CN106995805A - A kind of engineering bacteriophage quick detection microorganism of lysozyme mark - Google Patents
A kind of engineering bacteriophage quick detection microorganism of lysozyme mark Download PDFInfo
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- CN106995805A CN106995805A CN201710185606.0A CN201710185606A CN106995805A CN 106995805 A CN106995805 A CN 106995805A CN 201710185606 A CN201710185606 A CN 201710185606A CN 106995805 A CN106995805 A CN 106995805A
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Abstract
This work have developed microorganism in the engineering bacteriophage kit Direct Analysis environmental and biological materialses that a lysozyme is marked, and the engineering bacteriophage of this lysozyme mark can be with specific recognition pathogenic microorganism.After bacteriophage identification combination microorganism is engineered, the DNA that its genetic engineering is transformed is injected in microbial body, the substantial amounts of bacteriophage with antalzyme protein newly of recombination expression amplification in microbial body.Project main contents are：Key design is engineered bacteriophage and microorganism responsive materials recognition mechanism and kinetics, investigates these and recognizes the functional module responded action rule in microbial rapid detection and the instant expression analysis of microorganism.This work is related in microorganism detection method " online ", the electromechanical engineering of " portable " and " full-automatic " and biology Complex Problem for solution and provides reference and reference.
Description
Technical field
A kind of engineering bacteriophage quick detection microorganism of lysozyme mark of present invention design.
Background technology
In marine environment, microbiological contamination, body eutrophication, microbiologic(al) corrosion, microorganism, which are stained, etc. is all
The apparent form of microbial threats human being's production life, is also the objective condition of rapid microbial detection technical need.Existing number
According to showing, the loss of microbiological contamination and microbial identification species speed are closely related, and the identification determination time is longer, loss
Also it is bigger.Because, one side microorganism growth and the speed propagated are fast, aggravate environmental pollution and human diseases；Separately
On the one hand microbe species can not be specified to lead to not implement specific aim protectiving scheme, and then cause some drugses or antiseptic
Abuse.In ISO4883-2003 standards, the microbial identification time is identified as the important of microorganism detection industrial grade division
Parameter.Therefore, taking effective method quick detection microorganism microorganism disease and is stained effective in reduction marine environment
Means.
Microorganism detection and the theory of identification are extensively dependent on the response of microorganism feature product（Such as ATP responses luciferase,
Beta galactosidase response chloro- 3- indoles-β-D galactosides of the bromo- 4- of 5- and H2S responses Fe2+ of E. coli secretion etc.）、
The identification of microbial cell surface antigentic specificity (such as antibody-microbial cell, agglutinin-antimicrobial surface glycogen, antibiotic-
Antimicrobial surface specific site and aptamers-antimicrobial surface recognition site etc.) and microbial gene analysis（DNA and 16s
RNA）.For feature product response detection microorganism, it determines the microbe quantity in water sample, and its specificity is very weak.Pass through
Basionic is added in water sample, ATP in microbial cell is discharged, luciferase, this enzyme energy is added
Enough and ATP effects cause photochemical reaction, produce quantitative light intensity.To antimicrobial surface antigentic specificity identification for, itself in order to avoid
Microorganism is recognized and detects based on epidemic disease reaction, the data that this method is obtained are accurate, but can not be used as a kind of online live body
Detection instrument, is easily influenceed by extraneous factor.Our seminars have carried out a series of work in terms of microorganism detection：Explore
No signal mark based on enzyme linked immunoassay is quickly examined with the electro-chemistry immunity biology sensor of nano material signal mark
Survey marine microorganism.The expansion and comparative study of the two aspect work, new approaches are provided for the development of microorganism detection technology.It is right
For microbial gene analytical technology, it is widely used in microbial identification, and target spot primer used is generally according to micro- life
The 16S rRNA gene expression characteristicses sequences of thing and design, its test limit can reach 1 cfu mL-1, and weak point is to need spy
Different sample treatment and purifying, complex operation step have higher technical requirements.This project is from mentioned microorganism detection technique
Weak point is set out, and researches and develops portable microorganism care diagnostic instrument.
Lysozyme（lysozyme）Also known as muramidase（muramidase）Or N-acetylmuramide lycanohydrlase（N-
acetylmuramide glycanohydrlase）, it is a kind of alkaline enzyme that can be hydrolyzed and polysaccharide is sticked in pathogenic bacteria.Mainly by broken
The β between -acetylmuramic acid and NAG-Isosorbide-5-Nitrae glycosidic bond in bad cell wall, makes cell membrane insoluble glutinous many
Sugar decomposition causes cell wall rupture content to escape and make bacterolysis into soluble glycopeptide.Lysozyme can also with it is negatively charged
Virus protein directly in conjunction with, with DNA, RNA, apoprotein formation double salt, make virally inactivated.Therefore, the enzyme have antibacterial,
Anti-inflammatory, antiviral etc. are acted on.Recently, our laboratories find that there is lysozyme signal to mark and detection function.
Microorganism detection technology and imperfections, in addition it is also necessary to sustainable development and innovation.Microorganism detection field innovative point may
Based in terms of following four：One is to utilize the application of micro-nano Novel electronic devices and photoelectric device in microorganism detection.Two are
Multifunction device integrated system is separated to microorganism, screened and tested and analyzed.Three be the analysis system combination of Multi-example
Multimode detecting system is analyzed microorganism or synchronized to multichannel data acquisition system.Four be based on smart mobile phone just
Formula real-time test (point-of-care testing, POCT) technology is taken, makes the portable device and mobile Internet of personalization
With reference to.These are all the directions of innovation and development microorganism detection research.This work is reflected using biomolecule functionalization micron openings
Fixed and analysis microorganism, its signal detected, which mostlys come from biomolecule and target spot microorganism detection and can extend microorganism, to be passed through
The time of micron openings, and then obtain related microbial standard curve.
The content of the invention
To achieve the above object, the present invention use technical scheme for：
A kind of engineering bacteriophage quick detection microorganism of lysozyme mark, it is characterised in that：Including specific bacteriophage,
The lysozyme gene of functionalization.
Such as the bacteriophage of specificity microorganism in right 1, specificity microorganism includes bacteriophage, the gold for being directed to Escherichia coli
The bacteriophage of staphylococcus aureus, the bacteriophage of salmonella, the bacteriophage of vibrios, the bacteriophage of Enterobacter sakazakii, small intestine knot
The bacteriophage of enteritis Yersinia ruckeri, the bacteriophage of C.perfringens, the bacteriophage of clostridium botulinum, the bacteriophage of anaerobic bacteria,
The bacteriophage of bacillus cereus.
The enzyme functionalized bacteriophage such as bacteriolyze in right 2：Its feature includes：T4 bacteriophages, T7 bacteriophages, P2 bacteriophages,
P22 bacteriophages, bacteriophage lambda, the bacteriophages of φ 29.
The enzyme functionalized bacteriophage such as bacteriolyze in right 3, its feature is included in gp10B GFP terminal fusion lysozymes
Gene.
Brief description of the drawings
Fig. 1:The enzyme functionalized bacteriophage of bacteriolyze
（1）It is engineered bacteriophage；（2）Head；（3）It is coated gp-10B albumen；（4）Interior nucleoprotein；（5）Gp16 albumen；（6）gp15
Albumen；（7）Gp14 albumen；（8）Connector gp8；（9）Gp10B- lysozyme fusion proteins；（10）Gp6 and gp7 albumen；（11）
Gp11 and gp12 albumen；（12）Tail protein line gp17；（13）Afterbody；（14）For specified microorganisms host's associated proteins（Greatly
Enterobacteria, staphylococcus aureus, salmonella etc.）.
Fig. 2：Microbiological analysis system based on the enzyme functionalized bacteriophage of bacteriolyze.
Fig. 3：The standard curve of sulfate reducing bacteria detection.
Fig. 4：The standard curve of E. coli detection.
Fig. 5：The standard curve of staphylococcus aureus detection.
Fig. 6：The standard curve of salmonella detection.
Embodiment
Below by embodiment, the present invention will be further described.
Embodiment 1：
The detection of sulfate reducing bacteria：
The microorganism used in experiment utilizes bacteriolyze meat soup（Peptone 1%, sodium chloride 1%, yeast extract 0.5%, the mL of water 100）Suspend
Culture, single bacterium colony is centrifuged ten minutes in 4500 revs/min after incubated overnight under the conditions of 30 DEG C, 200 turns of shaking tables, and is buffered with PBS
Solution is diluted to various concentrations.
By 100 μ L concentration（10 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（102 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（103 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（104 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（105 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（106 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（107 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（108 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
The data of summary, draw the standard curve of microorganism under various concentrations.
Embodiment 2：
E. coli detection
The microorganism used in experiment utilizes bacteriolyze meat soup（Peptone 1%, sodium chloride 1%, yeast extract 0.5%, the mL of water 100）Suspend
Culture, single bacterium colony is centrifuged ten minutes in 4500 revs/min after incubated overnight under the conditions of 30 DEG C, 200 turns of shaking tables, and is buffered with PBS
Solution is diluted to various concentrations.
By 100 μ L concentration（10 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（102 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（103 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（104 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（105 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（106 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（107 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（108 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
The data of summary, draw the standard curve of microorganism under various concentrations.
Embodiment 3：
Staphylococcus aureus detects
The microorganism used in experiment utilizes bacteriolyze meat soup（Peptone 1%, sodium chloride 1%, yeast extract 0.5%, the mL of water 100）Suspend
Culture, single bacterium colony is centrifuged ten minutes in 4500 revs/min after incubated overnight under the conditions of 30 DEG C, 200 turns of shaking tables, and is buffered with PBS
Solution is diluted to various concentrations.
By 100 μ L concentration（10 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（102 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（103 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（104 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（105 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（106 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（107 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（108 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
The data of summary, draw the standard curve of microorganism under various concentrations.
Embodiment 4：
Salmonella detection detection
The microorganism used in experiment utilizes bacteriolyze meat soup（Peptone 1%, sodium chloride 1%, yeast extract 0.5%, the mL of water 100）Suspend
Culture, single bacterium colony is centrifuged ten minutes in 4500 revs/min after incubated overnight under the conditions of 30 DEG C, 200 turns of shaking tables, and is buffered with PBS
Solution is diluted to various concentrations.
By 100 μ L concentration（10 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur base,
And reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, is cultivated 30 minutes,
With the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains the microorganism letter under the concentration
Number.
By 100 μ L concentration（102 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（103 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（104 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（105 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（106 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（107 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
By 100 μ L concentration（108 cfu ml-1）The bacteriophage of bacterium solution and lysozyme mark is added to batch cultur
Base, and reacted 8 hours in 37 °C.The oligosaccharides fluorescein molecule of the marked by coumarin of lysozyme response is added, 30 are cultivated
Minute, with the excitation source actuated sensor platform of microbe diagnosis instrument, optical signal is produced, measurement obtains micro- life under the concentration
Thing signal.
The data of summary, draw the standard curve of microorganism under various concentrations.
Claims (4)
1. a kind of engineering bacteriophage quick detection microorganism of lysozyme mark, it is characterised in that：Including specific phagocytosis
The lysozyme gene of body, functionalization.
2. such as the bacteriophage of specificity microorganism in right 1, specificity microorganism includes bacteriophage for Escherichia coli, golden yellow
The staphylococcic bacteriophage of color, the bacteriophage of salmonella, the bacteriophage of vibrios, the bacteriophage of Enterobacter sakazakii, small intestine colon
The bacteriophage of scorching Yersinia ruckeri, the bacteriophage of C.perfringens, the bacteriophage of clostridium botulinum, the bacteriophage of anaerobic bacteria, wax
The bacteriophage of sample bacillus.
3. the enzyme functionalized bacteriophage such as bacteriolyze in right 2：Its feature includes：T4 bacteriophages, T7 bacteriophages, P2 bacteriophages, P22
Bacteriophage, bacteriophage lambda, the bacteriophages of φ 29.
4. the enzyme functionalized bacteriophage such as bacteriolyze in right 3, its feature is included in gp10B GFP terminal fusion lysozyme bases
Cause.
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