CN107177697A - Food-borne pathogens Visual retrieval probe and visible detection method - Google Patents
Food-borne pathogens Visual retrieval probe and visible detection method Download PDFInfo
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- CN107177697A CN107177697A CN201710600610.9A CN201710600610A CN107177697A CN 107177697 A CN107177697 A CN 107177697A CN 201710600610 A CN201710600610 A CN 201710600610A CN 107177697 A CN107177697 A CN 107177697A
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Abstract
The embodiment of the present invention provides a kind of food-borne pathogens Visual retrieval probe and visible detection method, and the Visual retrieval probe includes：In the first area design and the SmartAmp primed probes of synthesis of the previously selected gene as object bacteria couple candidate detection site, 5 ' ends of the SmartAmp primed probes connect golden nanometer particle and the predetermined fluorescent particles corresponding with the object bacteria by the first active group；And in the second area design and the fluorescence-encoded nano-probe of synthesis of the previously selected gene as object bacteria couple candidate detection site, 5 ' ends of the fluorescence-encoded nano-probe connect golden nanometer particle and the predetermined fluorescent nano particles corresponding with the object bacteria by the second active group.The embodiment of the present invention, which need not add fluorescent dye, to observe result by uviol lamp, be hybridized by fluorescence-encoded nano-probe and detected sample, realize that signal amplifies, and can further improve food-borne pathogens detection sensitivity and detection flux.
Description
Technical field
The present invention relates to food-borne pathogens detection technique field, and in particular to a kind of food-borne pathogens Visual retrieval
Probe and visible detection method.
Background technology
The detection means of food-borne pathogens is varied, can summarize and be divided into following three major types：
1st, traditional cultural method, by the Selective Separation of bacterium, macroscopic characteristic bacterium colony on flat board is carried out true
Recognize, and to the bacterium colony isolate using triple sugar iron agar, lysine decarboxylase experiment, peptone water, urea agar, potassium cyanide etc.
Carry out biochemical test and multivalence somatic antigen（O）, multivalence flagellar antigen（H）Deng Serologic detection, finally to make identification.Typically
Overall process takes at least 4~7 days, can just draw clear and definite diagnostic result.Although, the method take it is cumbersome, because its cost compared with
It is low and relatively stable, as the goldstandard of inspection and quarantine industry.
2nd, based on immunologic detection method, generally as prescreening method, detected positive finally also need through
Traditional cultural method is crossed further to be confirmed, and it is larger by such environmental effects, and the result between batch may be poor
Different larger, its detection sensitivity also tends to relatively low.Colloidal gold immunochromatographimethod technology has the advantage of itself in quick detection, its side
Method has obtained U.S. official setting analysis chemist association（AOAC International）Official recognition, can be to the sramana in sample
Salmonella, Escherichia coli O 157 etc. are quickly detected.The method is more quick compared with traditional cultivation, ELISA etc., but its
Sensitivity is not so good as PCR method, and the detection based on immunological method, is influenceed larger by environment, sample substrate etc., can only be used as one
Plant prescreening method.
3rd, the detection method based on molecular biology, this method carries out nucleic acid extraction firstly the need of to detection sample, even if
There is the application of Full automatic instrument for extracting nucleic acid, still some are cumbersome in the pretreatment process for carrying out a large amount of samples, and this instrument
Device is expensive, operates not simple enough, application has inconvenience.Round pcr is ratio in food-borne pathogens detection
Relatively successfully, a series of commercially available reagent box have been developed, such as comma bacillus, salmonella, Shigella, Bacillus cereus,
The PCR detection kits such as ETEC, part has obtained AOAC official recognitions.In addition to Standard PCR technology, multi-fluorescence
The application of round pcr also increasingly increases.
At present, though the method for detection food-borne pathogens has a variety of, still had in terms of easy to operate, sensitivity
Some shortcomings：It is long, higher if desired for valuable instrument, cumbersome operating procedure, complicated sample pre-treatments, detection cycle
False positive, actual sample detection there is sensitivity deficiency etc..Particularly with laboratories, conventional selective training is also rested on
The method of supporting is detected to pathogenic bacteria, lacks a kind of accurate, the highly sensitive Fast Detection Technique of simplicity.
Therefore, cumbersome sample pre-treatments are not needed, the quick, highly sensitive of pathogenic bacteria, high specific detection can be realized, and
Valuable instrument is not needed, the quick determination method that can carry out popularization and application in laboratories becomes the weight of this area research
Point direction.Need to possess several factors to solve the above problems：1）Sample simplifies processing, without carry out the extraction of nucleic acid with it is pure
Change；2）The highly sensitive and height of detection method is special；3）Various pathogens can be detected simultaneously；4）Without expensive instrument, Neng Gou
Laboratories carry out related work.
2007, Mitani etc. existed《Nature Methods》On report a kind of new isothermal amplification technique, i.e.,
SmartAmp technologies（Smart Amplification Process Technology）, it uses 5 primers and DNA mismatch knot
Hop protein（MutS）The amplification of target product is carried out, " definitely special " of amplified production is can guarantee that, the current technology is mainly used in
SNP（Single Nucleotide Polymorphisms, SNPs）Parting and the detection of mutation.2013
Year, Military Medical Science Institute Liu Xue woodss, Song Hongbin et al. have delivered a summary for introducing SmartAmp technologies, and this article is pointed out
Pathogen detection method in food, environment, although there is not yet correlative study is reported, but the principle of SmartAmp methods is equally fitted
With.SmartAmp technologies are from the thick end for being prepared into amplification of sample, and whole detection process 20~60 minutes is not in non-spy
The opposite sex, if there is amplified signal to mean that the presence of object, and also the amplification ability of SmartAmp technologies is regular-PCR technology
100 times[14,15].Its " absolute " specificity gives the credit to the uniqueness of design of primers（5 primers TP, FP, OP1, OP2, BP）, and
DNA mismatch associated proteins（MutS）The absolute special of amplification procedure even more being strengthened, non-specific letter is caused without may proceed to amplification
Number appearance.
SmartAmp technologies and other kinds of isothermal amplification technique, such as ring mediated isothermal amplification（Loop-mediated
Isothermal amplification, LAMP）, rely on nucleotide sequence amplification（Nucleic acid sequence-based
Amplification, NASBA）, chain substitute amplification（Strand displacement amplification, SDA）, rolling ring expand
Increase（Rolling circle amplification, RCA）Etc. being entirely different concept.It is the most similar with SmartAmp technologies
Be exactly LAMP isothermal duplications, the maximum advantage of SmartAmp technologies is can to avoid the appearance of primer dimer, makes background signal
It is preferably minimized, reaches theoretic " absolute " specifically, and LAMP technology causes to produce the production of dumbbell shaped using 2 TP primer pairs
Thing, easily causes primer dimer.Current isothermal amplification technique has been widely applied to based on nucleic acid-templated various types of
In detection technique, but it is exactly all the time of a relatively high to template purity requirement as Standard PCR, can equally produces non-specific expansion
The problems such as increasing, and SmartAmp technologies require low to sample quality, DNA only needs simple purification, even without purifying, and it can overcome the disadvantages that
To the deficiency of above general type isothermal amplification technique.
Traditional SmartAmp technologies are to use fluorescent dye, directly observation detection signal.Although SmartAmp technologies can
To avoid the generation of primer dimer to the full extent, but the sample matrices of complexity are run into, using the just easily production of fluorescent dye pattern
Raw false sun, causes testing result accuracy not enough.
The content of the invention
The technical problem to be solved of the embodiment of the present invention is there is provided a kind of food-borne pathogens Visual retrieval probe,
To simplify testing process, and improve detection efficiency, sensitivity and accuracy.
The technical problem further to be solved of the embodiment of the present invention is that there is provided a kind of food-borne pathogens Visual retrieval
Method, to simplify testing process, and improves detection efficiency, sensitivity and accuracy.
In order to solve the above technical problems, the technical scheme that the embodiment of the present invention is used is：A kind of food-borne pathogens are provided
Visual retrieval probe, including：
Draw in the SmartAmp of first area design and the synthesis of the previously selected gene as object bacteria couple candidate detection site
Physical prospecting pin, 5 ' end introducings of the primed probe have the first active group and connect Jenner's grain of rice by first active group
The sub and predetermined fluorescent particles corresponding with the object bacteria；And
Received in the second area design of the previously selected gene as object bacteria couple candidate detection site and the fluorescence-encoded of synthesis
Rice probe, 5 ' end introducings of the fluorescence-encoded nano-probe have the second active group and connected by second active group
Golden nanometer particle and the predetermined fluorescent nano particles corresponding with the object bacteria.
Further, designed in the second area of the fluorescence-encoded nano-probe of design of the gene in the couple candidate detection site
There are multiple fluorescence-encoded nano-probe binding sites, design and synthesize a fluorescence-encoded nanometer in each binding site and visit
Pin and constitute the fluorescence-encoded nano-probe in many sites.
Further, first active group and the second active group are selected from following group：Amino, sulfydryl or carboxyl.
Further, the base number of the fluorescence-encoded nano-probe in many sites is 10-30.
Further, the fluorescent nano particles use rare-earth fluorescent nano-particle.
On the other hand, the embodiment of the present invention also provides a kind of food-borne pathogens visible detection method, including：
The gene in the couple candidate detection site of the Yu Xianxuanding food-borne pathogens as object bacteria；
SmartAmp primed probes are separately designed and synthesize in the different zones of the gene in the couple candidate detection site and fluorescence is compiled
Code nano-probe obtains Visual retrieval probe, and 5 ' ends of the SmartAmp primed probes are connected by means of the first active group
Golden nanometer particle and the predetermined fluorescent nano particles corresponding with the object bacteria, the fluorescence-encoded nano-probe
5 ' ends be connected with golden nanometer particle and predetermined corresponding with the object bacteria glimmering by means of the second active group
Light nano-particle；
SmartAmp amplifications are carried out to measuring samples using Visual retrieval probe, then observed under ultra violet lamp,
The fluorescence signal arrived according to the observation, determines whether contain the food-borne pathogens in the measuring samples；
When failing to detect containing food-borne pathogens after being expanded, further using the Visual retrieval probe with treating
Sample product are hybridized, and are then observed again under ultra violet lamp, the fluorescence signal arrived according to the observation, it is determined that described treat
Whether contain the food-borne pathogens in sample product.
Using above-mentioned technical proposal, the embodiment of the present invention at least has the advantages that：The present invention is by improving
SmartAmp technologies, novelty introduces fluorescence-encoded nano-probe, and the hypersensitive Visual retrieval of food-borne pathogens can be achieved.
By holding mark fluorescent nano-particle the 5 ' of SmartAmp primed probes, i.e., probe is also served as primer, so expand it
Afterwards, its product carries fluorophor, and result can be observed by uviol lamp without adding fluorescent dye.And it is another in gene
Fluorescence-encoded nano-probe is also designed in region, is hybridized by fluorescence-encoded nano-probe and detected sample, realizes that signal amplifies,
It can further improve food-borne pathogens detection sensitivity and detection flux.The embodiment of the present invention is amplified by signal twice,
Set up a kind of fluorescence-encoded nano-probe technologies of new primed probe type SmartAmp- and realize that the signal of quick " PCR type " is put
Greatly, the high special pathogenic bacteria Visual retrieval of hypersensitive is carried out.
The technical scheme of the embodiment of the present invention can synchronously detect a variety of food-borne pathogens that may be present in food, can
Detected, the corresponding probe of each pathogenic bacteria can be yet mixed so that the corresponding probe of each pathogenic bacteria is realized in different reaction tubes respectively
Detected together, due to without cumbersome Sample pretreatment process, only the pre- of collective media need to be carried out to detection sample
Amplification, simple process can carry out the amplification of signal, and the process expanded is the process of detection, i.e. the technology amplified signal
" definitely special ".Because SmartAmp amplifications and Visual retrieval are without expensive instrument, the water-bath of common laboratory and ultraviolet
Lamp can be met, and be highly suitable for laboratories and carried out substantial amounts of pathogenic bacteria screening.
In addition, design primed probe of the embodiment of the present invention and fluorescence-encoded nano-probe are used for SmartAmp amplification systems,
Because secondary singal amplifies strategy, for the extremely low pathogenic bacteria of content so that Visual retrieval can be realized.And it is higher for content
Pathogenic bacteria, without hybridizing with fluorescence-encoded nano-probe, because visualization can be achieved based on primed probe type SmartAmp amplifications
Detection.
The embodiment of the present invention also further can form multiple fluorescence-encoded nanometers using many site multiple labelings in detection zone
Probe, the quantitative analysis of terminal product can be realized by hybridization, can further improve detection sensitivity and detection flux.With
This many site nano-probe design concept derived, can promote the use of other detection architectures, further improve detection sensitive
Degree.
Brief description of the drawings
Fig. 1 is the design structure schematic diagram of food-borne pathogens Visual retrieval probe one embodiment of the present invention.
Fig. 2 is the schematic flow sheet of food-borne pathogens visible detection method one embodiment of the present invention.
Fig. 3 is that food-borne pathogens Visual retrieval probe of the present invention screens effect ratio in candidate drugs in the design process
The schematic flow sheet of preferable primer.
Embodiment
The present invention is described in further detail with specific embodiment below in conjunction with the accompanying drawings.It should be appreciated that following signal
Property embodiment and illustrate only to be used for explaining the present invention, it is not as a limitation of the invention, moreover, in the case where not conflicting,
The feature in embodiment and embodiment in the present invention can be combined with each other.
As shown in figure 1, one embodiment of the invention provides a kind of food-borne pathogens Visual retrieval probe, including：
Draw in the SmartAmp of first area design and the synthesis of the previously selected gene as object bacteria couple candidate detection site
Physical prospecting pin, 5 ' end introducings of the SmartAmp primed probes have the first active group and connected by first active group
Golden nanometer particle and the predetermined fluorescent particles corresponding with the object bacteria；And
Received in the second area design of the previously selected gene as object bacteria couple candidate detection site and the fluorescence-encoded of synthesis
Rice probe, 5 ' end introducings of the fluorescence-encoded nano-probe have the second active group and connected by second active group
Golden nanometer particle and the predetermined fluorescent nano particles corresponding with the object bacteria.
In specific embodiment, first active group and the second active group are selected from following group：Amino, sulfydryl or carboxylic
Base.First active group and the second active group can build complete fluorescence probe for identical group.
And the fluorescent nano particles use rare-earth fluorescent nano-particle, furthermore, it is possible to which the mixed proportion by adjusting Eu/Tb, is realized
Color adjustment function.
The present invention introduces fluorescence-encoded nano-probe by improving SmartAmp technologies, novelty, and food-borne can be achieved and causes
The hypersensitive Visual retrieval of germ.By holding mark fluorescent nano-particle the 5 ' of SmartAmp primed probes, that is, it is used as and draws
Thing also serves as probe, after so expanding, and its product carries fluorophor, can be by ultraviolet without adding fluorescent dye
Lamp observes result.And fluorescence-encoded nano-probe is also designed in another region of gene, by fluorescence-encoded nano-probe with it is to be checked
Sample hybridization is surveyed, realizes that signal amplifies, food-borne pathogens detection sensitivity and detection flux is can further improve.The present invention
Embodiment is amplified by signal twice, sets up a kind of new fluorescence-encoded nano-probe technologies of primed probe type SmartAmp-
The signal amplification of quick " PCR type " is realized, the high special pathogenic bacteria Visual retrieval of hypersensitive is carried out.
In fig. 1 it is illustrated that having respectively with enterohemorrhagic escherichia coli（Ecoli）O157:H7, salmonella
（Salmonella）Specific gene and Listeria monocytogenes（Listeria）Specific gene is designed corresponding Visual retrieval and visited
The structural representation of pin.
With reference to shown in Fig. 1, in one embodiment, in the fluorescence-encoded nanometer of design of the gene in the couple candidate detection site
The second area of probe is designed with multiple fluorescence-encoded nano-probe binding sites, designs and synthesizes in each binding site
One fluorescence-encoded nano-probe and constitute the fluorescence-encoded nano-probe in many sites.In the specific implementation, many site fluorescence
The base number of coding nano-probe is controllable to 10-30, base number with specific reference to purpose product hybridization temperature（45-60
℃）To determine.Base number is few, can simplify the design of probe.
The present embodiment can realize terminal product by hybridization when in use by forming multiple fluorescence-encoded nano-probes
Quantitative analysis, can further improve detection sensitivity with detection flux.The many site nano-probes design derived with this
Theory, can promote the use of other detection architectures, further improve detection sensitivity.
On the other hand, as shown in Fig. 2 the embodiment of the present invention also provides a kind of based on the visualization inspection of above-mentioned food-borne pathogens
The visible detection method of probing pin, including：
S1, the gene in the couple candidate detection site of the preselect food-borne pathogens as object bacteria；
S2, SmartAmp primed probes and fluorescence are separately designed and synthesize in the different zones of the gene in the couple candidate detection site
Encode nano-probe and obtain Visual retrieval probe, 5 ' ends of the SmartAmp primed probes are connected by means of the first active group
Golden nanometer particle and the predetermined fluorescent nano particles corresponding with the object bacteria are connect, the fluorescence-encoded nanometer is visited
5 ' ends of pin are connected with golden nanometer particle and predetermined corresponding with the object bacteria by means of the second active group
Fluorescent nano particles；
Measuring samples are carried out SmartAmp amplifications using Visual retrieval probe, then seen under ultra violet lamp by S3
Examine, the fluorescence signal arrived according to the observation, determine whether contain the food-borne pathogens in the measuring samples；
S4, when failing to detect containing food-borne pathogens after being expanded, further utilizes the Visual retrieval probe
Hybridized with measuring samples, then observed again under ultra violet lamp, the fluorescence signal arrived according to the observation determines institute
State in measuring samples and whether contain the food-borne pathogens.
Illustrate by way of example below the design and building-up process of food-borne pathogens Visual retrieval probe of the present invention with
And carry out the method and step of Visual retrieval.
First, the design and synthesis of SmartAmp primers
1）Common food-borne pathogens are chosen for research object, for example：Salmonella, enterorrhagia Bacillus coil 0157：H7, list
Increase Listeria etc., salmonella is with reference to GB/T4789.4, Escherichia coli O 157:H7 is according to GB/T4789.6, single increasing Liszt
Bacterium is carried out according to GB/T4789.30 methods.
According to the specific gene of the report of document and the NCBI above-mentioned food-borne pathogens announced, the time of object bacteria is selected
Detection site, i.e. target gene are selected, for example：Enterorrhagia Bacillus coil 0157:H7 rfbE genes（Coded product：O157 antigens,
Genbank sequence numbers S83460）, hlyA genes（Coded product：Hemolysin, Genbank sequence numbers X94129）, slt1 genes
（Coded product：Shiga-like toxin 1, Genbank sequence numbers M19473）, slt2 genes（Coded product：Shiga-like toxin 2,
Genbank sequence numbers Z37725）Deng.The common target gene and its primer sequence of part food-borne pathogens can be as shown in the table：
2）According to selected target gene, using SmartAmp primer-design softwares, candidate drugs are designed.Can according to software scoring
To obtain multipair primer, obtained successively after multipair primer from high score to low point, carry out preliminary screening.The primer that screens and its
Complementary series is designed to primed probe, introduces the first active group at its 5 ' end, for example：Amino, sulfydryl, carboxyl etc., then pass through
First active group and gold nano, fluorescence nano, conventional fluorescent dyestuff（For example：FAM, Texas Red, TAMRA etc.）Knot
Close.
2nd, the design and synthesis of the fluorescence-encoded nano-probe in many sites
Many fluorescence-encoded nano-probe designs in site are consistent with SmartAmp design of primers region, after selected target gene, you can
It is designed in this target genetic region, the design of many fluorescence-encoded nano-probes in site is visited than traditional molecular beacon, TaqMan
Pin is simpler, alternatively, many fluorescence-encoded nano-probe base numerical control systems in site within 10-30, with specific reference to purpose
Products thereof temperature（45-60℃）To determine its base number.Obtain after its sequence and its complementary series, carry out fluorescence-encoded receive
Rice probe synthesis, and the second active group is introduced at 5 ' ends of fluorescence-encoded nano-probe, and pass through second active group
Connect gold nano and fluorescence nano.The realization of fluorescence-encoded function passes through multicolor fluorescence nanometer and designed many site nucleic acid sequences
Row are combined.Multicolor fluorescence nanometer, by adjusting Eu/Tb mixed proportion, realizes polychrome function using rare-earth fluorescent nanometer,
For salmonella, Escherichia coli O 157:The exclusive gene of the pathogenic bacteria such as H7, Listeria monocytogenes designs specific fluorescence coding
Nano-probe, multiple probe binding sites are designed in target genetic region.The many fluorescence-encoded nano-probes in site and detected sample
After hybridization, fluorescence report sequence can be isolated, so as under ultra violet lamp, realize secondary amplification, Neng Gouyong
The fluorescence signal being observed visually, realizes Visual retrieval.
3rd, the research of the direct amplification system of nucleic acid extraction is exempted from
The advantage of SmartAmp technologies is " amplification procedure i.e. testing result ", and SmartAmp systems will to the purity of sample
Ask not high, the detection so for food-borne pathogens is even more important, detecting step can be greatly simplified, strengthen the reality of technology
Application potential.On SmartAmp technical advantages, the improved SmartAmp technologies of the embodiment of the present invention are compared to document report
SmartAmp technologies have more preferable environmental resistance.Mainly set about from the following aspects：1）Carried out to above-mentioned primer pair
After simple experiment, that group of primer that amplification rate is very fast, product amount is larger is chosen（TP、FP）It is used as the excellent of direct amplification system
Change primer, take purified bacterium solution DNA and not purified bacterium solution DNA to carry out contrast test respectively experiment every time；2）With
Different PCR additives are grouped, and are divided into DMSO groups, Betaine groups, MgCl2Group, trehalose group, formamide group,
Tween20 groups etc., prepare the diluted concentration of series, synchronize amplification respectively；3）Learn from else's experience the bacterium solution DNA, not purified of purifying
Bacterium solution, the bacterium solution through simply centrifuging and pure water group tested, with reference to above-mentioned PCR additives, draw in simple TP, FP
Under the conditions of thing pair, the optimization tested using Orthogonal Method.
4th, SmartAmp technology for detection food-borne pathogens substance system
Preliminary identification is carried out using primed probe type SmartAmp detection architectures, and detects corresponding pathogenic bacteria：
1）To the primed probe designed, the experiment of series is carried out, the relatively good primer of effect in candidate drugs is filtered out and carries out
Follow-up experiment, as shown in figure 3, the step of screening includes：
A, candidate target primer screening；
B, synthesis and the designed primed probe of experiment, in the case of no Taq MutS presence, are first carried out to FP and TP primers
Experiment, tests all primers, including BP, OP1, OP2 afterwards；
C, selection yield and fastest primer are tested；
D, further Optimum Experiment condition, if necessary from step (b) since new；
E, use Taq MutS, the specificity of test reaction system；
F, return to step (d), the specificity of further optimization system.
2）The result of experiment is embodied with reference to directly amplification, the ideal additive of its optimization is added to SmartAmp
In system, including it is all to primer in the presence of, carry out preliminary experiment, and according to circumstances finely tune corresponding concentration.
5th, multiple simultaneous detection architecture
The nanometer color marked by the fluorescence-encoded nano-probe of primed probe and many sites is different, to realize that variety classes is caused
The identification of germ.Such as Escherichia coli O 157:On H7, the fluorescence-encoded nano-probe of its primed probe and many sites mark have
The nano-particle of green fluorescence, and quencher is gold nano.The either fluorescence-encoded nanometer of primed probe or many sites
Probe, when in static systems, occurs FRET, green is glimmering because gold nano and fluorescence nano are closer to the distance
Light is quenched by gold nano.When product amplification in system, its product binding ability is even more than primed probe and many sites are fluorescence-encoded
Nano-probe, so that gold nano departs from probe, makes probe light, realizes detection.
6th, actual sample detection is evaluated
Be intended to can be used among real work for set up system, to sample it is actually detected from checking sensitivity, it is special
Property, set about in terms of reappearance, accuracy, determined using classical selective cultivation, fluorescent PCR method, automatic luciferase mapping
Experiment is compared in method Mini-VIDAS.It is different from the detection technique based on nucleic acid amplification in the past, it is provided in an embodiment of the present invention
The technical scheme that SmartAmp primed probes combine the fluorescence-encoded nano-probe in many sites can greatly simplify the pre-treatment of sample
Journey, is being expanded in advance to sample, is carried out that after increasing bacterium the extraction of nucleic acid need not be carried out, is only needed simple centrifugal treating, can
Carry out the SmartAmp amplifications of sample.Simplify the sample operation program of processing, manpower, thing can be saved in actual sample detection
Power cost, greatly improves detection efficiency.
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of changes, modification can be carried out to these embodiments, replace without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention limits by appended claims and its equivalency range.
Claims (10)
1. a kind of food-borne pathogens Visual retrieval probe, it is characterised in that：Including：
Draw in the SmartAmp of first area design and the synthesis of the previously selected gene as object bacteria couple candidate detection site
Physical prospecting pin, 5 ' end introducings of the SmartAmp primed probes have the first active group and connected by first active group
Golden nanometer particle and the predetermined fluorescent particles corresponding with the object bacteria；And
Received in the second area design of the previously selected gene as object bacteria couple candidate detection site and the fluorescence-encoded of synthesis
Rice probe, 5 ' end introducings of the fluorescence-encoded nano-probe have the second active group and connected by second active group
Golden nanometer particle and the predetermined fluorescent nano particles corresponding with the object bacteria.
2. food-borne pathogens Visual retrieval probe according to claim 1, it is characterised in that：In the couple candidate detection
The second area of the gene in site is designed with multiple fluorescence-encoded nano-probe binding sites, is designed in each binding site
And synthesize a fluorescence-encoded nano-probe and constitute the fluorescence-encoded nano-probe in many sites.
3. food-borne pathogens Visual retrieval probe according to claim 1, it is characterised in that：First active group
Group and the second active group are selected from following group：Amino, sulfydryl or carboxyl.
4. food-borne pathogens Visual retrieval probe according to claim 2, it is characterised in that：Many site fluorescence
It is 10-30 to encode the base number of nano-probe.
5. food-borne pathogens Visual retrieval probe according to claim 1, it is characterised in that：The fluorescence nano grain
Son uses rare-earth fluorescent nano-particle.
6. a kind of food-borne pathogens visible detection method, it is characterised in that：Including：
The gene in the couple candidate detection site of the Yu Xianxuanding food-borne pathogens as object bacteria；
The spy of SmartAmp primers is separately designed and synthesized in the first area of the gene in the couple candidate detection site and second area
Pin and fluorescence-encoded nano-probe obtain Visual retrieval probe, and 5 ' ends of the SmartAmp primed probes are lived by means of first
Property the group connection golden nanometer particle and predetermined fluorescent nano particles corresponding with the object bacteria, the fluorescence volume
5 ' ends of code nano-probe are connected with golden nanometer particle and the predetermined and object bacteria by means of the second active group
Corresponding fluorescent nano particles；
SmartAmp amplifications are carried out to measuring samples using Visual retrieval probe, then observed under ultra violet lamp,
The fluorescence signal arrived according to the observation, determines whether contain the food-borne pathogens in the measuring samples；
When failing to detect containing food-borne pathogens after being expanded, further using Visual retrieval probe with treating sample
Product are hybridized, and are then observed again under ultra violet lamp, the fluorescence signal arrived according to the observation, it is determined that described treat sample
Whether contain the food-borne pathogens in product.
7. food-borne pathogens visible detection method according to claim 6, it is characterised in that：In the couple candidate detection
The second area of the gene in site is designed with multiple fluorescence-encoded nano-probe binding sites, is designed in each binding site
And synthesize a fluorescence-encoded nano-probe and constitute the fluorescence-encoded nano-probe in many sites.
8. food-borne pathogens visible detection method according to claim 6, it is characterised in that：First active group
Group and the second active group are selected from following group：Amino, sulfydryl or carboxyl.
9. food-borne pathogens visible detection method according to claim 7, it is characterised in that：Many site fluorescence
It is 10-30 to encode the base number of nano-probe.
10. food-borne pathogens visible detection method according to claim 6, it is characterised in that：The fluorescence nano
Particle uses rare-earth fluorescent nano-particle.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402272A (en) * | 2018-08-24 | 2019-03-01 | 暨南大学 | Primer sets, kit and method based on intelligent constant-temperature amplification technique detection Listeria monocytogenes |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324542A (en) * | 2008-04-25 | 2008-12-17 | 山东出入境检验检疫局检验检疫技术中心 | Method for electrochemical detecting pig streptococcus by ring mediated isothermal amplification-micropore plate hybridization nanometer |
| CN102816855A (en) * | 2012-09-03 | 2012-12-12 | 华南师范大学 | Method for detecting food-borne pathogenic bacteria by using nucleic acid test strip based on hyper-branched rolling cycle amplification and kit |
| CN103642924A (en) * | 2013-12-10 | 2014-03-19 | 华南师范大学 | Method for quickly identifying food pathogenic bacteria subtype based on asymmetric polymerase chain reaction (PCR) combined test strip platform and kit |
| CN103993078A (en) * | 2014-05-09 | 2014-08-20 | 南京大学 | Nanoprobe for in situ imaging of telomerase activity in cells and preparation method thereof |
| CN104232784A (en) * | 2014-10-10 | 2014-12-24 | 山东农业大学 | Multiplex PCR (polymerase chain reaction) method for testing three main pathogens in beef |
| WO2017010001A1 (en) * | 2015-07-16 | 2017-01-19 | 森永乳業株式会社 | Microorganism detection method and microorganism detection kit |
-
2017
- 2017-07-21 CN CN201710600610.9A patent/CN107177697A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324542A (en) * | 2008-04-25 | 2008-12-17 | 山东出入境检验检疫局检验检疫技术中心 | Method for electrochemical detecting pig streptococcus by ring mediated isothermal amplification-micropore plate hybridization nanometer |
| CN102816855A (en) * | 2012-09-03 | 2012-12-12 | 华南师范大学 | Method for detecting food-borne pathogenic bacteria by using nucleic acid test strip based on hyper-branched rolling cycle amplification and kit |
| CN103642924A (en) * | 2013-12-10 | 2014-03-19 | 华南师范大学 | Method for quickly identifying food pathogenic bacteria subtype based on asymmetric polymerase chain reaction (PCR) combined test strip platform and kit |
| CN103993078A (en) * | 2014-05-09 | 2014-08-20 | 南京大学 | Nanoprobe for in situ imaging of telomerase activity in cells and preparation method thereof |
| CN104232784A (en) * | 2014-10-10 | 2014-12-24 | 山东农业大学 | Multiplex PCR (polymerase chain reaction) method for testing three main pathogens in beef |
| WO2017010001A1 (en) * | 2015-07-16 | 2017-01-19 | 森永乳業株式会社 | Microorganism detection method and microorganism detection kit |
Non-Patent Citations (10)
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402272A (en) * | 2018-08-24 | 2019-03-01 | 暨南大学 | Primer sets, kit and method based on intelligent constant-temperature amplification technique detection Listeria monocytogenes |
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