CN105974044B - The method of N- acyl-homoserine lactone types of populations induction signal molecule in test sample - Google Patents
The method of N- acyl-homoserine lactone types of populations induction signal molecule in test sample Download PDFInfo
- Publication number
- CN105974044B CN105974044B CN201610309598.1A CN201610309598A CN105974044B CN 105974044 B CN105974044 B CN 105974044B CN 201610309598 A CN201610309598 A CN 201610309598A CN 105974044 B CN105974044 B CN 105974044B
- Authority
- CN
- China
- Prior art keywords
- film
- gal
- acyl
- semisolid
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention proposes a kind of methods of N- acyl-homoserine lactone types of populations induction signal molecule in test sample; this method comprises: previously prepared semisolid LB culture medium film is placed in the upper surface of TLC chromatographic sheet by (1); contain reporting bacterial strain in the semisolid LB culture medium film, the sample is unfolded on the chromatographic sheet in advance；(2) film is made to carry out chromogenic reaction using X-gal；And (3) based on the chromogenic reaction as a result, determining in the sample whether contain N- acyl-homoserine lactone types of populations induction signal molecule.The experimental result repeatability for the detection method that the embodiment of the present invention is proposed significantly improves, the spot indiffusion that develops the color, colour developing is clear, high resolution, X-gal dosage is few, testing result fidelity is high.
Description
Technical field
The present invention relates to field of biotechnology, in particular it relates to N- acyl-homoserine lactone in test sample
The method of types of populations induction signal molecule.
Background technique
Quorum sensing (Quorum sensing, QS) is the Auto-regulating System of Density of Heavy Medium signal between bacterium, is considered as the communication of bacterium
Language, it has very heavy on the generation of bioluminescence, the formation of biomembrane, the secretion of the toxicity factor and environmental suitability
The adjustment effect wanted.QS system is made of self-induction molecule, induction molecule and Downstream regulatory albumen.According to bacterium property, synthesis
Signaling molecule and induction mechanism difference, QS system mainly includes three classes: LuxR/AI-I system (mainly Gram-negative
Bacterium), LuxS/AI-2 and oligopeptides system (based on gram-positive bacteria, supplemented by negative bacterium), and it is adapted to inter-species exchange
(intra-communication) AI-3 system (such as microorganism and plant).AI-1 and AI-2 are primarily adapted for use in microorganism
Kind in exchange (inter-communication).So far, the multi-signal point including AI-1 and AI-2 has been had been acknowledged
Son, such as acyl homoserine lactones class compound (N-acyl homoserine lactones, AHLs), oligopeptides compound,
Aromatic alcohol compound, diketopiperazine compound (di-keto-piperazines, DKP), 2- heptyl -3- hydroxyl -4- quinoline
(2-heptyl-3-hydroxy-4-quinolone PQS) and furan boronic acid diester (Furanosyl borate
Diester) etc..These signals there are the group behaviors of adjustable arthrobacter itself, to realize the ecological functions under specific environment.
Currently, being the most commonly used one kind of research by the AI-1 class signal object of representative of AHL molecule, medicine is focused at first
Field, such as novel antibiotic substitution drug is developed, to reduce the drug resistance of microorganism.In addition, can be hindered using AHL system
The only expression of virulence gene generates the degrading enzyme or disabling signal for inactivating signaling molecule by composite signal molecular mimics
The synthesis of molecule carrys out block induced system, makes the expression of virulence gene that can not be activated, to reduce the infection energy of bacterium
Power.In addition, being exactly in medicine using biomembrane, because microbial film is by AHL molecule using another typical case of AHL system
It adjusts, AHL system plays sizable effect in the formation of bacterial biofilm.The signal point of AHL system is confirmed
Son participates in the formation, development and maturation of bacterial biofilm.If interfering the system, pathogen will be made to synthesize bacterial biofilm
Ability reduce, then the bacterial biofilm formed is relatively thin and attack vulnerable to antibiotic.Therefore, the operation of system is blocked
Also there is sizable effect to the formation for preventing biofilm, the shape of bacterial biofilm is interfered by regulation AHL system
At to control pathogen.In the past 10 years, with deep and subject crossing the development of AHL understanding, the range of AHL application is more next
It is more extensive, it has gradually been applied to waste water control, marine organisms fouling resistance, the culture of energy algae and aquaculture etc..
To, in order to various industries can utilize this system, for AHL it is effective detect become instantly urgently
A technical problem being concerned about.
Summary of the invention
The application is to be made based on inventor to the discovery of following facts and problem and understanding:
The existing side using thin-layer chromatography (TLC) detection N- acyl-homoserine lactone types of populations induction signal molecule
Method is that the semisolid LB culture medium containing reporting bacterial strain and X-gal is poured directly on chromatographic sheet, passes through semisolid LB
The free diffusing of culture medium is fixed on formation semisolid LB culture medium film on chromatographic sheet, therefore, existing skill to tiling
In art, it is difficult to ensure that different operation personnel are in each experiment, film in homogeneous thickness can be formed on chromatographic sheet, because
This it is difficult to ensure that experimental result repeatability；In addition, in the prior art, semisolid LB culture medium freely expands on thin layer plate
Dissipate and process of setting in, the infiltration of moisture generates tension on thin layer plate, so AHL substance to be checked can be generated dilution and
Diffusion, causing colour developing point to have, infiltration, colour developing circle be excessive, colour developing point has that diffusion, visual effect are poor, resolution ratio is low；In another aspect, existing
Have in technology, be to mix X-gal with semisolid LB culture medium in advance, X-gal dosage is big, and X-gal and AHL to be checked
It directly contacts, there is certain false positive results.
Discovery based on inventor to existing thin layer chromatography problem, inventor have attempted previously prepared semisolid LB training
Base rubber piece is supported, then it is contacted with chromatographic sheet, inventors have found that in this way, gained film stock thickness is uniform, glue surface
Smooth, experimental result is repeatable to be significantly improved, the spot diameter that develops the color is small, different colour developing point discriminations are good, colour developing is clear, resolution ratio
It is high；And the mode that inventor has attempted to be coated with X-gal on the semisolid LB culture medium film of formation carries out color developing detection
AHL, inventors have found that in this way, X-gal dosage substantially reduces, testing result fidelity is significantly improved.
In the present invention, the invention proposes N- acyl-homoserine lactone types of populations induction letters in a kind of test sample
The method of number molecule.According to an embodiment of the invention, the described method includes: previously prepared semisolid LB is cultivated base rubber by (1)
Piece is placed in the upper surface of chromatographic sheet, contains reporting bacterial strain in the semisolid LB culture medium film, the sample exists in advance
It is unfolded on the chromatographic sheet；(2) film is made to carry out chromogenic reaction using X-gal；And (3) are based on the colour developing instead
It is answering as a result, determining in the sample whether contain N- acyl-homoserine lactone types of populations induction signal molecule.The present invention is real
The method for applying N- acyl-homoserine lactone types of populations induction signal molecule in the test sample that example is proposed, experimental result can
Repeated height, colour developing point focusing, different colour developing point discriminations are good, develop the color clear, high resolution, X-gal dosage are few, testing result
Fidelity is high.
According to an embodiment of the invention, N- acyl-homoserine lactone types of populations inductive signal point in above-mentioned test sample
The method of son can further include at least one following additional technical feature:
According to an embodiment of the invention, the difference in height of the different location of the film is no more than 0.5mm.The embodiment of the present invention
Detection method LB semisolid culturemedium film smooth surface, bubble-free, flatness it is good, thickness is uniform, further
Improve the stability and repeatability of proposed method of the embodiment of the present invention.
According to an embodiment of the invention, the size of the film is 20.5*20.5cm, the thickness of the film is 0.3~
0.4cm.Film in the embodiment of the present invention is under above-mentioned size and thickness, N- acyl-homoserine lactone types of populations in sample
The colour developing of induction signal molecule is more clear, resolution ratio is higher.
According to an embodiment of the invention, the film is prepared by plastic tank, the plastic tank is by removably making
Offset plate and cutting composition, optionally, the size of the plastic plate is 22*22cm, and optionally, the height of the cutting is 0.5cm.
Film in the detection method of the embodiment of the present invention is prepared using plastic tank, the thickness of gained film is uniform, flatness is high,
It is easily operated, and then the repeatability of the experimental result of detection method that the embodiment of the present invention is proposed further increases, and develops the color
Point indiffusion, clear spot, resolution ratio further increase.
According to an embodiment of the invention, the reporting bacterial strain is Agrobacterium tumefaciems A136 bacterial strain.Carbon chain lengths are C6~C14
AHL molecule starting Agrobacterium tumefaciems A136 bacterial strain reporter gene expression, and then can make X-gal be shown as blue, thus
The presence or absence of the AHL molecule of C6~C14 can be determined by the significant changes more intuitive and convenient of chromogenic substrate color.
According to an embodiment of the invention, the film is by following operation preparation: (1) reporting bacterial strain and temperature
It is mixed for 40~50 DEG C of semisolid LB culture medium；And gained mixed liquor is carried out forming processes by (2).The present invention is implemented
The detection method that example is proposed prepares film by aforesaid operations, and reporting bacterial strain activity is high in gained film, is evenly distributed, in turn
Further improve the sensitivity and accuracy of detection method of the embodiment of the present invention.
According to an embodiment of the invention, the expansion is carried out in the mobile phase of first alcohol and water, it is optionally, described to be
Methanol: the volume ratio of water is 6:4.The good dispersion degree of the sample to be tested AHL molecule of the embodiment of the present invention, the spot that develops the color it is discrete
It spends, high resolution, further improves the sensitivity and accuracy of the detection method of the embodiment of the present invention.
According to an embodiment of the invention, being based on 1L semisolid LB culture medium, the dosage of the X-gal is 13~33mg/L.
The detection method of existing thin-layer chromatography, in the detection method of the embodiment of the present invention, X-gal is shown under the dosage of 13~33mg/L
Color identification is good, colour developing point resolution is high.
According to an embodiment of the invention, the chromogenic reaction is in 28~30 DEG C of 12~18h of progress.It is carried out at 28~30 DEG C
The chromogenic reaction of 12~18h, chromogenic reaction result good resolution, accuracy are high.
According to an embodiment of the invention, further comprising: the size based on locus coeruleus linear transport value determines N- acyl in sample
Base-homoserine lactone types of populations induction signal molecule parting.According to an embodiment of the invention, there are N- acyl groups-in sample
Homoserine lactone class (AHL) colony induction signaling molecule can make X-gal be shown as blue, in semisolid LB culture medium film
Formed locus coeruleus, meanwhile, be based on locus coeruleus linear transport value (Rf) size, due to linear transport value (Rf) only with AHL signaling molecule
Structure it is related, therefore the size based on locus coeruleus linear transport value, the method that the embodiment of the present invention is proposed can further really
Determine the parting of AHL signaling molecule.
Detailed description of the invention
Fig. 1 is to be divided according to embodiments of the present invention using existing method and method proposed by the invention the standard items of AHL
Not carry out point sample testing result figure；
Fig. 2 be in existing method according to an embodiment of the present invention and method proposed by the invention the dosage of X-gal and
The comparison result figure of the linear degree of linear transport value；
Fig. 3 is according to an embodiment of the present invention using existing method and method proposed by the invention is to the mixing mark of AHL
The result figure that quasi- product are detected；And
Fig. 4 is according to an embodiment of the present invention using existing method and method proposed by the invention is to pseudomonas aeruginosa
The result figure that P.aeruginosa PAO1AHL signaling molecule extract is detected.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
In the present invention, the invention proposes N- acyl-homoserine lactone types of populations induction letters in a kind of test sample
The method of number molecule.According to an embodiment of the invention, this method comprises: (1) is by previously prepared semisolid LB culture medium film
It is placed in the upper surface of chromatographic sheet, contains reporting bacterial strain in semisolid LB culture medium film, sample is in advance in chromatographic sheet
Upper expansion；(2) film is made to carry out chromogenic reaction using X-gal；And (3) based on chromogenic reaction as a result, determine sample in be
It is no to contain N- acyl-homoserine lactone types of populations induction signal molecule.N- in the test sample that the embodiment of the present invention is proposed
The method of acyl-homoserine lactone types of populations induction signal molecule, experimental result repeatability is high, colour developing spot diameter is small, no
With the easy differentiation of colour developing point, colour developing is clear, high resolution, X-gal dosage is few, testing result fidelity is high.
According to a particular embodiment of the invention, the difference in height of above-mentioned film is no more than 0.5mm.The detection of the embodiment of the present invention
The prepared film compared with the prior art of half culture medium film of LB obtained by method, smooth surface, bubble-free are smooth
Degree is more secure, and thickness is uniform, and different operation personnel are easy to keep the repeatability of experimental result, Jin Erben in different experiments
The stability and repeatability for the method that inventive embodiments are proposed significantly improve.
According to an embodiment of the invention, the size of above-mentioned film is 20.5*20.5cm, the thickness of film is 0.3~
0.4cm.Film in the embodiment of the present invention is under above-mentioned size and thickness, N- acyl-homoserine lactone types of populations in sample
The colour developing of induction signal molecule is more clear, resolution ratio is higher.
According to an embodiment of the invention, above-mentioned film is by following operation preparation: (1) reporting bacterial strain and temperature
It is mixed for 40~50 DEG C of semisolid LB culture medium；And gained mixed liquor is carried out forming processes by (2).The present invention is implemented
In the detection method of example, reporting bacterial strain is mixed in advance with the semisolid LB culture medium that temperature is 40~50 DEG C, reporting bacterial strain
Activity in the semisolid LB culture medium that temperature is 40~50 DEG C is high, and LB culture medium is in semi-solid state, reports bacterium
Strain is evenly distributed in LB culture medium, and the mixed liquor of gained reporting bacterial strain and LB culture medium is then carried out forming processes, gained
Reporting bacterial strain activity is high in film, is evenly distributed, and further improves the sensitivity of detection method of the embodiment of the present invention and accurate
Degree.
According to an embodiment of the invention, above-mentioned film is prepared by plastic tank, plastic tank is by dismountable plastic plate
It is formed with cutting, optionally, the size of the plastic plate is 22*22cm, and optionally, the height of the cutting is 0.5cm.Invention
People has found through a large number of experiments, advances with plastic tank and prepares film, and the thickness of gained film is uniform, flatness is high, and
It is easily operated.Plastic tank employed in the embodiment of the present invention is made of dismountable plastic plate and cutting, is both conveniently torn open in this way
It washes, and ease of assembly glue, the selection of the size of plastic plate is to determine that the present invention is real according to the size of chromatographic sheet to be spread
The size for applying chromatographic sheet to be spread in example is 20*20cm, and then the size of the used plastic plate of the embodiment of the present invention is
22*22cm；On the other hand, the selection of the height of the cutting of plastic plate is determined according to the thickness of film to be prepared, this
The film to be prepared of inventive embodiments with a thickness of 0.3~0.4cm, and then the cutting of the embodiment of the present invention is designed as 0.5cm, slightly
It higher than the thickness of film to be prepared, both saves and prepares material, also ensure the thickness of film.To sum up, the embodiment of the present invention
Film in detection method is prepared using plastic tank, and the thickness of gained film is uniform, flatness is high, easily operated, in turn
The repeatability of the experimental result for the detection method that the embodiment of the present invention is proposed further increases, and colour developing spot diameter is small, different
Colour developing spot does not interfere with each other mutually, develops the color clearly, and resolution ratio further increases.
In addition, according to a particular embodiment of the invention, the reporting bacterial strain in the embodiment of the present invention is Agrobacterium tumefaciems A136
Bacterial strain.Agrobacterium tumefaciems A136 bacterial strain reporter gene LacZ, the AHL molecule that carbon chain lengths are C6~C14 start crown gall agriculture
The reporter gene LacZ of bacillus A136 bacterial strain, and then beta galactosidase is expressed, beta galactosidase digests X-gal, makes X-gal
Color change occurs, therefore, under the conditions of existing for the chromogenic substrate X-gal of beta galactosidase, carbon chain lengths are C6~C14
AHL molecule can make X-gal be shown as blue, and then can pass through chromogenic substrate color variation determine C6~C14 AHL molecule
Presence or absence.
In addition, according to a particular embodiment of the invention, expansion is carried out in the mobile phase of first alcohol and water, optionally,
Methanol: the volume ratio of water is 6:4.The mobile phase that the sample to be tested of the embodiment of the present invention is 6:4 in the volume ratio of first alcohol and water
Under be unfolded, the good dispersion degree of AHL molecule in sample, so the spot that develops the color dispersion is good, high resolution, further increase
The sensitivity and accuracy of the detection method of the embodiment of the present invention.
In addition, according to a particular embodiment of the invention, being based on 1L semisolid LB culture medium, the dosage of the X-gal is 13
~33mg/L.The detection method of existing thin-layer chromatography is based on 1L semisolid LB culture medium, and the dosage of X-gal is 15~50mg/L,
Compared with the prior art, using finally coating X-gal by the way of rather than mixed in advance with semisolid LB culture medium, X-
Gal about saves 1/3 dosage.In the detection method of the embodiment of the present invention, X-gal is shown under the dosage of 13~33mg/L
Color effect is good, colour developing point resolution is high, and X-gal dosage is lower than 13mg/L, and color change is unobvious and influences result judgement, X-
Gal dosage is higher than 33mg/L, and X-gal is excessive, causes to waste.
In addition, according to a particular embodiment of the invention, chromogenic reaction is in 28~30 DEG C of 12~18h of progress.28~30 DEG C
It is the optimum temperature of reporting bacterial strain A136, at 28~30 DEG C, reporting bacterial strain is further proliferated, after 12~18h, sample to be tested
Middle AHL colony induction signaling molecule can make chromogenic substrate X-gal that apparent color change occur, and chromogenic reaction is lower than 12h, colour developing
Substrate X-gal color change is unobvious, and chromogenic reaction is longer than 18h, and chromogenic reaction result is there is no significant change, overlong time,
It will cause the raising of false positive results probability again.The chromogenic reaction of 12~18h, chromogenic reaction result resolution ratio are carried out at 28~30 DEG C
Height, accuracy are secure.
Finally, according to a particular embodiment of the invention, this method further comprises: based on the big of locus coeruleus linear transport value
It is small, determine the parting of N- acyl-homoserine lactone types of populations induction signal molecule in sample.There are N- acyl group-height in sample
Serine lactone (AHL) colony induction signaling molecule can make X-gal be shown as blue, in semisolid LB culture medium film shape
At locus coeruleus, meanwhile, be based on locus coeruleus linear transport value (Rf) size, due to linear transport value (Rf) only with AHL signaling molecule
Structure is related, therefore the size based on locus coeruleus linear transport value, can determine the parting of AHL signaling molecule.It is according to the present invention
Specific embodiment, inventor are determined the parting of AHL signaling molecule by parallel laboratory test, divided in parallel laboratory test known AHL signal
The standard items of sub- parting carry out the thin-layer chromatography under equal conditions, and the thin layer chromatogram analysis of sample to be tested and standard items is compared
Compared with, and then judge the parting of AHL signaling molecule in sample to be tested, while sample can be qualitatively judged according to the shade of locus coeruleus
The relative amount of middle AHL signaling molecule.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
The preparation of embodiment 1 bacterial strain uses therefor, material, reagent
In the examples below, N- acyl-homoserine lactone class (AHL) colony induction signaling molecule used detects bacterial strain
For bacillus radicicola Agrobacterium tumefaciens A136 (pCF218) (pCF372), condition of culture is that LB+ is grand mould
Plain Sp (50 micrograms/ml)+tetracycline Tc (4.5 micrograms/ml).
Another used bacterial strain is pseudomonas aeruginosa P.aeruginosa PAO1, it, which has, produces C4 and 3-
The ability of oxo-C12AHL.
Spectinomycin Sp, tetracycline Tc, X-gal (5- bromine 4- chlorine 3- indoles beta galactose glycosides), AHL standard items C6 (article No.
10940-25MG), C8 (article No. 17247-25MG), C11 (article No. 10937-25MG) and C14-HSL (article No. 09139-25MG) purchase
From Sigma company, RP-C18F254s chromatographic sheet (TLC plate) is purchased from Merck company of Germany.
The preparation process of LB liquid (Luria-Bertani) culture medium is as described below: accurately weighing peptone (Typtone)
1.0g, yeast extract (Yeast Extract) 0.5g, sodium chloride nacl 1.0g add distilled water (ddH2O it) to 100ml, fills
Divide and mixes dissolution, high pressure steam sterilization 15 minutes at 121 DEG C.
The preparation process of semisolid LB culture medium (soft culture medium) is as described below: being added in above-mentioned LB liquid medium
High pressure steam sterilization after the agar powder of 10.0g/L.
Other chemical reagent used include dimethyl sub-maple (DMSO) (purchased from Sangon, Dalian), and ethyl acetate (divides
Analyse it is pure, be purchased from Tianjin Zhi Yuan chemical reagent Co., Ltd), methanol, ethyl alcohol (analysis it is pure, purchased from Tianjin recover fine chemistry industry
Research institute) and acetic acid etc. (analysis is pure, is purchased from Shanghai Chinese medicines group).
Embodiment 2 is compared with the thin-layered chromatography of AHL signaling molecule in existing test sample
The thin-layered chromatography of AHL signaling molecule is as described below in existing test sample:
(1) by 2~3 microlitres of point samples of AHL standard items on RP-C18F254s chromatographic sheet (20*20cm), with methanol/
After water (60 ﹕ 40, v/v) is sufficiently spread out as mobile phase, solvent is volatilized.
(2) (Kendra P.Rumbaugh (ed.), Quorum Sensing:Methods according to recorded in classical documents
and Protocols,Methods in Molecular Biology,vol.692,DOI 10.1007/978-1-60761-
971-0_1, Springer Science Business Media, LLC 2011) thin-layer chromatography method preparation upper layer half it is solid
Body culture medium is laid on TLC plate.Preparation method is as described below: configuration 50mL semisolid LB culture medium, high pressure steam sterilization it
Afterwards, to be cooled to 45 DEG C or so (agar is avoided to be condensed into blocks), the 3mL A136 bacterial strain being incubated overnight is connect by sterile working
Enter semisolid culturemedium, shake up, then add the X-gal of 50 microlitres of 60mg/mL, jog mix after, be laid in advance expansion have it is to be measured
On the TLC plate of sample.
(3) upper layer semisolid culturemedium and TLC plate are placed in 28 DEG C of incubators, observation colour developing result after 24~48h.
The method of AHL signaling molecule is as described below in the test sample that the embodiment of the present invention is proposed:
(1) by 2~3 microlitres of point samples of AHL standard items on RP-C18F254s chromatographic sheet (20*20cm), with methanol/
After water (60 ﹕ 40, v/v) is sufficiently spread out as mobile phase, solvent is volatilized.
(2) the 100mL semisolid LB culture medium for being cooled to 45 DEG C or so is mixed with 6mL A136 bacterial strain by sterile working
It closes, gently shakes up rear bed board, plastic tank used in bed board is the plastic plate of 22*22cm and the cutting that height is 0.5cm by size
Composition, is prepared into the Thin cell layer base that thickness is about 3-4mm, forms culture medium film in room temperature cooled and solidified.
(3) cutting to be pulled up, holds up the whole film solidified with a sterile hard plastic sheet, one end gently contacts TLC plate,
Slowly covering is tiled forward in a manner of similar sticking film for mobile phone, is fitted in above TLC plate, was tiled until whole film is intact
Extract sterile hard plastic sheet in journey out, operating process pays attention to excluding bubble.
(4) after the completion of fitting process, using microorganism spreading rod by 100 microlitres of concentration be 20 micrograms/microlitre X-gal it is equal
It is even to be coated on above film.
(5) after x-gal liquid air-dries, 28 DEG C of incubators are placed in, observation colour developing result after 12-18h.
AHL signaling molecule with different side chains is having region existing for AHL, upper layer after thin layer chromatography is unfolded
Reporting bacterial strain experience, and blue spot is presented in the presence of X-gal.According to the size of spot, position, mobility
The relative amount and its classification of AHL are qualitatively judged with shade.
Inventor is utilized respectively the C6, C9, C11,3-o- of method that existing method and the embodiment of the present invention are proposed to AHL
C8,3-o-C14 standard items are detected, wherein C6, C9, C11,3-o-C8, and 3-o-C14 standard items are respectively labeled as 1,2,3,4,
5, as shown in Figure 1, wherein A shows the colour developing result figure using existing method detection AHL standard items, B is shown testing result
Utilize the colour developing result figure for the method examination criteria product that the embodiment of the present invention is proposed.It can be with by comparing the colour developing result of A and B
Find out, the diameter ratio A figure of the colour developing spot of B figure reduces 1.4~1.9 times, and spot diffusion is small, and gray value also obviously mentions
Height develops the color apparent, and the linear degree of linear transport value Rf is higher, and the comparison result of the linear degree of linear transport value Rf is such as
Shown in Fig. 2.
Meanwhile inventor is utilized respectively method that existing method and the embodiment of the present invention are proposed to the hybrid standard of AHL
Product are detected, testing result as shown in figure 3, Fig. 3 show to the AHL hybrid standard product of 5 kinds of carbon chain lengths (C6, C8, C11,
C14 and 3-o-C8)) testing result, inventors have found that the embodiment of the present invention propose method to mixing sample colour developing result
Colour developing resolution ratio it is higher, visual effect becomes apparent from.
In addition, the dosage of X-gal is compared in the method that inventor proposes existing method and the embodiment of the present invention
Analysis, it is found that the dosage of X-gal saves one third, the dosage statistical result of X-gal is as shown in Figure 2.
The extracting of 3 pseudomonas aeruginosa P.aeruginosa PAO1 AHL signaling molecule of embodiment and to this extract
Detection
Inventor has extracted pseudomonas aeruginosa P.aeruginosa PAO1 AHL signaling molecule by following operation:
(1) by monoclonal PAO1 strain inoculated in 10mL LB liquid medium, it is incubated overnight acquisition first order seed, later
First order seed is added in the conical flask containing the sterile LB liquid medium of 500mL and is expanded culture, expand culture be
It 30 DEG C, is cultivated for 24 hours under conditions of 220r/min.
(2) zymocyte liquid is centrifuged 20min, centrifugal speed 6000g/min at 4 DEG C.
(3) supernatant is collected, the supernatant after centrifugation is filtered with 0.22 micron of filter membrane, is further cleaned.
(4) ratio for being 1:1 according to volume ratio by the fermented supernatant fluid being collected into and ethyl acetate in the separatory funnel of 1L
Example mixing, turns upside down for several times, sufficiently shakes up for every 5 minutes, extracts 30 minutes.
(5) (temperature is controlled at 45 DEG C) is distilled, on a rotary evaporator to remove whole ethyl acetate.
(6) it is dissolved after drying extract with 2mL methanol, and is filtered with 1.0 microns of organic phase filter membrane, obtained
AHL signaling molecule extract.
In turn, inventor is utilized respectively detection method that existing method and the embodiment of the present invention are proposed to P. aeruginosa
Bacterium P.aeruginosa PAO1 AHL signaling molecule extract is detected.Testing result is as shown in Figure 4, wherein digital 1-4
For the potential AHL secretion of PAO1, including C4 and 3-O-C12, the detection method that the embodiment of the present invention is proposed as the result is shown is aobvious
Color vision is more attractive, and effect is apparent, shows that the method that the embodiment of the present invention is proposed is not only applicable to standard items with this,
Suitable for natural AHL extract.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (2)
1. a kind of method of N- acyl-homoserine lactone types of populations induction signal molecule in test sample, which is characterized in that packet
It includes:
(1) previously prepared semisolid LB culture medium film is placed in the upper surface of chromatographic sheet, the semisolid LB culture
Contain reporting bacterial strain in base rubber piece, the reporting bacterial strain is Agrobacterium tumefaciems A136 bacterial strain, and the sample is in advance in the thin layer
It is unfolded on chromatosheet；
(2) film is made to carry out chromogenic reaction using X-gal, the X-gal is coated on the upper surface of the film, is based on 1L
Semisolid LB culture medium, the dosage of the X-gal are 20mg/L；And
(3) based on the chromogenic reaction as a result, determining in the sample whether contain N- acyl-homoserine lactone types of populations
Induction signal molecule,
Wherein, the difference in height of the film different location is no more than 0.5mm,
The size of the film is 20.5*20.5cm, and the thickness of the film is 0.3~0.4cm,
The film is prepared by plastic tank, and the plastic tank is made of dismountable plastic plate and cutting,
The size of the plastic plate is 22*22cm,
The height of the cutting is 0.5cm,
The film is by following operation preparation:
(1) reporting bacterial strain is mixed with the semisolid LB culture medium that temperature is 40~50 DEG C；And
(2) gained mixed liquor is subjected to forming processes,
The expansion is carried out in the mobile phase of first alcohol and water, and the methanol: the volume ratio of water is 6:4,
The chromogenic reaction is in 28~30 DEG C of 12~18h of progress.
2. the method according to claim 1, wherein further comprising: the size based on locus coeruleus linear transport value,
Determine the parting of N- acyl-homoserine lactone types of populations induction signal molecule in sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610309598.1A CN105974044B (en) | 2016-05-10 | 2016-05-10 | The method of N- acyl-homoserine lactone types of populations induction signal molecule in test sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610309598.1A CN105974044B (en) | 2016-05-10 | 2016-05-10 | The method of N- acyl-homoserine lactone types of populations induction signal molecule in test sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105974044A CN105974044A (en) | 2016-09-28 |
| CN105974044B true CN105974044B (en) | 2018-12-25 |
Family
ID=56992088
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610309598.1A Active CN105974044B (en) | 2016-05-10 | 2016-05-10 | The method of N- acyl-homoserine lactone types of populations induction signal molecule in test sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105974044B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662948A (en) * | 2020-05-14 | 2020-09-15 | 青海大学 | Method for detecting homoserine lactone substance signal molecule in bacteria |
| CN113322269B (en) * | 2021-06-01 | 2022-08-09 | 中国人民解放军军事科学院军事医学研究院 | Biosensor based on bacterial population density sensing protein LuxR and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1743842A (en) * | 2004-09-03 | 2006-03-08 | 中国科学院沈阳应用生态研究所 | A kind of thin-layer chromatography coloration method |
| CN101067600A (en) * | 2007-06-14 | 2007-11-07 | 中国农业科学院饲料研究所 | Aquatic pathogenic bacterium colony induction signaling molecule detecting method and special reagent kit thereof |
| CN102925511A (en) * | 2012-06-19 | 2013-02-13 | 文才艺 | Staurosporine preparation method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6395282B1 (en) * | 1998-04-16 | 2002-05-28 | University Of Rochester | Immunogenic conjugates of Gram-negative bacterial autoinducer molecules |
| US7888120B2 (en) * | 2006-04-13 | 2011-02-15 | Wisconsin Alumni Research Foundation | Global regulator of morphogenesis and pathogenicity in dimorphic fungi and uses thereof |
-
2016
- 2016-05-10 CN CN201610309598.1A patent/CN105974044B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1743842A (en) * | 2004-09-03 | 2006-03-08 | 中国科学院沈阳应用生态研究所 | A kind of thin-layer chromatography coloration method |
| CN101067600A (en) * | 2007-06-14 | 2007-11-07 | 中国农业科学院饲料研究所 | Aquatic pathogenic bacterium colony induction signaling molecule detecting method and special reagent kit thereof |
| CN102925511A (en) * | 2012-06-19 | 2013-02-13 | 文才艺 | Staurosporine preparation method |
Non-Patent Citations (4)
| Title |
|---|
| Biofilm Formation, Extracellular Polysaccharide Production, and Cell-to-Cell Signaling in Various Enterobacter sakazakii Strains: Aspects Promoting Environmental Persistence;ANGELIKA LEHNER 等;《Journal of Food Protection》;20051231;第68卷(第11期);第2287–2294页 * |
| HPLC-MS法检测N-酰基-高丝氨酸内酯类信号分子;綦国红;《分析测试学报》;20070531;第26卷(第3期);第417-419页 * |
| 副溶血弧菌群体感应与调控的初步研究;汪映;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑 》;20150115(第01期);第B024-55页 * |
| 食源假单胞菌群体感应信号分子的产生及其对食品腐败的影响;綦国红;《中国优秀博士学位论文全文数据库 工程科技Ⅰ辑 》;20110315(第03期);第B024-11页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105974044A (en) | 2016-09-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jadulco et al. | New metabolites from sponge-derived fungi curvularia l unata and cladosporium h erbarum | |
| McKerrow et al. | A simple HPLC method for analysing diaminopimelic acid diastereomers in cell walls of Gram‐positive bacteria | |
| Joux et al. | Use of fluorescent probes to assess physiological functions of bacteriaat single-cell level | |
| Selvameenal et al. | Antibiotic pigment from desert soil actinomycetes; biological activity, purification and chemical screening | |
| Pandey et al. | Studies on the antibacterial activity of the Actinomycetes isolated from the Khumbu Region of Nepal | |
| Decker | The ability of direct fluorescence‐based, two‐colour assays to detect different physiological states of oral streptococci | |
| Betina et al. | Antimicrobial properties of cytochalasins and their alteration of fungal morphology | |
| Swanson et al. | In situ quantification of a natural settlement cue and recruitment of the Australian sea urchin Holopneustes purpurascens | |
| Motohashi et al. | A new angucycline and a new butenolide isolated from lichen-derived Streptomyces spp. | |
| Stargel et al. | Modification of the Minitek miniaturized differentiation system for characterization of anaerobic bacteria | |
| CN105974044B (en) | The method of N- acyl-homoserine lactone types of populations induction signal molecule in test sample | |
| CN100562742C (en) | The detection method of aquatic pathogenic bacterium colony induction signaling molecule and dedicated kit thereof | |
| Pettit et al. | Isolation and Structure of Hemibastadinols 1− 3 from the Papua New Guinea Marine Sponge Ianthella basta | |
| Saito et al. | Discovery of “heat shock metabolites” produced by thermotolerant actinomycetes in high-temperature culture | |
| Hollmann et al. | Fluorescent nanosensors reveal dynamic pH gradients during biofilm formation | |
| Ohshiro et al. | New verticilides, inhibitors of acyl-CoA: cholesterol acyltransferase, produced by Verticillium sp. FKI-2679 | |
| Thao et al. | Discovering potential Streptomyces hormone producers by using disruptants of essential biosynthetic genes as indicator strains | |
| Battu et al. | Isolation of secondary metabolites from Pseudomonas fluorescens and its characterization | |
| Thines et al. | Trichoflectin, a bioactive azaphilone from the ascomycete Trichopezizella nidulus | |
| Coisne et al. | Actinorhodin production by Streptomyces coelicolor A3 (2) in iron‐restricted media | |
| Young et al. | Serotyping of Pseudomonas aeruginosa | |
| Biswas et al. | In vitro motility assays for Acinetobacter species | |
| Syal | Novel method for quantitative estimation of biofilms | |
| Lee et al. | Isolation and antifungal activity of 4-phenyl-3-butenoic acid from Streptomyces koyangensis strain VK-A60 | |
| Prasad et al. | Synthesis, spectroscopic studies, antibacterial activity, and colorimetric evaluation of the time-killing assay for newly synthesized chalcones using resazurin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |