB403 thalline luminous, if it is luminous promptly positive, otherwise negative;
In above-mentioned four experiments,, judge that then being tried bacterium produces N-acidylate homoserine lactone colony induction signaling molecule if it is positive to have at least an experiment to show, otherwise, judge that then being tried bacterium does not produce induction signal molecule.
5, method according to claim 4; it is characterized in that: in the described method; also comprise detection is shown that the bacterium of being tried that produces N-acidylate homoserine lactone colony induction signaling molecule cultivates in a large number, extract N-acidylate homoserine lactone signaling molecule and carry out qualitative detection with thin-layered chromatography.
6, method according to claim 5 is characterized in that: being tried bacterium, to cultivate with nutrient culture media in a large number be the ABT nutrient culture media, and described ABT nutrient culture media is for containing 0.4g/L (NH
4)
2SO
4, 0.6g/L Na
2HPO
4, 0.3g/L KH
2PO
4, 0.3g/L NaCl, 1mmol/L MgCl
2, 0.1mmol/L CaCl
2, 0.01mmol/L FeCl
3, 2.5mg/L thiamines, quality percentage composition be that 0.5% glucose and quality percentage composition are the nutrient culture media of 0.5% acid hydrolysis casein; The method of described extraction N-acidylate homoserine lactone signaling molecule is to be used in to add ethyl acetate extraction in the supernatant that is tried bacteria culture fluid and obtain N-acidylate homoserine lactone.
7, method according to claim 6 is characterized in that: in the described method, described to be tried bacterium be from sick fish and/or breeding water body and/or culture to separate the bed mud and obtain.
8, method according to claim 7 is characterized in that: in the described method, described solid plate is a solid LB flat board; Described to be tried bacterium and the reporting bacterial strain cultured method of ruling jointly be to make the bacterium colony distance of being tried bacterium and reporting bacterial strain for below the 1cm, is preferably two strain bacterium parallel scribings; The temperature of described cultivation is 25 ℃, and the time of cultivation is 24 hours.
9, method according to claim 8 is characterized in that: the concentration of X-Gal is 30-80 μ g/ml in the described solid plate that adds X-Gal, is preferably 50.0 μ g/ml; The concentration of ortho-nitrophenol β-D-galactoside (ONPG) is 40-80 μ g/ml in the described solid plate that adds ortho-nitrophenol β-D-galactoside (ONPG), is preferably 60 μ g/ml; The concentration of having added N-acidylate homoserine lactone in the solid plate of N-acidylate homoserine lactone is 400-600nmol/L, is preferably 500nmol/L; Described N-acidylate homoserine lactone is one or more combination in any among 3-OH-C6-HSL, 3-OH-C8-HSL, 3-OH-C10-HSL, 3-Oxo-C10-HSL, C8-HSL, 3-Oxo-C8-HSL, 3-Oxo-C12-HSL, 3-Hydroxy-7-C14-HSL, C4-HSL, 3-Hydroxy-C4-HSL and the 3-OH-C12-HSL.
10, method according to claim 9 is characterized in that: in the described method, also comprise and described four control experiments that experiment is carried out synchronously, in described four experiments, all rule separately with described reporting bacterial strain and cultivate negative contrast; Described 1) and 3) in, rule separately to cultivate or rule jointly with Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 and cultivate positive contrast with described reporting bacterial strain, described 2) in, rule separately to cultivate or rule jointly with liquefied Serratia (Serratia Liquefaciens) MG1 and cultivate positive contrast with described reporting bacterial strain, described 4) in, rule separately to cultivate or rule jointly with Fei Shi vibrios (Vibrio fischeri) MJ1 and cultivate positive contrast with described reporting bacterial strain.
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