CN105974044A - Method for detecting N-acyl-homoserine lactone quorum sensing signal molecules in sample - Google Patents
Method for detecting N-acyl-homoserine lactone quorum sensing signal molecules in sample Download PDFInfo
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Abstract
The invention provides a method for detecting N-acyl-homoserine lactone quorum sensing signal molecules in a sample. The method comprises the following steps: (1) placing a pre-prepared semi-solid LB culture medium film on the upper surface of a TLC thin layer chromatoplate, wherein the semi-solid LB culture medium film contains report strains, and the sample is pre-outspreaded on the thin layer chromatoplate; (2) subjecting the film to a chromogenic reaction by utilizing X-Gal; and (3), confirming whether the sample contains the N-acyl-homoserine lactone quorum sensing signal molecules or not based on the results of the chromogenic reaction. The detection method provided by the embodiment of the invention has the advantages of obviously improved repeatability of experimental results, non-diffusion of chromogenic spots, clear color development, high resolution, low amount of X-gal, and high fidelity of detection results.
Description
Technical field
The present invention relates to biological technical field, in particular it relates to N-acyl-homoserine lactone in detection sample
The method of types of populations induction signal molecule.
Background technology
Quorum sensing (Quorum sensing, QS) is the Auto-regulating System of Density of Heavy Medium signal between antibacterial, is considered the communication of antibacterial
Language, it has the heaviest on the generation of bioluminescence, biomembranous formation, the secretion of the toxicity factor and environmental suitability
The regulation effect wanted.QS system is made up of self-induction molecule, sensing molecule and Downstream regulatory albumen.According to antibacterial character, synthesis
Signaling molecule and the difference of induction mechanism, QS system mainly includes three classes: LuxR/AI-I system (mainly Gram-negative
Bacterium), LuxS/AI-2 and oligopeptides system (gram positive bacteria be main, negative bacterium be auxiliary), and exchange between being adapted to kind
(intra-communication) AI-3 system (such as microorganism and plant).AI-1 and AI-2 is primarily adapted for use in microorganism
Kind in exchange (inter-communication).So far, it has been acknowledged that the multi-signal including AI-1 and AI-2 divides
Son, such as acyl homoserine lactones compounds (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..The group behavior of the existence scalable antibacterial of these signals self, to realize the ecological functions under specific environment.
At present, the AI-1 class signal thing with AHL molecule as representative is to study most commonly used one, pays close attention to medical science at first
Field, such as, develop novel antibiotic and substitute medicine, to reduce the drug resistance of microorganism.Additionally, application AHL system can hinder
The only expression of virulence gene, by composite signal molecular mimics, produces the digestive enzyme or disabling signal making signaling molecule inactivate
The synthesis of molecule, carrys out block induced system, makes the expression of virulence gene to be activated, thus reduces the infection energy of antibacterial
Power.It addition, apply another typical case of AHL system to utilize biomembrane exactly, because microbial film is by AHL molecule in medical science
Regulation, AHL system plays sizable effect in the formation of bacterial biofilm.Confirm that the signal of AHL system divides
Son participates in the formation of bacterial biofilm, grows with ripe.If disturbing this system, pathogen will be made to synthesize bacterial biofilm
Ability reduce, then the bacterial biofilm formed is relatively thin and is vulnerable to the attack of antibiotic.Therefore, the operation of system is blocked
Formation to stoping biofilm also has sizable effect, is disturbed the shape of bacterial biofilm by regulation and control AHL system
Become thus control pathogen.Over nearly 10 years, along with going deep into and the development of subject crossing of AHL understanding, the scope of AHL application is more come
The most extensive, the most gradually it is applied to the aspects such as waste water control, marine organisms fouling resistance, the cultivation of energy algae and aquaculture.
Thus, in order to this system can be utilized at industry-by-industry, for the effectively detection of AHL become instantly urgently
The technical problem being concerned about.
Summary of the invention
The application is to make following facts and the discovery of problem and understanding based on inventor:
The existing side utilizing thin layer chromatography (TLC) detection N-acyl-homoserine lactone types of populations induction signal molecule
Method, is will to be poured directly on chromatographic sheet containing the semi-solid LB culture medium of reporting bacterial strain and X-gal, by semi-solid LB
The free diffusing of culture medium thus tiling is fixed on chromatographic sheet the semi-solid LB culture medium film of formation, therefore, existing skill
In art, it is difficult to ensure that different operating personnel are in experiment every time, film in uniform thickness can be formed on chromatographic sheet, because of
This is it is difficult to ensure that the repeatability of experimental result；It addition, in prior art, semi-solid LB culture medium freely expands on thin layer flat board
Dissipating and in process of setting, the infiltration of moisture produces tension force on thin layer flat board, so AHL material to be checked can be produced dilution with
Spreading, infiltration, colour developing circle are excessive, the point that develops the color has diffusion, poor visual effect, resolution are low to cause colour developing point to have；Another further aspect, existing
Having in technology, be to be mixed with semi-solid LB culture medium in advance by X-gal, X-gal consumption is big, and X-gal and AHL to be checked
Directly contact, has certain false positive results.
Based on inventor's discovery to existing thin layer chromatography problem, inventor has attempted previously prepared semi-solid LB training
Supporting base film, then it contacted with chromatographic sheet, inventor finds, adopting in this way, gained film stock thickness is homogeneous, glue surface
Smooth, experimental result is repeatable to be significantly improved, the spot diameter that develops the color colour developing little, different some discrimination is good, colour developing is clear, resolution
High；And inventor has attempted being coated with the mode of X-gal on the semi-solid LB culture medium film formed and has carried out color developing detection
AHL, inventor finds, adopting in this way, X-gal consumption substantially reduces, and testing result fidelity significantly improves.
In the present invention, N-acyl-homoserine lactone types of populations sensing letter during the present invention proposes a kind of detection sample
The method of number molecule.According to embodiments of the invention, described method includes: (1) is by previously prepared semi-solid LB culture medium glue
Sheet is placed in the upper surface of chromatographic sheet, and containing reporting bacterial strain in described semi-solid LB culture medium film, described sample exists in advance
Launch on described chromatographic sheet；(2) X-gal is utilized to make described film carry out chromogenic reaction；And (3) are anti-based on described colour developing
The result answered, determines and whether contains N-acyl-homoserine lactone types of populations induction signal molecule in described sample.The present invention is real
Executing the method for N-acyl-homoserine lactone types of populations induction signal molecule in the detection sample that example is proposed, experimental result can
Repeatability is high, colour developing point focusing, difference develop the color, and some discrimination is good, colour developing is clear, resolution height, X-gal consumption are few, testing result
Fidelity is high.
According to embodiments of the invention, in above-mentioned detection sample, N-acyl-homoserine lactone types of populations induced signal divides
The method of son can further include at least one following additional technical feature:
According to embodiments of the invention, the difference in height of the diverse location of described film is less than 0.5mm.The embodiment of the present invention
The surfacing of LB semisolid culturemedium film of detection method smooth, bubble-free, flatness are good, thickness is homogeneous, further
Improve stability and the repeatability of embodiment of the present invention proposed method.
According to embodiments of the invention, the size of described film is 20.5*20.5cm, the thickness of described film be 0.3～
0.4cm.Film in the embodiment of the present invention under above-mentioned size and thickness, N-acyl-homoserine lactone types of populations in sample
The colour developing of induction signal molecule becomes apparent from, resolution is higher.
According to embodiments of the invention, described film is prepared by glue groove, and described glue groove is by dismountable system
Offset plate and cutting composition, optionally, the size of described glue plate is 22*22cm, and optionally, the height of described cutting is 0.5cm.
Film in the detection method of the embodiment of the present invention uses glue groove to be prepared, the thickness of gained film is homogeneous, flatness is high,
Easily operated, and then the repeatable of the experimental result of detection method that the embodiment of the present invention is proposed improve further, colour developing
Point indiffusion, clear spot, resolution improves further.
According to embodiments of the invention, described reporting bacterial strain is Agrobacterium tumefaciems A136 bacterial strain.Carbon chain lengths is C6～C14
AHL molecule start the expression of reporter gene of Agrobacterium tumefaciems A136 bacterial strain, and then X-gal can be made to be shown as blue, thus
Can be by the presence or absence significantly changing the AHL molecule more judging to intuitive and convenient C6～C14 of chromogenic substrate color.
According to embodiments of the invention, described film is by following operation preparation: (1) described reporting bacterial strain and temperature
It is that the semi-solid LB culture medium of 40～50 DEG C mixes；And gained mixed liquor is shaped processing by (2).The present invention implements
The detection method that example is proposed prepares film by aforesaid operations, and in gained film, reporting bacterial strain activity is high, be evenly distributed, and then
Further increase sensitivity and the accuracy of embodiment of the present invention detection method.
According to embodiments of the invention, described expansion is to carry out in the flowing mutually of first alcohol and water, optionally, described is
Methanol: the volume ratio of water is 6:4.The good dispersion degree of the detected sample AHL molecule of the embodiment of the present invention, develop the color the discrete of speckle
Spend, resolution high, further increase sensitivity and the accuracy of the detection method of the embodiment of the present invention.
According to embodiments of the invention, based on 1L semisolid LB culture medium, the consumption of described 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 under the consumption of 13～33mg/L, aobvious
Color identification is good, colour developing point resolution is high.
According to embodiments of the invention, described chromogenic reaction is to carry out 12～18h at 28～30 DEG C.Carry out at 28～30 DEG C
The chromogenic reaction of 12～18h, chromogenic reaction result good resolution, accuracy are high.
According to embodiments of the invention, farther include: size based on locus coeruleus linear transport value, determine N-acyl in sample
The typing of base-homoserine lactone types of populations induction signal molecule.According to embodiments of the invention, sample exists N-acyl group-
Homoserine lactone class (AHL) colony induction signaling molecule, can make X-gal be shown as blue, at semi-solid LB culture medium film
Formed locus coeruleus, meanwhile, size based on locus coeruleus linear transport value (Rf), due to linear transport value (Rf) only with AHL signaling molecule
Structure be correlated with, therefore size based on locus coeruleus linear transport value, the method that the embodiment of the present invention is proposed can be the most true
Determine the typing of AHL signaling molecule.
Accompanying drawing explanation
Fig. 1 is to utilize existing method and method proposed by the invention to divide the standard substance of AHL according to embodiments of the present invention
Do not carry out the testing result figure of point sample；
Fig. 2 be X-gal in existing method according to embodiments of the present invention and method proposed by the invention consumption and
The comparative result figure of the linear degree of linear transport value；
Fig. 3 be according to embodiments of the present invention utilize existing method and the method proposed by the invention mixing mark to AHL
Quasi-product carry out the result figure detected；And
Fig. 4 be according to embodiments of the present invention utilize existing method and method proposed by the invention to Pseudomonas aeruginosa
P.aeruginosa PAO1AHL signaling molecule extract carries out the result figure detected.
Detailed description of the invention
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this
Bright, and be not considered as limiting the invention.
In the present invention, N-acyl-homoserine lactone types of populations sensing letter during the present invention proposes a kind of detection sample
The method of number molecule.According to embodiments of the invention, the method includes: (1) is by previously prepared semi-solid LB culture medium film
Being placed in the upper surface of chromatographic sheet, containing reporting bacterial strain in semi-solid LB culture medium film, sample is in advance at chromatographic sheet
Upper expansion；(2) X-gal is utilized to make film carry out chromogenic reaction；And (3) result based on chromogenic reaction, determine in sample and be
No containing N-acyl-homoserine lactone types of populations induction signal molecule.N-in the detection 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 little, no
Easily distinguish with colour developing point, colour developing is clear, resolution is high, X-gal consumption is few, testing result fidelity is high.
According to a particular embodiment of the invention, the difference in height of above-mentioned film is less than 0.5mm.The detection of the embodiment of the present invention
LB half culture medium film obtained by method is compared to the film prepared by prior art, and surfacing is smooth, bubble-free, smooth
Spending more secure, thickness is homogeneous, and different operating personnel are prone to keep the repeatability of experimental result, Jin Erben in different experiments
Stability and the repeatability of the method that inventive embodiments is proposed significantly improve.
According to embodiments of the invention, the size of above-mentioned film is 20.5*20.5cm, the thickness of film be 0.3～
0.4cm.Film in the embodiment of the present invention under above-mentioned size and thickness, N-acyl-homoserine lactone types of populations in sample
The colour developing of induction signal molecule becomes apparent from, resolution is higher.
According to embodiments of the invention, above-mentioned film is by following operation preparation: (1) described reporting bacterial strain and temperature
It is that the semi-solid LB culture medium of 40～50 DEG C mixes；And gained mixed liquor is shaped processing by (2).The present invention implements
In the detection method of example, reporting bacterial strain mixes in advance with the semi-solid LB culture medium that temperature is 40～50 DEG C, reporting bacterial strain
Activity in the semi-solid 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, is then shaped the mixed liquor of gained reporting bacterial strain Yu LB culture medium processing, gained
In film, reporting bacterial strain activity is high, be evenly distributed, and further increases the sensitivity and accurately of embodiment of the present invention detection method
Degree.
According to embodiments of the invention, above-mentioned film is prepared by glue groove, and glue groove is by dismountable glue plate
Forming with cutting, optionally, the size of described glue plate is 22*22cm, and optionally, the height of described cutting is 0.5cm.Invention
People is found by substantial amounts of experiment, and advancing with glue groove and prepare film, the thickness of gained film is homogeneous, flatness is high, and
Easily operated.Glue groove employed in the embodiment of the present invention is made up of dismountable glue plate and cutting, the most conveniently tears open
Washing, again ease of assembly glue, the selection of the size of glue plate is that the size according to chromatographic sheet to be spread determines, the present invention is real
Executing the size of chromatographic sheet to be spread in example is 20*20cm, and then the size of the glue plate used of the embodiment of the present invention is
22*22cm；On the other hand, the selection of the height of the cutting of glue plate is that the thickness according to film to be prepared is determined, this
The thickness of the film to be prepared of inventive embodiments is 0.3～0.4cm, and then the cutting of the embodiment of the present invention is designed as 0.5cm, slightly
Higher than the thickness of film to be prepared, both saved and prepared material, in turn ensure that the thickness of film.To sum up, the embodiment of the present invention
Film in detection method uses glue groove to be prepared, and the thickness of gained film is homogeneous, flatness is high, easily operated, and then
The repeatable of the experimental result of the detection method that the embodiment of the present invention is proposed improves further, and colour developing spot diameter is little, different
Colour developing speckle does not interfere with each other mutually, develops the color clearly, and resolution improves further.
It 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, carbon chain lengths is that the AHL molecule of C6～C14 starts crown gall agriculture
The reporter gene LacZ of bacillus A136 bacterial strain, and then express beta galactosidase, beta galactosidase digestion X-gal, make X-gal
Color change occurs, and therefore, under conditions of the chromogenic substrate X-gal of beta galactosidase exists, carbon chain lengths is C6～C14
AHL molecule X-gal can be made to be shown as blue, and then the AHL molecule of C6～C14 can be judged by the change of chromogenic substrate color
Presence or absence.
It addition, according to a particular embodiment of the invention, expansion is to carry out in the flowing mutually of first alcohol and water, optionally,
Methanol: the volume ratio of water is 6:4.The detected sample of the embodiment of the present invention is in the flowing phase that volume ratio is 6:4 of first alcohol and water
Under launch, the good dispersion degree of AHL molecule in sample, so colour developing speckle dispersion good, resolution is high, improve further
The sensitivity of the detection method of the embodiment of the present invention and accuracy.
It addition, according to a particular embodiment of the invention, based on 1L semisolid LB culture medium, the consumption of described X-gal is 13
～33mg/L.The detection method of existing thin layer chromatography, based on 1L semisolid LB culture medium, the consumption of X-gal is 15～50mg/L,
Compared to prior art, use the mode of last coating X-gal rather than mix with semi-solid LB culture medium in advance, X-
Gal about saves the consumption of 1/3.In the detection method of the embodiment of the present invention, X-gal is under the consumption of 13～33mg/L, aobvious
Chromatic effect is good, colour developing point resolution is high, and X-gal consumption is less than 13mg/L, and color change is inconspicuous and affects result and judges, X-
Gal consumption, higher than 33mg/L, X-gal excess, causes waste.
It addition, according to a particular embodiment of the invention, chromogenic reaction is to carry out 12～18h at 28～30 DEG C.28～30 DEG C
Being the optimum temperature of reporting bacterial strain A136, at 28～30 DEG C, reporting bacterial strain is bred further, behind 12～18h, and detected sample
Middle AHL colony induction signaling molecule can make chromogenic substrate X-gal that the change of obvious color occurs, and chromogenic reaction is less than 12h, colour developing
The change of substrate X-gal color is inconspicuous, and chromogenic reaction is longer than 18h, and chromogenic reaction result no longer has a significant change, overlong time,
False positive results probability can be caused again to raise.28～30 DEG C carry out 12～18h chromogenic reaction, chromogenic reaction result resolution
Height, accuracy are secure.
Finally, according to a particular embodiment of the invention, the method farther includes: based on locus coeruleus linear transport value big
Little, determine the typing of N-acyl-homoserine lactone types of populations induction signal molecule in sample.Sample exists N-acyl group-height
Serine lactone (AHL) colony induction signaling molecule, can make X-gal be shown as blue, in semi-solid LB culture medium film shape
Become locus coeruleus, meanwhile, size based on locus coeruleus linear transport value (Rf), due to linear transport value (Rf) only with AHL signaling molecule
Structure is correlated with, therefore size based on locus coeruleus linear transport value, it may be determined that the typing of AHL signaling molecule.According to the present invention's
Specific embodiment, inventor determines the typing of AHL signaling molecule by parallel laboratory test, in parallel laboratory test divides known AHL signal
The standard substance of sub-typing carry out the thin layer chromatography under equal conditions, are compared by the thin layer chromatogram analysis of testing sample with standard substance
Relatively, and then judge the typing of AHL signaling molecule in testing sample, sample can be qualitatively judged according to the shade of locus coeruleus simultaneously
The relative amount of middle AHL signaling molecule.
Below in conjunction with embodiment, the solution of the present invention is explained.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.Unreceipted concrete technology or bar in embodiment
Part, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or instrument
Device unreceipted production firm person, be can by city available from conventional products.
Embodiment 1 bacterial strain uses therefor, material, the preparation of reagent
In the examples below, N-acyl-homoserine lactone class (AHL) colony induction signaling molecule used detection bacterial strain
For bacillus radicicola Agrobacterium tumefaciens A136 (pCF218) (pCF372), condition of culture is that LB+ is grand mould
Element Sp (50 micrograms/ml)+tetracycline Tc (4.5 micrograms/ml).
The bacterial strain that another one is used is Pseudomonas aeruginosa P.aeruginosa PAO1, and it has product C4 and 3-
The ability of oxo-C12AHL.
Spectinomycin Sp, tetracycline Tc, X-gal (5-bromine 4-chlorine 3-indole beta galactose glycosides), AHL standard substance 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 set-up procedure of LB liquid (Luria-Bertani) culture medium is as described below: accurately weigh peptone (Typtone)
1.0g, yeast extract (Yeast Extract) 0.5g, sodium chloride nacl 1.0g, add distilled water (ddH2O) to 100ml, fill
Mixing is divided to dissolve, high pressure steam sterilization 15 minutes at 121 DEG C.
The set-up procedure of semi-solid LB culture medium (soft culture medium) is as described below: add in above-mentioned LB fluid medium
High pressure steam sterilization after the agar powder of 10.0g/L.
Other chemical reagent used includes that DMSO (DMSO) (purchased from Sangon, Dalian), ethyl acetate (divide
Analyse pure, purchased from Tianjin Zhi Yuan chemical reagent company limited), (analytical pure recovers fine chemistry industry purchased from Tianjin for methanol, ethanol
Institute), and acetic acid etc. (analytical pure, purchased from Shanghai traditional Chinese medicines group).
Embodiment 2 compares with the thin layer chromatography of AHL signaling molecule in existing detection sample
In existing detection sample, the thin layer chromatography of AHL signaling molecule is as described below:
(1) by AHL standard substance 2～3 microlitre point sample on RP-C18F254s chromatographic sheet (20*20cm), with methanol/
After water (60 40, v/v) is sufficiently spread out mutually as flowing, volatilize solvent.
(2) according to (Kendra P.Rumbaugh (ed.), Quorum Sensing:Methods described 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) the method for thin layer chromatography to prepare upper strata half 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
After, to be cooled to about 45 DEG C (avoiding agar to condense in bulk), by sterile working, the 3mL A136 bacterial strain of incubated overnight is connect
Entering semisolid culturemedium, shake up, then add the X-gal of 50 microlitre 60mg/mL, after jog mixing, being laid on expansion in advance has to be measured
On the TLC plate of sample.
(3) upper strata semisolid culturemedium and TLC plate are placed in 28 DEG C of incubators, after 24～48h, observe colour developing result.
In the detection sample that the embodiment of the present invention is proposed, the method for AHL signaling molecule is as described below:
(1) by AHL standard substance 2～3 microlitre point sample on RP-C18F254s chromatographic sheet (20*20cm), with methanol/
After water (60 40, v/v) is sufficiently spread out mutually as flowing, volatilize solvent.
(2) by sterile working, the 100mL semisolid LB culture medium being cooled to about 45 DEG C is mixed with 6mL A136 bacterial strain
Closing, shake up rear bed board gently, the glue groove used by bed board is the glue plate of 22*22cm and the cutting that height is 0.5cm by size
Composition, is prepared as the Thin cell layer base that thickness is about 3-4mm, forms culture medium film in room temperature cooled and solidified.
(3) pulling up cutting, hold up whole the film solidified with an aseptic hard plastic sheet, one end contacts TLC plate gently,
Slowly cover, tile forward in the way of similar sticking film for mobile phone, fit in above TLC plate until whole film is intact, tiled
Extracting aseptic hard plastic sheet in journey out, operating process notes getting rid of bubble.
(4), after laminating process completes, use microorganism spreading rod by equal for the X-gal that 100 lli are 20 micrograms/microlitre
Even coat above film.
(5) after x-gal liquid air-dries, it is placed in 28 DEG C of incubators, after 12-18h, observes colour developing result.
There is the AHL signaling molecule of different side chain after thin layer chromatography launches, on the region with the presence of AHL, upper strata
Reporting bacterial strain experience, and in the presence of X-gal, present blue spot.Size according to speckle, position, mobility
With relative amount and the classification thereof that shade qualitatively judges AHL.
The method that inventor is utilized respectively existing method and the embodiment of the present invention the is proposed C6 to AHL, C9, C11,3-o-
C8,3-o-C14 standard substance detect, wherein C6, and C9, C11,3-o-C8,3-o-C14 standard substance are respectively labeled as 1, and 2,3,4,
5, testing result as it is shown in figure 1, wherein A show utilize existing method detection AHL standard substance colour developing result figure, B shows
Utilize the colour developing result figure of the method examination criteria product that the embodiment of the present invention proposed.Permissible by comparing the colour developing result of A and B
Finding out, the diameter of the colour developing speckle of B figure reduces 1.4～1.9 times than A figure, and speckle diffusion is little, and gray value the most substantially carries
Height, develops the color apparent, and the linear degree of linear transport value Rf is higher, and the comparative result of the linear degree of linear transport value Rf is such as
Shown in Fig. 2.
Meanwhile, the method that inventor is utilized respectively existing method and the embodiment of the present invention the is proposed hybrid standard to AHL
Product detect, testing result as it is shown on figure 3, Fig. 3 show the AHL hybrid standard product to 5 kinds of carbon chain lengths (C6, C8, C11,
C14 and 3-o-C8)) testing result, inventor finds, the method colour developing result to biased sample that the embodiment of the present invention proposes
Colour developing resolution higher, visual effect becomes apparent from.
It addition, the consumption of X-gal compares in inventor's method of being proposed existing method and the embodiment of the present invention
Analyzing, find that the consumption of X-gal saves 1/3rd, the consumption statistical result of X-gal is as shown in Figure 2.
The extracting of embodiment 3 Pseudomonas aeruginosa P.aeruginosa PAO1 AHL signaling molecule and to this extract
Detection
Inventor has extracted Pseudomonas aeruginosa P.aeruginosa PAO1 AHL signaling molecule by following operation:
(1) by monoclonal PAO1 inoculation in 10mL LB liquid medium, incubated overnight obtains first order seed, afterwards
First order seed is joined the conical flask containing 500mL aseptic LB fluid medium is enlarged cultivate, amplification culture be
30 DEG C, under conditions of 220r/min, cultivate 24h.
(2) by zymocyte liquid centrifugal 20min, centrifugal speed 6000g/min at 4 DEG C.
(3) collect supernatant, with the filter membrane of 0.22 micron, the supernatant after being centrifuged is filtered, further remove impurity.
(4) in the separatory funnel of 1L by the fermented supernatant fluid collected and ethyl acetate according to the ratio that volume ratio is 1:1
Example mixes, and within every 5 minutes, turns upside down for several times, fully shakes up, extracts 30 minutes.
(5) carry out on a rotary evaporator distilling (temperature controls at 45 DEG C), to remove whole ethyl acetate.
(6) dissolve with 2mL methanol after extract being dried, and filter with the organic facies filter membrane of 1.0 microns, obtain
AHL signaling molecule extract.
And then, the detection method that inventor is utilized respectively existing method and the embodiment of the present invention is proposed is to P. aeruginosa
Bacterium P.aeruginosa PAO1 AHL signaling molecule extract detects.Testing result as shown in Figure 4, wherein, numeral 1-4
For AHL secretions potential for PAO1, including C4 and 3-O-C12, the detection method that the result display embodiment of the present invention is proposed shows
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 substance, also with this
It is applicable to the AHL extract of natural origin.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
Example " or the description of " some examples " etc. means to combine this embodiment or example describes specific features, structure, material or spy
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.And, the specific features of description, structure, material or feature can be in office
One or more embodiments or example combine in an appropriate manner.Additionally, in the case of the most conflicting, the skill of this area
The feature of the different embodiments described in this specification or example and different embodiment or example can be tied by art personnel
Close and combination.
Although above it has been shown and described that embodiments of the invention, it is to be understood that above-described embodiment is example
Property, it is impossible to being interpreted as limitation of the present invention, those of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, revises, replaces and modification.
Claims (10)
1. one kind is detected the method for N-acyl-homoserine lactone types of populations induction signal molecule in sample, it is characterised in that bag
Include:
(1) previously prepared semi-solid LB culture medium film is placed in the upper surface of chromatographic sheet, and described semi-solid LB cultivates
Containing reporting bacterial strain in base film, described sample launches in advance on described chromatographic sheet；
(2) X-gal is utilized to make described film carry out chromogenic reaction；And
(3) result based on described chromogenic reaction, determines and whether contains N-acyl-homoserine lactone types of populations in described sample
Induction signal molecule.
Method the most according to claim 1, it is characterised in that the difference in height of described film diverse location is less than 0.5mm.
Method the most according to claim 1, it is characterised in that the size of described film is 20.5*20.5cm, described film
Thickness be 0.3～0.4cm.
Method the most according to claim 3, it is characterised in that described film is prepared by glue groove, described glue
Groove is made up of dismountable glue plate and cutting,
Optionally, the size of described glue plate is 22*22cm,
Optionally, the height of described cutting is 0.5cm.
Method the most according to claim 1, it is characterised in that described reporting bacterial strain is Agrobacterium tumefaciems A136 bacterial strain.
Method the most according to claim 1, it is characterised in that described film is by following operation preparation:
(1) described reporting bacterial strain mixes with the semi-solid LB culture medium that temperature is 40～50 DEG C；And
(2) it is shaped gained mixed liquor processing.
Method the most according to claim 1, it is characterised in that described expansion is to carry out in the flowing mutually of first alcohol and water
,
Optionally, described for methanol: the volume ratio of water is 6:4.
Method the most according to claim 1, it is characterised in that based on 1L semisolid LB culture medium, the consumption of described X-gal
It is 13mg/L～33mg/L.
Method the most according to claim 1, it is characterised in that described chromogenic reaction is to carry out 12～18h at 28～30 DEG C.
Method the most according to claim 1, it is characterised in that farther include: based on locus coeruleus linear transport value big
Little, determine the typing of N-acyl-homoserine lactone types of populations induction signal molecule in sample.
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Cited By (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 |
| CN113322269A (en) * | 2021-06-01 | 2021-08-31 | 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 (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040156856A1 (en) * | 1998-04-16 | 2004-08-12 | Kende Andrew S. | Immunogenic conjugates of gram-negative bacterial autoinducer molecules and antibodies raised against the same |
| 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 |
| US20080038201A1 (en) * | 2006-04-13 | 2008-02-14 | Klein Bruce S | Global regulator of morphogenesis and pathogenicity in dimorphic fungi and uses thereof |
| CN102925511A (en) * | 2012-06-19 | 2013-02-13 | 文才艺 | Staurosporine preparation method |
-
2016
- 2016-05-10 CN CN201610309598.1A patent/CN105974044B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040156856A1 (en) * | 1998-04-16 | 2004-08-12 | Kende Andrew S. | Immunogenic conjugates of gram-negative bacterial autoinducer molecules and antibodies raised against the same |
| 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 |
| US20080038201A1 (en) * | 2006-04-13 | 2008-02-14 | Klein Bruce S | Global regulator of morphogenesis and pathogenicity in dimorphic fungi and uses thereof |
| 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 |
|---|
| ANGELIKA LEHNER 等: "Biofilm Formation, Extracellular Polysaccharide Production, and Cell-to-Cell Signaling in Various Enterobacter sakazakii Strains: Aspects Promoting Environmental Persistence", 《JOURNAL OF FOOD PROTECTION》 * |
| 汪映: "副溶血弧菌群体感应与调控的初步研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑 》 * |
| 綦国红: "HPLC-MS法检测N-酰基-高丝氨酸内酯类信号分子", 《分析测试学报》 * |
| 綦国红: "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", 《中国优秀博士学位论文全文数据库 工程科技Ⅰ辑 》 * |
Cited By (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 |
| CN113322269A (en) * | 2021-06-01 | 2021-08-31 | 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 |
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