CN106970213A - A kind of method for detecting parasiticin - Google Patents
A kind of method for detecting parasiticin Download PDFInfo
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- CN106970213A CN106970213A CN201710356333.1A CN201710356333A CN106970213A CN 106970213 A CN106970213 A CN 106970213A CN 201710356333 A CN201710356333 A CN 201710356333A CN 106970213 A CN106970213 A CN 106970213A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2415/00—Assays, e.g. immunoassays or enzyme assays, involving penicillins or cephalosporins
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Abstract
The invention provides a kind of method for detecting parasiticin, the detection of parasiticin is realized in homogeneous phase solution in the present invention, the amplification of signal is realized by way of the circulation of two steps, so as to realize the highly sensitive detection of parasiticin, and relatively low Monitoring lower-cut is obtained.
Description
Technical field
The present invention relates to a kind of detection method, and in particular to detects benzyl mould based on the aptamer conformation change that target is induced
The method of element.
Background technology
Parasiticin is one kind of antibiotic, is extract by Penicillium notatum, due to containing penam in molecule, can destroy thin
The cell membrane of bacterium and from the breeding period of bacterial cell bactericidal action a class antibiotic.Penicillin is smaller to human toxicity, but
It is that can cause serious allergic reaction, occurs the probability of allergy in 5%-10%, symptom is expiratory dyspnea, cyanosis, drop in blood pressure, dusk
Fan, limbs are tetanic, finally faint from fear, and rescue can cause death not in time.And it is anaphylactoid generation with drug dose size without
Close, patient allergic to penicillin, even micro can also cause shock.The method for the detection parasiticin reported at present includes
High performance liquid chromatography, capillary electrophoresis, surface plasma body resonant vibration method and fluorescence resonance method.These methods have detection
Cost is high, and instrumentation is complicated, it is necessary to the shortcomings of professional operator, therefore, the method for establishing a sensitive high selectivity
Clinical diagnosis and in Food Safety Analysis detect parasiticin be vital.
The content of the invention
In order to solve, the above detects the method specificity of parasiticin in the prior art and sensitivity is all higher than relatively low, cost asks
Topic, is fitted the invention provides the nucleic acid based on object induction that a species specificity and sensitivity are high, cost is low, detection speed is fast
The method of ligand conformational change detection parasiticin.
What the present invention was obtained through the following steps：
4 DNAs are used altogether in the present invention, its sequence is respectively：
HAP1：5′-GCG GGC GGT TGT ATA GCG GTT TTT TTT TTG CCC GCA TTT AGT TT -3’
（SEQ ID NO.1）
S1：5’- AAA AGC TGA GGA CTA TAG C -3’(SEQ ID NO.2)
HAP2：5’- TAG TCC TCA GCT TTT GGG TTT TGG GTT TTG GGT TTT GGG ACT A -3’
(SEQ ID NO.3)
S2：5’- GCT ATA GTC CTC AAT CTA ATA AAC TAA ATG CG -3’(SEQ ID NO.4)
Wherein HAP1 underscore part is the restriction endonuclease of underscore mark in the aptamer of object parasiticin, HAP2Nt.BbvCIIdentification cutting sequence.Lower stroke of boldface is the sequence rich in G in HAP2, passes through the sequence formation G tetra- rich in G
Conjuncted, catalysis oxidation hydrogen peroxide and ABTS produce color change quantitatively to detect parasiticin.
Two kinds of enzymes have been used in the present invention：Phi29 archaeal dna polymerases and Nt.BbvCI restriction endonucleases.Phi29 archaeal dna polymerases are drawing
Thing along template strand from 5 ' with the presence of template, holding to 3 ' ends and growing, polymerize the series with template strand base complete complementary.
Nt.BbvCI can realize and the specificity of DNA double chain is cut that its specific cutting recognition sequence is in specific position：CC
TCA GC, cleavage site is above between second and the 3rd base.
The detection of parasiticin is realized in homogeneous phase solution in the present invention, and letter is realized by way of the circulation of two steps
Number amplification, so as to realize the highly sensitive detection of parasiticin, and obtain relatively low Monitoring lower-cut.
The reaction occurred in homogeneous mainly has：HAP1 is made up of two parts：Aptamer, and 3 with S2,Hold complementary series.
In the presence of having parasiticin, due to the specific recognition between aptamer and object and combination, HAP1 can be opened,
Make on HAP1 and 3 in S2,The complementary series at end is exposed to outside in single-stranded form.3 of S2 in then homogeneous,End can lead to
The HAP1 that base pair complementarity is crossed with opening is hybridized to certain double-strand.
The 5 of S1 and S2,Base pair complementarity is held, certain double-strand is hybridized to.In the presence of phi29DNA polymerases, S2
3,Hold using heteroduplexs of the HAP1 opened as template growth into complete complementary, and discharge object parasiticin, dissociate
Parasiticin out can continue to open other HAP1, then repeatedly said process.This is that the first step circulates amplification（Object
The circulation amplification of induction）.
Equally in the presence of phi29DNA polymerases, the HAP1 of opening by template growth of S2 into complete complementary hybridization
Double-strand, and discharge S1, free S1 can open more HAP2, then repeatedly said process.This is that second step circulation is put
Greatly.The amplification of this two step is to carry out simultaneously, and Phi29DNA polymerases are acted simultaneously.
In homogeneous reaction, reaction condition is 37 DEG C, and the reaction time is 4h.
Specific detecting step is as follows：
（1）DNA is pre-processed；
（2）Mixed in homogeneous phase solution；
（3）By developer ABTS with mixed solution is warm is detected.
Described preparation method, concrete operation step is as follows：
（1）Will be equipped with the centrifuge tube of DNA, be put into 12000R/min in centrifuge, centrifuge 10min, after centrifugation is finished, according to from
Ratio on heart pipe, adds appropriate aqua sterilisa, then puts and shake 30s in an oscillator, makes chain warm uniform；
（2）By aqua sterilisa, 10 × buffer buffer solutions, HAP1 and object to be measured add in centrifuge tube, shake 30s, be put into
1h is incubated in 37 DEG C of insulating box；
（3）By step（2）The centrifuge tube of middle incubation is taken out, then by S1 and S2, phi29 archaeal dna polymerase, dNTPs add this from
In heart pipe, shake in 30s, the insulating box for being put into 37 DEG C and be incubated 1h；
（4）By step（3）The centrifuge tube of middle incubation takes out addition HAP2 and Nt.BbvCI restriction endonucleases in centrifuge tube, shakes 30s,
1h is incubated in the insulating box for being put into 37 DEG C；
（5）By step（4）The centrifuge tube of middle incubation is taken out, and adds iron chloride ferroheme in centrifuge tube, shakes 30s, be put into 37
DEG C insulating box in be incubated 1h, incubation after add H2O2Detected with ABTS；
The detection mode of the invention is colorimetric detection, is detected using respective sample and the color change of blank sample.
Using ultraviolet specrophotometer, quantitative detection can be realized to detection sample.Realized by the difference of absorbance quantitative
Detection, the position of absworption peak is 415 or so.Specific recognition of the invention based on aptamer and object, puts with chain
The phi29DNA polymerases of function are changed, endonuclease is put in dissection of the specific position to nucleic acid chains, strand displacement isothermal
The redox characteristic of big and G tetrads constructs colorimetric bio sensor.The sensor has detection speed fast, test limit
It is low, the advantages of specific high, the shortcomings and deficiencies of the existing detection method of parasiticin can be made up, realization is quick to its, accurately
Quantitative detection.
Beneficial effects of the present invention：
1st, the Idiotype of aptamer make use of to recognize, the aptamer by the use of parasiticin is realized pair as identification material
The high specific detection of object parasiticin；
2nd, using the phi29 archaeal dna polymerases with strand displacement function, recycling for object and primer strand is realized, is amplified
Detection signal, improves the sensitivity of detection；
3rd, the identification using core restriction endonuclease to specific sequence and dissection, signal are produced；
4th, the reaction condition of the sensor is gentle, and reaction speed is fast；
5th, all processes of Cleaning Principle are realized in homogeneous, are improved reaction speed, are reduced the complicated journey of operation
Degree, realizes the quick of object, simply, sensitive detection；
6th, preparation method is simple, and performance is stable, it is adaptable to the detection of parasiticin and biology sensor industrialization in food security
Practical application；
7th, the process costs for making sensor are low, it is adaptable to inexpensive requirement in industrialization.
Brief description of the drawings
Fig. 1 is the schematic diagram of the experiment；
Fig. 2 is the assist probes S1 concentration optimization testing result figures of embodiment 1；
Fig. 3 is the desired specificities testing result figure of embodiment 2；
Fig. 4 is the working curve that sensor is detected.
Embodiment
The present invention is further described with reference to specific embodiment.
The preparation method of described biology sensor, comprises the following steps：
（1）DNA is pre-processed；
（2）Mixed in homogeneous phase solution；
（3）By developer ABTS with mixed solution is warm is detected.
Described preparation method, concrete operation step is as follows：
（1）Will be equipped with the centrifuge tube of DNA, be put into 12000R/min in centrifuge, centrifuge 10min, after centrifugation is finished, according to from
Ratio on heart pipe, adds appropriate aqua sterilisa, then puts and shake 30s in an oscillator, makes chain warm uniform.
（2）By aqua sterilisa, 10 × buffer buffer solutions, HAP1 and object to be measured add in centrifuge tube, shake 30s,
1h is incubated in the insulating box for being put into 37 DEG C；
（3）The centrifuge tube being incubated in step 2 is taken out, then by S1 and S2, phi29DNA polymerase, dNTPs adds this centrifugation
Guan Zhong, shakes in 30s, the insulating box for being put into 37 DEG C and is incubated 1h；
（4）The centrifuge tube being incubated in step 3 is taken out into addition HAP2 and Nt.BbvCI restriction endonucleases in centrifuge tube, 30s is shaken,
1h is incubated in the insulating box for being put into 37 DEG C；
（5）The centrifuge tube being incubated in step 4 is taken out, adds iron chloride ferroheme in centrifuge tube, shakes 30s, be put into 37 DEG C
Insulating box in be incubated 1h, incubation after add H2O2Detected with ABTS；
The detection mode of the invention is colorimetric detection, is detected using respective sample and the color change of blank sample.
Using ultraviolet specrophotometer, quantitative detection can be realized to detection sample.Realized by the difference of absorbance quantitative
Detection, the position of absworption peak is 415 or so.Specific recognition of the invention based on aptamer and object, puts with chain
The phi29DNA polymerases of function are changed, endonuclease is put in dissection of the specific position to nucleic acid chains, strand displacement isothermal
The redox characteristic of big and G tetrads constructs colorimetric bio sensor.The sensor has detection speed fast, test limit
It is low, the advantages of specific high, the shortcomings and deficiencies of the existing detection method of parasiticin can be made up, realization is quick to its, accurately
Quantitative detection.Schematic diagram is as shown in Figure 1.
Embodiment 1
The key step of specific operation process is as follows：
A, the centrifuge tube that will be equipped with DNA, are put into 12000R/min in centrifuge, centrifuge 10min, after centrifugation is finished, according to from
Ratio on heart pipe, adds appropriate aqua sterilisa, then puts and shake 30s in an oscillator, makes chain warm uniform.
B, by aqua sterilisa, 10 × buffer buffer solutions, HAP1（10 μM）, object to be measured（10 μM）, add centrifugation
Guan Zhong, shakes in 30s, the insulating box for being put into 37 DEG C and is incubated 1h.
C, the centrifuge tube being incubated in b taken out, then by the S1 of various concentrations（2 μM, 5 μM, 10 μM, 15 μM）And S2（5μ
M）, phi29 archaeal dna polymerases（2 μL）, dNTPs（2 μL）Add in this centrifuge tube, shake 30s, be put into 37 DEG C of insulating box
Middle incubation 1h；
D, general（c）The centrifuge tube of middle incubation is taken out, and adds HAP2（5μM）And Nt.BbvCI（2μL）Restriction endonuclease is in centrifuge tube, shake
30s is swung, 1h is incubated in the insulating box for being put into 37 DEG C；
E, the centrifuge tube being incubated in d taken out, add iron chloride ferroheme（1mM）In centrifuge tube, 30s is shaken, 37 DEG C are put into
Insulating box in be incubated 1h, incubation after add H2O2(27 μM) and ABTS (0.1 μM) are detected；
F, detected with ultraviolet specrophotometer, detection peak value is quantitatively detected 420 or so according to the change of absorbance
The content of object.
10X buffer solution（buffer）Buy, can be used directly with polymerase.
The ultra-pure water of configuration is both needed to carry out high-temperature sterilization processing.Specific method is to be individually positioned in ultra-pure water different
In conical flask, then sealed with masking foil and newspaper.Sterilize 20 min in high-pressure sterilizing pot at a temperature of 120 DEG C.
As a result Fig. 2 is seen, it can be seen that the blank detected and response signal are as S1 concentration is in 2-10 μM of area
In increase and increase, after concentration is more than 10 μM, blank signal also strengthens, and causes blank and the signal absorption peak value responded
Gap value diminishes.So S1 optium concentration is 10 μM.
Embodiment 2
The key step of experimentation is as follows：
A, the centrifuge tube that will be equipped with DNA, are put into 12000R/min in centrifuge, centrifuge 10min, after centrifugation is finished, according to from
Ratio on heart pipe, adds appropriate aqua sterilisa, then puts and shake 30s in an oscillator, makes chain warm uniform.
B, by aqua sterilisa, 10 × buffer buffer solutions, HAP1（10 μM）, object parasiticin to be measured（10 μM）,
Streptomysin（10 μM）, terramycin（10 μM）, kanamycins（10 μM）Add in centrifuge tube, shake 30s, be put into 37 DEG C of perseverance
1h is incubated in incubator.
C, the centrifuge tube being incubated in b taken out, then by the S1 of various concentrations（10 μM）And S2（5μM）, phi29 DNA gather
Synthase（2 μL）, dNTPs（2 μL）Add in this centrifuge tube, shake in 30s, the insulating box for being put into 37 DEG C and be incubated 1h；
D, general（c）The centrifuge tube of middle incubation is taken out, and adds HAP2（5μM）And Nt.BbvCI（2μL）Restriction endonuclease is in centrifuge tube, shake
30s is swung, 1h is incubated in the insulating box for being put into 37 DEG C；
E, the centrifuge tube being incubated in d taken out, add iron chloride ferroheme（1mM）In centrifuge tube, 30s is shaken, 37 DEG C are put into
Insulating box in be incubated 1h, incubation after add H2O2(27 μM) and ABTS (0.1 μM) are detected；
F, detected with ultraviolet specrophotometer, detection peak value is quantitatively detected 420 or so according to the change of absorbance
The content of object.
As a result see Fig. 3, by image it can be seen that, only add parasiticin sample have obvious absworption peak,
The absorbance and blank sample gap for adding the sample of other antibiotic are little, so this sensor has specificity.
Embodiment 3
A kind of preparation method of electrochemica biological sensor of the present invention：
A, the centrifuge tube that will be equipped with DNA, are put into 12000R/min in centrifuge, centrifuge 10min, after centrifugation is finished, according to from
Ratio on heart pipe, adds appropriate aqua sterilisa, then puts and shake 30s in an oscillator, makes chain warm uniform.
B, by aqua sterilisa, 10 × buffer buffer solutions, HAP1（10 μM）, the object benzyl mould to be measured of various concentrations
Element（100pM, 1nM, 10nM, 100nM, 1 μM, 10 μM）Add in centrifuge tube, shake 30s, be put into 37 DEG C of insulating box and be incubated
1h。
C, the centrifuge tube being incubated in b taken out, then by the S1 of various concentrations（10 μM）And S2（5μM）, phi29 DNA gather
Synthase（2 μL）, dNTPs（2 μL）Add in this centrifuge tube, shake in 30s, the insulating box for being put into 37 DEG C and be incubated 1h；
D, general（c）The centrifuge tube of middle incubation is taken out, and adds HAP2（5μM）And Nt.BbvCI（2μL）Restriction endonuclease is in centrifuge tube, shake
30s is swung, 1h is incubated in the insulating box for being put into 37 DEG C；
E, the centrifuge tube being incubated in d taken out, add iron chloride ferroheme（1mM）In centrifuge tube, 30s is shaken, 37 DEG C are put into
Insulating box in be incubated 1h, incubation after add H2O2(27 μM) and ABTS (0.1 μM) are detected；
Detected using ultraviolet specrophotometer, set wave-length coverage from 300 to 500, go out peak position between 415 to 420, lead to
The absorbance that detects is crossed to carry out quantitative detection.Testing result is as shown in figure 4, a to f is respectively 100pM, 1nM,
10nM, 100nM, 1 μM, 10 μM.It will be seen that when the concentration of ampicillin is at 100pM to 10 μM, benzyl is blue or green in figure
The logarithm of mycin concentration and size of current are proportional, meanwhile, we continue on the basis of 100pM concentration to lower dense
Degree detection, after testing when concentration is less than 100pM, the relation of absorbance and concentration is no longer complies with matched curve rule just,
The Monitoring lower-cut that therefore minimum point of absorbance i.e. in figure can obtain this method is 100pM.
。
<110>University Of Ji'nan
<120>A kind of method for detecting parasiticin
<160>2
<210>1
<211>44
<212>DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(44)
<223>Primer
<400>1
GCG GGC GGT TGT ATA GCG GTT TTT TTT TTG 30
CCC GCA TTT AGT TT 44
<210>2
<211>19
<212>DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(19)
<223>Primer
<400>2
AAA AGC TGA GGA CTA TAG C 19
<210> 3
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(43)
<223>Primer
<400> 3
TAG TCC TCA GCT TTT GGG TTT TGG GTT TTG 30
GGT TTT GGG ACT A 43
<210> 4
<211> 32
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(32)
<223>Primer
<400> 4
GCT ATA GTC CTC AAT CTA ATA AAC TAA ATG 30
CG 32
Claims (1)
1. a kind of method for detecting parasiticin, it is characterised in that comprise the following steps：
（1）The centrifuge tube of each DNA is will be equipped with, 12000R/min in centrifuge is put into, 10min is centrifuged, aqua sterilisa is added, then put
30s is shaken in an oscillator, makes chain warm uniform；
（2）By aqua sterilisa, 10 × buffer buffer solutions, 10 μM of HAP1 and object to be measured add in centrifuge tube, shake 30s,
1h is incubated in the insulating box for being put into 37 DEG C；
（3）The centrifuge tube being incubated in step (2) is taken out, then by 10 μM of S1 and 5 μM of S2,2 μ Lphi29 archaeal dna polymerases, 2 μ L
DNTPs is added in this centrifuge tube, is shaken in 30s, the insulating box for being put into 37 DEG C and is incubated 1h；
（4）By step（3）The centrifuge tube of middle incubation takes out 5 μM of HAP2 of addition and 2 μ L Nt.BbvCI restriction endonucleases in centrifuge tube,
Shake in 30s, the insulating box for being put into 37 DEG C and be incubated 1h；
（5）By step（4）The centrifuge tube of middle incubation is taken out, and adds iron chloride ferroheme, shakes 30s, is put into 37 DEG C of insulating box
Middle incubation 1h, H is added after incubation2O2And ABTS, using UV spectrophotometer measuring；
The nucleotide sequence of the HAP1 is as shown in SEQ ID NO.1；
The nucleotide sequence of the S1 is as shown in SEQ ID NO.2；
The nucleotide sequence of the HAP2 is as shown in SEQ ID NO.3；
The nucleotide sequence of the S2 is as shown in SEQ ID NO.4.
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|---|---|---|---|
| CN201710356333.1A CN106970213B (en) | 2017-05-19 | 2017-05-19 | A method of detection parasiticin |
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|---|---|---|---|
| CN201710356333.1A CN106970213B (en) | 2017-05-19 | 2017-05-19 | A method of detection parasiticin |
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| CN106970213A true CN106970213A (en) | 2017-07-21 |
| CN106970213B CN106970213B (en) | 2018-10-19 |
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| CN107561071A (en) * | 2017-08-31 | 2018-01-09 | 贵州大学 | A kind of quick determination method of animal product streptomycin residual |
| CN109283147A (en) * | 2018-09-20 | 2019-01-29 | 湖北师范大学 | It is a kind of detect kanamycins homogeneous bio-sensing method and its application |
| CN112505320A (en) * | 2020-11-17 | 2021-03-16 | 新乡学院 | Ampicillin residue detection method and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107561071A (en) * | 2017-08-31 | 2018-01-09 | 贵州大学 | A kind of quick determination method of animal product streptomycin residual |
| CN109283147A (en) * | 2018-09-20 | 2019-01-29 | 湖北师范大学 | It is a kind of detect kanamycins homogeneous bio-sensing method and its application |
| CN112505320A (en) * | 2020-11-17 | 2021-03-16 | 新乡学院 | Ampicillin residue detection method and application |
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| Publication number | Publication date |
|---|---|
| CN106970213B (en) | 2018-10-19 |
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