CN110205394A - Biosensor and method for detecting salmonella - Google Patents
Biosensor and method for detecting salmonella Download PDFInfo
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- CN110205394A CN110205394A CN201910389304.4A CN201910389304A CN110205394A CN 110205394 A CN110205394 A CN 110205394A CN 201910389304 A CN201910389304 A CN 201910389304A CN 110205394 A CN110205394 A CN 110205394A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention relates to the technical field of molecular biology, in particular to a biosensor and a method for detecting salmonella. The biosensor comprisesRPA reaction reagent, Lambda exonuclease cleavage reaction reagent, HCR reaction reagent and color reagent. The invention amplifies double-stranded nucleic acid of salmonella by a multienzyme system to obtain dsDNA phosphorylated at the 5' end. Lambda exonuclease digests the 5' -phosphorylated strand in dsDNA and the RPA product becomes single stranded. The universal linker is exposed, HCR reaction is started, and the released G-quadruplex is combined with Hemin to form G4 DNAzyme which can catalyze H2O2And oxidizing the colorless TMB into macroscopic blue oxTMB, and further detecting the salmonella through color change and light absorption value change. The method has the characteristics of visualization, universality, rapidness, high sensitivity and the like.
Description
Technical field
It is the present invention relates to technical field of molecular biology, in particular to a kind of for detecting the biosensor of salmonella
And method.
Background technique
Salmonellosis refers to as caused by various types salmonella to the mankind, domestic animal and wild brutish not similar shape
The general name of the disease of formula.The people of salmonella or the fecal pollution food of carrier are infected, can make one to poison by food.According to system
It counts in the bacterial species food poisoning of countries in the world, the salmonellal normal column umber one of food poisoning.
The detection technique of salmonella relies primarily on Enzyme-multiplied immune technique, DNA molecular nucleic acid probe or gene probe at present
Technology, biochemical identification technology, biosensor technology.Biosensor technology is most important excellent relative to traditional detection method
Point is that it has very high sensitivity, highly sensitive can be detected to target substance, this is mainly due to its to detection signal into
Gone amplification, it mainly includes two kinds that signal, which amplifies mode: one is the nucleic acid amplification technologies based on toolenzyme, as round pcr,
RCA technology, SDA technology and RPA technology etc..Another kind is not depend on enzyme signal amplifying technique, and such as hybridizing chain reaction, (HCR is anti-
It answers).
Relative to a series of amplification techniques of mistake mentioned above, the maximum difference HCR be do not need the participation of enzyme can
It reacts.Hybridization chain reaction is a kind of signal amplification technique of external self assembly, is that Dirks et al. was mentioned for the first time in 2004
Out.Its principle is to cause two different nucleic acid hair clips using a single stranded DNA alternately to identify, hybridization reaction occurs, is formed
The dsDNA nano wire of overlength, amplifies transducing signal.HCR's is easy to operate, and cost is relatively low and has compatibility well, will
Hybridization chain reaction is combined with other technologies, can construct many biosensors, as electrochemical sensor, biological pass
Sensor, Sidestream chromatography biosensor, visual biosensor etc. realize the detection to biomolecule such as nucleic acid, albumen.Mesh
Before, the Salmeterol fluticasone propionate technology based on HCR is more rarely seen.
Summary of the invention
In view of this, the present invention provides a kind of for detecting the biosensor and method of salmonella.Present invention tool
There is the features such as visualization, versatility, quick and hypersensitivity.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of for detecting the biosensor of salmonella, including RPA reaction reagent, Lambda core
Sour excision enzyme cleavage reaction reagent, HCR reaction reagent and colour reagent；RPA reaction reagent includes buffer, sequence such as SEQ ID
The downstream primer as shown in SEQ ID NO:2 of upstream primer, sequence shown in NO:1, sequence are led to as shown in SEQ ID NO:3
With primer, magnesium acetate and water.
In HCR technology, nucleic acid amplification product is mostly dsDNA, due to dsDNA have very high stability, be difficult with
Other technologies combine, and compatibility and detection sensitivity are all lower.In contrast, ssDNA has higher hybridization efficiency and detection
Sensitivity.Therefore, how to realize that conversion of the dsDNA to ssDNA is always the hot issue that scientists are paid close attention to.Lambda nucleic acid
Excision enzyme can complete this conversion, act on double-stranded DNA, gradually cut 5 ' mononucleotides along 5 ' -3 ' directions.Most suitable bottom
Object is the double-stranded DNA of 5 ' phosphorylations, and can also slowly degrade single stranded DNA and phosphorylated substrate, but the cutting speed at the end 5 '-OH
Than 5 '-PO4End is about 20 times slow, digests 100 times slower than double-stranded DNA of single stranded DNA or so.
As specific function nucleic acid, the sequence rich in guanine (G) can when combining with hemin (Hemin)
It is formed co-ordination complex (G4 DNAzyme), with peroxidase catalytic activity.The catalytic activity of the compound is than single
About 10 times of only Hemin high, the significant catalytic performance for improving Hemin itself.G4 DNAzyme is widely used in catalysis H2O2It adjusts
The redox reaction of section.For example, it can be catalyzed H2O2Colourless 3,3 ', 5,5 '-tetramethyl benzidines (TMB) are oxidized to indigo plant
The oxidation TMB (oxTMB) of color.The colour developing principle is simple, quickly and does not need enzymatic, is widely used to colorimetric bio sensing
The building of device.
The present invention provides a kind of Detection Methods of Salmonella, by the group of recombinase, polymerase and lambda exonuclease
It closes (RPL principle) and hybridization chain reaction (HCR) combines, construct a kind of chimeric based on universal joint triggering G- tetrad
The unmarked visual biosensor of the dual signal amplification of HCR detects salmonella.By multienzyme system to salmonella
Double-strandednucleic acid expanded, obtain largely 5 ' end phosphorylations dsDNA.Largely the amplification with phosphorylation produces in order to obtain
Object joined the universal sequence that one section of 5 ' end is phosphorylated in RPA reaction system, and the complementation of this section of universal sequence
Chain is named as universal joint.Lambda excision enzyme selectively digests 5 '-phosphorylation chains in dsDNA, in unwanted chain quilt
After digestion, RPA product becomes single-stranded.Universal joint is exposed, starting HCR reaction, and the G- tetrad in H1 hair clip is released
It releases.G- tetrad can form the G4 DNAzyme with peroxidase activity in conjunction with Hemin.This analogue enztme
H can be catalyzed2O2Colourless TMB is oxidized to macroscopic blue oxTMB, and then passes through the change of color change and light absorption value
Salmonella is detected.
In the present invention, sequence downstream primer as shown in SEQ ID NO:2 is the sequence such as SEQ ID of 5 ' end phosphorylations
Downstream primer shown in NO:2.
In the present invention, sequence universal primer as shown in SEQ ID NO:3 is the sequence such as SEQ ID of 5 ' end phosphorylations
Universal primer shown in NO:3.
In the present invention, Lambda exonuclease cleavage reaction reagent include Lambda exonuclease, buffer and
Water.
In the present invention, HCR reaction reagent includes hair clip H1, hair clip H2 and buffer.
Preferably, hair clip H1 sequence, as shown in SEQ ID NO:4, hair clip H2 sequence is as shown in SEQ ID NO:5.
In the present invention, colour reagent includes buffer, Hemin, TMB color developing agent and H2SO4。
In the present invention, TMB and H is contained in TMB color developing agent2O2。
The present invention also provides a kind of detection methods of salmonella, detect salmonella using above-mentioned biosensor,
Including RPA reaction, the enzyme reaction of Lambda Exonucleolytic, HCR reaction and chromogenic reaction.
Preferably, the system of RPA reaction is as follows:
Preferably, the system of RPA reaction is as follows:
Preferably, the condition of RPA reaction are as follows: 38~40 DEG C of 18~22min of reaction.
Preferably, the condition of RPA reaction are as follows: 39 DEG C of reaction 20min.
Preferably, the system of Lambda Exonucleolytic enzyme reaction are as follows:
It is preferred that the system of Lambda Exonucleolytic enzyme reaction are as follows:
Preferably, the condition of Lambda Exonucleolytic enzyme reaction are as follows: 36~38 DEG C of 18~22min of reaction.
Preferably, the condition of Lambda Exonucleolytic enzyme reaction are as follows: 37 DEG C of reaction 20min.
Preferably, the system of HCR reaction are as follows:
Preferably, the system of HCR reaction are as follows:
Preferably, the condition of HCR reaction are as follows: 36~38 DEG C of 28~32min of reaction.
Preferably, the condition of HCR reaction are as follows: 37 DEG C of reaction 30min.
Preferably, the system of chromogenic reaction are as follows:
Preferably, the system of chromogenic reaction are as follows:
Preferably, the condition of chromogenic reaction: HCR reaction product, Hemin, buffer be incubated for 19 at 36~38 DEG C~
21min is added after TMB color developing agent the 4~6min that develops the color at 36~38 DEG C, is eventually adding H2SO4Terminate reaction.
Preferably, the condition of chromogenic reaction: HCR reaction product, Hemin, buffer are incubated for 20min at 37 DEG C, are added
Develop the color at 37 DEG C 5min after TMB color developing agent, is eventually adding H2SO4Terminate reaction.
The present invention provides a kind of for detecting the biosensor and method of salmonella.The biosensor includes
RPA reaction reagent, Lambda exonuclease cleavage reaction reagent, HCR reaction reagent and colour reagent；RPA reaction reagent packet
Include buffer, sequence upstream primer as shown in SEQ ID NO:1, sequence downstream primer, sequence as shown in SEQ ID NO:2
Universal primer, magnesium acetate and water as shown in SEQ ID NO:3.The present invention is based on the visual inspections of two kinds of isothermal amplification technologies
The biosensor for surveying salmonella has the advantage that
(1) a large amount of dsDNA for having universal joint the design of universal joint: can be generated after RPA is expanded.By
The cutting of lambda exonuclease is, it can be achieved that conversion of the dsDNA to ssDNA, general to connect after the digestion of unwanted DNA chain
Head is exposed to realize the triggering to HCR.
(2) dsDNA is converted into ssDNA: double-strand RPA product is digested to ssDNA by Lambda excision enzyme, realizes dsDNA
Conversion to ssDNA.
(3) visualize: after HCR reaction occurs, the H1 hair clip comprising G- tetrad structure is opened.Alternately opening hair clip
During there is a large amount of G- tetrad to generate, can react with TMB, generate macroscopic blue realization to salmonella
Detection.More accurate experimental result in order to obtain, can be by measuring realization quantitative analysis to light absorption value.
(4) versatility: due to the introducing of universal joint, the present invention can be completed by changing the primer sequence of RPA amplification
Detection to different target substances.
(5) quick and hypersensitivity: this biosensor can realize the inspection to salmonella with the time of about 90min
It surveys, and the detection of individual cell level may be implemented.
Detailed description of the invention
Fig. 1 is that different proportion primer carries out RPA reaction result；Wherein, swimming lane 1 is RPA-F:RPA-R '=1:1；Swimming lane 2
It is RPA-F:RPA-R ': U-P=1:1:1；Swimming lane 3 is RPA-F:RPA-R ': U-P=2:2:1；Swimming lane 4 is RPA-F:RPA-R ':
U-P=3:2:1；Swimming lane 5 is RPA-F:RPA-R ': U-P=3:1:3；Swimming lane 6 is RPA-F:RPA-R=1:1；Swimming lane 7 is RPA-
F:RPA-R ': U-P=2:1:1；Swimming lane 8 is RPA-F:RPA-R ': U-P=3:1:2；
Fig. 2 is Lambda exonuclease concentration optimization result；Wherein, the concentration of swimming lane 1-8 is respectively as follows: 0.1 U/ μ L,
0.5 U/ μ L, 1 U/ μ L, 2 U/ μ L, 3 U/ μ L, 4 U/ μ L, 5 U/ μ L and 0 U/ μ L；
Fig. 3 is the different digestion times to carry out HCR reaction result；Wherein, the swimming lane 1-9 endonuclease reaction time be respectively 20min,
30min, 40min, 50min, 60min, 70min, 80min, 90min and 120min；
Fig. 4 is the test result verifying dsDNA and converting to ssDNA；(A) fluorescent value changes；Wherein, curve a is to pass through
Fluorescent value change curve after the cutting of lambda excision enzyme, curve b are without the fluorescent value change curve Jing Guo digestion；(B) and A
Corresponding agarose gel electrophoresis figure；(C) and the variation of (D) solubility curve；Wherein, curve 1 is not by lambda excision enzyme
The curve of cutting, curve 2 are the curve after the cutting of Lambda excision enzyme, and curve 3 is negative control；
Fig. 5 is the test result of different HCR hair fastener concentration and reaction time；(A) optimum results of HCR hair fastener concentration；Its
In, swimming lane 1-8 hair fastener concentration is respectively 10 μM, 8 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2 μM and 1 μM, and swimming lane 9 is negative control, nothing
Triggering carries out HCR reaction；(B) optimum results in difference HCR reaction time；Wherein, the reaction time of swimming lane 1-7 is respectively
20min, 30min, 40min, 50min, 60min, 90min, negative control；(C) the differential responses time carries out the suction of the product of HCR
Light value and colour developing result；
Fig. 6 is light absorption value of the various concentration salmonella at 450nm；
Fig. 7 is specificity analysis result.
Specific embodiment
The invention discloses a kind of for detecting the biosensor and method of salmonella, and those skilled in the art can be with
Present disclosure is used for reference, realization of process parameters is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications are to ability
It is it will be apparent that they are considered as being included in the present invention for field technique personnel.Method and application of the invention has been led to
Preferred embodiment is crossed to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to this paper institute
The methods and applications stated are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The present invention develop it is a kind of based on universal joint triggering G- tetrad be fitted into HCR dual signal amplification it is unmarked
Visual biosensor detects salmonella.[recombinase is expanded by double-strandednucleic acid of the multienzyme system to salmonella
Polymeric enzymatic amplification (Recombinase Polymerase Amplification, RPA)], it obtains largely in 5 ' end phosphorylations
dsDNA.The amplified production for largely having phosphorylation in order to obtain, joined one section of 5 ' end by phosphoric acid in RPA reaction system
The universal sequence of change, and the complementary strand of this section of universal sequence is named as universal joint.Lambda excision enzyme selectively disappears
Change 5 '-phosphorylation chains in dsDNA, after unwanted chain is digested, RPA product becomes single-stranded.Universal joint is exposed
Come, starting HCR reaction, the G- tetrad in H1 hair clip is released.G- tetrad can be in conjunction with Hemin, and formation has
The G4 DNAzyme of peroxidase activity.This analogue enztme can be catalyzed H2O2Colourless TMB is oxidized to macroscopic blue
OxTMB, and then salmonella is detected by the change of color change and light absorption value.
Agents useful for same or instrument can be by biosensor and method provided by the present invention for detecting salmonella
Market is bought.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1
1. experimental material
1.1 test strain
Salmonella (CGMCC 1.0090), Enterobacter sakazakii (CICC 21560), Listeria monocytogenes (CMCC
55004), staphylococcus aureus (ATCC 25923), vibrio parahemolyticus (CMCC20001), Escherichia coli (ATCC
43889) it is provided by China Agricultural University's Food Science and nutrition engineering college food safety laboratory.
1.2 main agents
Yeast extract, peptone, yeast extract, beef extract, glucose, dipotassium hydrogen phosphate, ammonium citrate, anhydrous acetic acid
Sodium, bitter salt, four hydrated sulfuric acids are violent, Tween 80, sodium chloride, agar, ethylenediamine tetra-acetic acid (EDTA), sodium hydroxide,
Trishydroxymethylaminomethane (Tris), glacial acetic acid, hemin (Hemin), agarose, 3,3 ', 5,5 '-tetramethyl biphenyls
Amine (TMB), RPA reaction kit, sulfuric acid, terminal deoxynucleotidyl transferase (TdT), double-stranded specific nuclease (DSN),
DGTP, dATP, DNA marker, DNA loading buffer, polyacrylamide, methylene diacrylamide, TEMED, SYBR
Gold, ammonium persulfate, boric acid, hydrochloric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium chloride, potassium chloride, Lambda exonuclease,
SYBR GreenⅠ。
2. design of primers
The universal sequence that outer plus one section of 5 ' end is phosphorylated on the primer of RPA, and G- tetrad sequence is embedded in core
RPA reverse primer (RPA-R ') and HCR hair fastener H1 are separately designed in sour hair fastener.All primers are by Invitrogen (Shanghai) trade
Co., Ltd's synthesis.
1 RPA primer of table and HCR hairpin
3.DNA is extracted
It takes 1mL bacterium solution to manage in 1.5mL EP, under the conditions of 4 DEG C, 5min is centrifuged with 12000r/min, discards supernatant liquid.Again
1mL ddH is added in Xiang Guanzhong2O, 12000r/min, 5min, 4 DEG C, centrifugation, discard supernatant liquid.Add 50 μ L ddH2O, it mixes,
Be placed in electric-heated thermostatic water bath 99 DEG C boil 10min after, set 20min on ice immediately.Then with 12000r/min, 4 DEG C from
Heart 5min takes supernatant, -20 DEG C of preservations.DNA profiling, which extracts, to be completed, and can be used for further detecting, all bacterium used in experiment
Class DNA is extracted with above method.
4. agarose gel electrophoresis
2 agarose gel electrophoresis method for detecting of table and step
5.RPA reaction
3 RPA reaction system of table
6.Lambda Exonucleolytic enzyme reaction
4 Lambda exonuclease reaction system of table
7.HCR reaction
5 HCR reaction system of table
8.TMB chromogenic reaction and uv-visible absorption spectra analysis
6 TMB color development system of table
9. Salmeterol fluticasone propionate
Present invention detection includes four reaction steps: (1) RPA is expanded；(2) Lambda exonuclease cleavage reaction；(3)
HCR reaction；(4) TMB chromogenic reaction and uv-visible absorption spectra analysis.Introduced into multienzyme system DNA profiling and primer into
Row amplification obtains the dsDNA of a large amount of 5 ' ends phosphorylation.Again under the circumscribed enzyme effect of Lambda, selectively digest in dsDNA
5 '-phosphorylation chains, after unwanted chain is digested, RPA product becomes single-stranded.Universal joint is exposed, and starting HCR is anti-
It answers, the G- tetrad in H1 hair clip is released.G- tetrad can be in conjunction with Hemin, and being formed has peroxidase activity
The G4 DNAzyme of property.This analogue enztme can be catalyzed H2O2Colourless TMB is oxidized to macroscopic blue oxTMB, Jin Ertong
The change for crossing color change and light absorption value detects salmonella.
Embodiment 2
The optimization of 1.RPA reaction condition
The present invention carries out RPA reaction first, and reaction condition is 39 DEG C, 20min.It is anti-that RPA is carried out with the primer of different proportion
It answers, finds out most suitable primer ratio and carry out constant-temperature amplification, produced after reaction with 2% agarose gel electrophoresis analysis amplification
Object.It is available, useful newly-designed primer carry out the product of RPA amplification will be than universal sequence (comparison sequence be not added
It is long to be shown in Table the DNA fragmentation 1) amplified by RPA-R:CCTCAATACTGAG CGGCTGCTCGCCTTTGCTGG；And when upstream is drawn
Object: downstream primer: when universal primer=3:1:2, electrophoretic band brightness ratio others band is bright (swimming lane 8), illustrates RPA product amount
Also at most (Fig. 1).
2. the optimization of Lambda exonuclease reaction condition
The optimization of 2.1 Lambda exonuclease reaction densities
First carry out RPA reaction first, 39 DEG C of reaction 20min, then by the lambda nucleic acid of RPA amplified production various concentration
After excision enzyme reacts 20min under the conditions of 37 DEG C, is analyzed with 2% agarose gel electrophoresis, compare various concentration
Product brightness after the cutting of lambda exonuclease.It obtains, after concentration increases to 3U/ μ L (swimming lane 5), enzyme concentration increases item
Band brightness does not change substantially, so the lambda Exonucleolytic enzyme concentration of 3U/ μ L is best (Fig. 2).
The optimization in 2.2 Lambda exonuclease reaction time
First progress RPA reaction first, 39 DEG C of reaction 20min, then by RPA amplified production outside 3 U/ μ L lambda nucleic acid
Enzyme cutting acts on lower 37 DEG C of reactions different time, and the cleaved products of differential responses time have then been carried out HCR reaction.Reaction produces
Object is analyzed with agarose gel electrophoresis, and the band disperse degree for comparing the bright dark and HCR reaction of product band is reacted
The selection of time.It can be clear that from figure, the reaction time extends to 120min from 20min, and HCR reaction product amount does not have substantially
It changes, therefore selects digestion time (Fig. 3) of the 20min (swimming lane 1) as lambda exonuclease.
2.3 Lambda exonucleases realize the verification result that dsDNA is converted to ssDNA
First progress RPA reaction first, 39 DEG C of reaction 20min, then by RPA amplified production outside 3 U/ μ L lambda nucleic acid
Enzyme cutting acts on lower 37 DEG C of reactions 20min, be added a certain amount of SYBR Green I fluorescent dye measure the fluorescence intensity of system into
Row, which is analyzed or analyzed using agarose gel electrophoresis, compares the variation of digestion front and back band brightness or in real-time fluorescence quantitative PCR
The measurement for carrying out solubility curve in instrument to digestion process, be added final concentration of 0.5 in the reaction system × SYBR Green I,
Compare the appearance position analysis experimental result of solubility curve to verify Lambda exonuclease and can realize that dsDNA turns to ssDNA
Change.In Fig. 4 result, curve a is the fluorescent value change curve after the cutting of lambda exonuclease, compared to not passing through
The curve b fluorescent value for crossing digestion significantly reduces, and band a brightness obviously weakens after the cutting of lambda exonuclease, and
There is Double-peak Phenomenon in solubility curve, and peak position shifts out, demonstrates lambda exonuclease and is acted on, and can incite somebody to action
DsDNA is converted into ssDNA (Fig. 4).
The optimization of 3.HCR reaction condition
First progress RPA reaction first, 39 DEG C of reaction 20min, then by RPA amplified production outside 3 U/ μ L lambda nucleic acid
Enzyme cutting acts on lower 37 DEG C of reactions 20min, then carries out HCR reaction, and reaction product carries out TMB chromogenic reaction and ultraviolet-ray visible absorbing
Spectrum analysis.The test result of each group HCR after reaction also carries out agarose electrophoretic analysis.HCR is as a kind of novel signal
Amplification method, its competitive hybridization between nucleic acid probe are self-assembled into a kind of nucleic acid nano structure as energy source, realize letter
Number amplification.Therefore, two hair clip H1 and H2 with specific nucleotide sequence and triggering for triggering this series reaction
There are vital effect in concentration ratio and reaction time to the number that final signal amplifies.Therefore, dense by comparing HCR hair fastener
Degree and HCR reaction time are systematically analyzed.As a result reaction effect preferably (figure when hair clip concentration is 10 μM (band 1) is proved
5A), the 20min reaction time (Fig. 5 B, 5C) is best.
4. visual biosensor sensitivity analysis
Salmonella is selected to carry out gradient dilution, selecting salmonella concentration is 101-108The sample of CFU/mL carries out DNA
Extraction, reacted under optimum reaction condition, by the measurement result of variation and light absorption value with TMB reaction color, into
The calculating of row detection sensitivity.In the reaction system, the light absorption value of experimental group increases with the increase of salmonella concentration, can
The yellow seen is also deeper and deeper, and salmonella concentration is 101Cfu/mL to 108When cfu/mL, it is linear for mapping, and phase
Close equation are as follows: A450nm=0.1571lg [salmonella (CFU/mL)]+0.4452, coefficient R2It is 0.9903, detection is limited to
4cfu/mL is suitble to quantitative detection (Fig. 6).
5. visual biosensor specificity is analyzed
The specificity analysis tested according to the peak optimization reaction system obtained and reaction condition of test, as shown in fig. 7,
Under the same test conditions, light absorption value of the salmonella at 450nm is apparently higher than other strains.The result shows that the inspection of building
The biosensor for surveying salmonella has excellent specificity (Fig. 7).
6. Salmeterol fluticasone propionate in ferment agent for sour milk
In order to verify the feasibility for the bioanalytical sensing platform that we build in practical applications, added in ferment agent for sour milk
The salmonella of various concentration is tested.The knot that the biosensor testing result and traditional bacterium colony for comparing building count
Fruit calculates the rate of recovery.The result shows that this method is close with the testing result obtained by colony counting method, actual sample is surveyed
The accuracy of examination shows good applicability of this method in reality in pathogen monitoring.
The rate of recovery of salmonella is added in 7 ferment agent for sour milk of table
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
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Claims (10)
1. a kind of for detecting the biosensor of salmonella, which is characterized in that including RPA reaction reagent, Lambda nucleic acid
Excision enzyme cleavage reaction reagent, HCR reaction reagent and colour reagent；The RPA reaction reagent includes buffer, sequence such as SEQ
Upstream primer shown in ID NO:1, sequence downstream primer as shown in SEQ ID NO:2, sequence are as shown in SEQ ID NO:3
Universal primer, magnesium acetate and water.
2. biosensor according to claim 1, which is characterized in that the Lambda exonuclease cleavage reaction examination
Agent includes Lambda exonuclease, buffer and water.
3. biosensor according to claim 1, which is characterized in that the HCR reaction reagent includes hair clip H1, hair clip
H2 and buffer.
4. biosensor according to claim 1, which is characterized in that the hair clip H1 sequence such as SEQ ID NO:4 institute
Show, hair clip H2 sequence is as shown in SEQ ID NO:5.
5. biosensor according to claim 1, which is characterized in that the colour reagent include buffer, Hemin,
TMB color developing agent and H2SO4。
6. a kind of detection method of salmonella, which is characterized in that using bio-sensing described in any one of claims 1 to 5
Device detects salmonella, including RPA reaction, the enzyme reaction of Lambda Exonucleolytic, HCR reaction and chromogenic reaction.
7. detection method according to claim 6, which is characterized in that the system of the RPA reaction is as follows:
The condition of the RPA reaction are as follows: 38~40 DEG C of 18~22min of reaction.
8. detection method according to claim 6, which is characterized in that the system of the Lambda Exonucleolytic enzyme reaction
Are as follows:
The condition of the Lambda Exonucleolytic enzyme reaction are as follows: 36~38 DEG C of 18~22min of reaction.
9. detection method according to claim 6, which is characterized in that the system of the HCR reaction are as follows:
The condition of the HCR reaction are as follows: 36~38 DEG C of 28~32min of reaction.
10. detection method according to claim 6, which is characterized in that the system of the chromogenic reaction are as follows:
The condition of the chromogenic reaction: HCR reaction product, Hemin, buffer are incubated for 19~21min at 36~38 DEG C, are added
Develop the color at 36~38 DEG C 4~6min after TMB color developing agent, is eventually adding H2SO4Terminate reaction.
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| CN116087508A (en) * | 2022-09-06 | 2023-05-09 | 宁波大学 | Preparation method and application of HCR-based salmonella multivalent aptamer |
| CN117568495A (en) * | 2023-11-20 | 2024-02-20 | 军事科学院军事医学研究院军事兽医研究所 | Kit for detecting salmonella typhimurium based on lambda RPA and CRISPR/Cas12a |
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