CN109837353A - Based on DNA nanometers of autonomous dresses to the method for detection of Salmonella invA genetic test - Google Patents
Based on DNA nanometers of autonomous dresses to the method for detection of Salmonella invA genetic test Download PDFInfo
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Abstract
The present invention provides one kind based on DNA nanometers of autonomous dresses to detection of SalmonellainvAThe method of genetic test, this method select detection of Salmonella highly conservedinvAGene designs the probe specifically bound with it, in conjunction with DNA self-assembling technique, is based on its signal amplification, detects detection of Salmonella using Colorimetric techniquesinvAGene.The present invention is with stem ring DNA(H0) for template, pass through opening H0 (complementary with part cyclic template) in conjunction with target nucleic acid, continuous hybrid then occurs with H1, H2 to react, a large amount of complexs containing rich G sequence are ultimately formed, in catalystic converter system (Hemin, ABTS2‑And H2O2) under color change occurs.Detection method does not need expensive instrument and equipment, has the characteristics that detection easy quickly, high sensitivity, testing cost be low and on-site test, suitable large-scale promotion.
Description
Technical field
The invention belongs to field of biological detection, and in particular to one kind is based on DNA nanometers of autonomous dresses to detection of Salmonella invA gene
The method of detection.
Background technique
Salmonella enterobacteriaceae is Grain-negative enteric bacilli, it is most important pathogenic bacteria in food origin disease
One of.Mainly cause food poisoning, gastroenteritis, Typhoid and paratyphoid after salmonella infection.Some researches show that salmonella
Invasin protein is pathogenic closely related with its, decides the ability that bacterium enters host epithelial cells.It is mainly by a series of bases
Because of coding, wherein invA is the gene for encoding detection of Salmonella and infecting surface epithelial cell albumen, closely related with the bacteria pathogenic, is
Detection of Salmonella is distinctive.
Currently, there are mainly three types of most common pathogenic entero becteria detection methods: traditional is separately cultured identification, enzyme linked immunological
Absorption method (ELISA) and polymerase chain reaction (PCR).Traditional identification method that is separately cultured is passed through after being separately cultured bacterium
Bacterium colony counts and the methods of colonial morphology, dyeing characteristic, biochemical reaction identify that bacterium, this method is the gold of Bacteria Identification
Standard method, but its time and effort consuming.ELISA method is the immunology detection based on antigen-antibody reaction, with traditional separation
Culture identification method compares, and shortens detection time, however, there remains this step of Bacteria Culture, at least needs 24~48 small
Shi Caineng obtain accurately as a result, and this method detection limit generally >=105CFU mL-1, it is difficult to meet low concentration bacterium
Detection.For round pcr compared with above two method, advantage is higher sensitivity, but PCR result is easy to appear false sun
Property；The segment of PCR amplification is mainly detected in laboratory with gel electrophoresis technology at present, but the resolution ratio of gel electrophoresis technology compared with
Thermal cycler that is low and needing higher accuracy, therefore round pcr is limited in the extensive utilization of laboratory testing bacterium.It is another
Method, without using amplification (NASBA) and self-sustained sequence replication system (ASR) that the nucleic acid of thermal cycler relies on, in specificity
It has a greatly reduced quality again, mainly due to that must be expanded with 40 DEG C of relatively low temperature.Strand displacement amplification art (SDA)
It is expanded under isothermal conditions using four kinds of primers, largely overcomes these defects, but there are still weak links:
It must be carried out amplification reaction using expensive modified nucleoside acid as substrate.
Summary of the invention
In order to solve the problems in the prior art, the purpose of the present invention is to provide one kind based on DNA nanometers of autonomous dresses pair
The method of detection of Salmonella invA genetic test, this method is good for detection of Salmonella invA genetic test high sensitivity, specificity, and operates
Simply, it is suitble to large-scale promotion.
The object of the present invention is achieved like this:
A method of based on DNA nanometers of autonomous dresses to detection of Salmonella invA genetic test, including signal iodine system
And catalystic converter system, it is characterised in that: include the stem ring for detection of Salmonella invA gene in the signal iodine system
Template H0, amplified reaction assist stem ring template H1 and H2, buffer system；The sequence such as SEQ ID NO:1 of the stem ring template H0
Shown, the sequence of the auxiliary stem ring template H1 is as shown in SEQ ID NO:2, the sequence such as SEQ of the auxiliary stem ring template H2
Shown in ID NO:3.
SEQ ID NO:1 (stem ring template H0), 5 '-TGGCAGCCCTTTCTCAATGCGGATTCCCAGTTGAGTGTGAG
AAAGG-3'；
SEQ ID NO:2 (auxiliary stem ring template H1), 5 '-GGGTAGGGCGGGTTGGGATGAGAAAGGGCTGCCACAT
CCCAACCCATA-3'；
SEQ ID NO:3 (auxiliary stem ring template H2): 5 '-TATGGGTTGGGATGTGGCAGCCATCCCAAC-3 '.
Testing principle: the present invention uses DNA nanoassemble technology to detection of Salmonella invA genetic test, using specific stem
Ring moulds plate (H0) is reacted with realizing the identification to the high specific of detection of Salmonella invA gene by DNA self assembly nucleic acid isothermal, and
The detection highly sensitive to trace detection of Salmonella invA gene is realized in the amplification of DNAzyme signal, and detection schematic diagram is as shown in Figure 1.
Specifically, stem ring template (H0) contains the a* segment specifically designed, with detection of Salmonella invA gene a complete complementary.Work as detection of Salmonella
In the presence of invA gene a, compound 1 is formed in conjunction with the a* segment in stem ring template (H0) and opens stem ring.H0 is opened by a
Afterwards, prior closed b is exposed, c segment can be further with the b* in auxiliary stem ring template (H1), and c* segment occurs specific
Hybridization reaction simultaneously opens auxiliary stem ring template H1.After H1 is opened, expose auxiliary stem ring template (H1) in d, c* segment,
Specific hybrid can further occur with auxiliary stem ring template (H2) and react and open H2, and compound 1 is anti-by strand displacement
It should displace.The compound 1 being replaced may thereafter continue to react with H1, move in circles.It assists in stem ring template (H1)
E, f segment (rich GDNA segment) can separate out.Functional nucleic acid GDNA be one section be rich in guanine nucleotide, when with
Hemin has the activity similar to horseradish peroxidase, the GDNA catalysis substrate ABTS in last system after combining2-It is shown
Colour response measures its inspection of absorbance change realization to detection of Salmonella invA gene at 418nm by ultraviolet specrophotometer
It surveys.
In one embodiment, in signal iodine system of the present invention, the buffer system is selected from TNaK
buffer.The catalystic converter system includes Hemin, ABTS2-And H2O2。
In one embodiment, in signal iodine system of the present invention, the stem ring template (H0) final concentration of 50
~150nM, most preferably 100nM.Auxiliary stem ring template (H1) the final concentration of 75~175nM, most preferably 125nM.
In one embodiment, the temperature of reaction system of the present invention is 4~55 DEG C, preferably 37 DEG C.DNA of the present invention is from group
The reaction cartridge time is 10~40min, preferably 20min.
Beneficial effect
The present invention provides a kind of method based on DNA nanometers of autonomous dresses to detection of Salmonella invA genetic test, and this method is selected
The highly conserved invA gene of detection of Salmonella designs the probe specifically bound with it, in conjunction with DNA self-assembling technique, based on its letter
Number amplification utilizes Colorimetric techniques to form colorimetric sensor and detects detection of Salmonella invA gene.The present invention is with stem ring DNA (H0)
Template is then occurred continuous hybrid with H1, H2 and is reacted by opening H0 (complementary with part cyclic template) in conjunction with target nucleic acid,
A large amount of complexs containing rich G sequence are ultimately formed, in catalystic converter system (Hemin, ABTS2-And H2O2) under occur color become
Change.The present invention is good to detection of Salmonella invA genetic test high sensitivity, specificity, and the range of linearity of detection reaches 50pM~200nM,
Sensitivity are as follows: 32pM.Detection method does not need expensive instrument and equipment, has detection easy quick, high sensitivity, inspection
The features such as surveying at low cost and on-site test is suitble to large-scale promotion.
Detailed description of the invention
Fig. 1 is detection schematic diagram；
Fig. 2 is each element and DNA self assembly product non-denaturing polyacrylamide involved in DNA self-assembling reaction
Gel electrophoresis carries out proof diagram；
Fig. 3 is to each element and DNA self assembly product ultraviolet specrophotometer involved in DNA self-assembling reaction
Carry out proof diagram；
Fig. 4 is the ultraviolet detection figure of various concentration H0 building detection of Salmonella invA gene colorimetric sensor；
Fig. 5 is the ultraviolet detection figure of various concentration H1 building detection of Salmonella invA gene colorimetric sensor；
Fig. 6 is the reaction buffer system ultraviolet detection figure of different temperatures；
Fig. 7 is the reaction buffer system ultraviolet detection figure of differential responses time；
Fig. 8 is the absorbance change figure of the detection of Salmonella invA gene of various concentration；
Fig. 9 is the Linear equations of detection of Salmonella invA gene.
Embodiment
In order to keep the purpose of the present invention and technical solution clearer, the preferred embodiment of the present invention is carried out below detailed
Description.It is noted that following embodiment is served only for that the present invention is further detailed, and should not be understood as to this hair
The limitation of bright protection scope.Those skilled in the art's above content according to the present invention make it is some it is nonessential improvement and
Adjustment all belongs to the scope of protection of the present invention.The raw materials used in the present invention and reagent are commercial product.
Embodiment 1
Extract the genomic DNA for being used as pcr template
Due to Salmonella infection be much it is food-borne, usually food or excrement can be used as detection source.With
Food inspection: aseptically being operated, and food to be checked is put into refiner and stirs into homogenate, takes 25g (or mL) to be checked
The homogenate of sample is diluted to 1: 10 homogeneous homogenate with 225mL buffered peptone water (BP).Take 1mL homogeneous homogenate be placed in from
In heart pipe, 12000r/min is centrifuged 5min, and sterile saline washs 2 times, finally distills aqueous suspension with 0.25mL, be placed in 100
It is incubated for 15 minutes in DEG C water-bath and is placed in ice immediately after.It is centrifuged after ten minutes at 4 DEG C with 12000r/min, supernatant is shifted
Into new pipe, i.e. containing the genomic DNA that can be directly used as pcr template in the supernatant.
It is detected with excrement: taking diarrhea patient stool sample 200mg, use QIAamp DNA Stool Mini Kit
(Qiagen Inc., Valencia, CA, USA, Cat No.51504) kit carries out the separation and Extraction of DNA, and specific steps are pressed
It is operated according to kit specification.
Embodiment 2
Amplification obtains PCR product sample to be checked
Prepare PCR reaction solution: from food or the supernatant 5.0 containing genomic DNA of excrement in Example 1
μ L, Premix Taq (1.25U the DNA polymerase, 2 × Taq of 20 μM of forward and reverse primer each 1.0 μ L, 25 μ L
Buffer, 0.4mM dNTPs) and 18 μ L water, the final volume of PCR reaction is 50 μ L.According to the invA that detection of Salmonella is highly conserved
Gene utilizes the amplimer of the Primer Blast module design invA gene of the website NCBI, forward primer sequence are as follows: 5 '-
GCATCCGCA TCAATAATACCG-3 ', reverse primer sequences are as follows: 5 '-TTCTCTGGATGGTATGCCC-3 '.PCR reacts item
Part is as follows: 95 DEG C initial denaturation 1 minute；Then 95 DEG C be denaturalized 30 seconds, 51 DEG C anneal 30 seconds, 72 DEG C primer extend 30 seconds, 35 are followed
Ring, last 72 DEG C extend 4 minutes.It is spare to get to measuring samples that amplified production is preserved in 4 DEG C of refrigerators.
Embodiment 3
The preparation of reaction system
The preparation of stem ring template: TE buffer (10mM Tris-HCl, pH 8.0 is used；1mM
Ethylenediaminetetraacetic acid (EDTA)) stem ring template (H0) and amplified reaction assisted into stem ring template
(H1, H2) is dissolved to 10 μM, 95 DEG C of denaturation 10min, slowly restore to room temperature, -20 DEG C freeze it is spare.
The use of colorimetric sensor: 0.5 μ L is diluted to the detection of Salmonella invA gene of 7 various concentrations with TE buffer
(50pM, 200pM, 500pM, 5nM, 10nM, 50nM, 200nM) is added in DNA self-assembling reaction system, 37 DEG C of incubation 20min.
Then ABTS is added with Hemin incubation at room temperature 40min is added again2-And H2O2Detect the variation of absorbance.
The verifying of colorimetric sensor: to each element and DNA self assembly product involved in DNA self-assembling reaction with non-
Denaturing polyacrylamide gel electrophoresis is verified, such as Fig. 2 (M:20-bp marker；1:H0；2:H1；3:H2；4:H0+H1；5:
H1+H2；6:T0+H0；7:H0+H1+H2；It 8:T0+H0+H1+H2) shows, (band in the presence of no target detection of Salmonella invA gene
7) a small amount of H1-H2 compound can, be generated.Only in the presence of target detection of Salmonella invA gene, a large amount of H1-H2 compounds could be generated,
Free richness GDNA segment out, to cause subsequent Catalytic color reaction.
Each element and DNA self assembly product involved in DNA self-assembling reaction are carried out with ultraviolet specrophotometer
Verifying, such as Fig. 3 ((a) H1；(b)H1+H2；(c)H0+H1+H2；(d)T0+H0+H1；And (e) T0+H0+H1+H2), illustration is pair
The chrominance response answered), only in the presence of target detection of Salmonella invA gene (e), apparent color change is just had, and at 418nm
There is an apparent absorption peak.
Embodiment 4
The optimization of colorimetric sensor use condition
To the concentration of H0 in experimentation, the concentration of H1, the temperature of reaction system, DNA self-assembling reaction time carry out into
The optimization of one step.1 points of progress a series of experiments are chosen respectively by low concentration to high concentration to each condition.
To investigate influence of the concentration of H0 to detection of Salmonella invA gene colorimetric sensor, this experiment uses various concentration (50-
150nM) H0 constructs detection of Salmonella invA gene colorimetric sensor, then carries out uv-spectrophotometric detection.As shown in figure 4, letter
It makes an uproar more different and different than the concentration with H0, when the concentration of H0 is 100nM, signal-to-noise ratio reaches maximum value, illustrates that this concentration is
Optium concentration.
Similarly, the influence for the concentration of H1 in investigation system to detection of Salmonella invA gene colorimetric sensor, this experiment use
Various concentration (75-175nM) H1 constructs detection of Salmonella invA gene colorimetric sensor, then carries out uv-spectrophotometric detection.Such as
Shown in Fig. 5, when the concentration of H1 is 125nM, signal-to-noise ratio reaches maximum value, illustrates that this concentration is optium concentration.
It similarly, is to investigate influence of the reaction temperature to sensor is used in amplification system, this experiment use difference (4,25,
37,42,55 DEG C) reaction buffer system, then carry out uv-spectrophotometric detection.As shown in fig. 6, the optimum response temperature of reaction
Degree is 37 DEG C.
Influence for the investigation amplification system reaction time to sensor is used, this experiment use time containing differential responses (0,
10,20,30,40min) then reaction buffer system carries out chrominance response detection.As shown in fig. 7, amplification system is best anti-
It is 20min between seasonable.
Embodiment 5
The performance evaluation of colorimetric sensor
In order to assess the performance of detection of Salmonella invA gene colorimetric sensor, to the difference being equipped with TE (pH 8.0) buffer
The detection of Salmonella invA gene of concentration is analyzed.Specifically, the sand of 7 various concentrations will be diluted to TE (pH 8.0) buffer
Door bacterium invA gene (50pM, 200pM, 500pM, 5nM, 10nM, 50nM, 200nM) is added in iodine system, 37 DEG C of incubations
20min.(2) 40min then is incubated at room temperature with addition Hemin again.ABTS is added2-And H2O2Detect variation (such as Fig. 8 of absorbance
It is shown), it draws standard curve (as shown in Figure 9).Experimental result is shown, when detection of Salmonella invA mrna concentration is in 50pM to 200nM
When, obtained signal and the logarithm of detection of Salmonella invA mrna concentration is linearly related, and linear equation is Y=0.1471lg (C/
PM) -0.1498, related coefficient 0.9994.Blank solution (is referred to that the molten of amplification system is added in no detection of Salmonella invA gene
Liquid continuous scanning 10 times, detection limit is estimated plus signal value corresponding to 3 times of standard deviations according to blank signal, calculates to obtain 32pM.
Claims (8)
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| CN111154843A (en) * | 2020-04-07 | 2020-05-15 | 中国农业大学 | Quantitative detection method based on overspeed PCR and functional nucleic acid color development |
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