CN106970213B - A method of detection parasiticin - Google Patents
A method of detection parasiticin Download PDFInfo
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- CN106970213B CN106970213B CN201710356333.1A CN201710356333A CN106970213B CN 106970213 B CN106970213 B CN 106970213B CN 201710356333 A CN201710356333 A CN 201710356333A CN 106970213 B CN106970213 B CN 106970213B
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- 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
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- 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 present invention provides a kind of methods of detection parasiticin, the detection of parasiticin is realized in homogeneous phase solution in the present invention, the amplification of signal is realized by way of two steps cycle, to realize the highly sensitive detection of parasiticin, and obtains lower Monitoring lower-cut.
Description
Technical field
The present invention relates to a kind of detection methods, and in particular to the aptamer conformation change based on target induction detects benzyl
The method of penicillin.
Background technology
Parasiticin is one kind of antibiotic, is extract by Penicillium notatum, due to containing penam in molecule, can break
The cell wall of bad bacterium and from the breeding period of bacterial cell bactericidal effect a kind of antibiotic.Penicillin to human toxicity compared with
It is small, but serious allergic reaction can be caused, the probability of allergy occurs in 5%-10%, symptom is under expiratory dyspnea, cyanosis, blood pressure
Drop, stupor, limbs are tetanic, finally faint from fear, and rescue can cause death not in time.And it is anaphylactoid occur it is big with drug dose
Small unrelated, allergic to penicillin patient, even micro can also cause to suffer a shock.The method for the detection parasiticin reported at present
Including high performance liquid chromatography, capillary electrophoresis, surface plasma body resonant vibration method and fluorescence resonance method.These methods have
Testing cost is high, and instrumentation is complicated, the shortcomings of needing professional operator, therefore, establish one it is sensitive highly selective
Method clinical diagnosis and in Food Safety Analysis detect parasiticin be vital.
Invention content
In order to which the method specificity and sensitivity that solve the above parasiticin of detection in the prior art are all relatively low, of high cost
The problem of, the present invention provides a species specificity and high sensitivity, the core based on object induction at low cost, detection speed is fast
The method of sour aptamers conformation change detection parasiticin.
What the present invention was obtained through the following steps：
4 DNA chain are used altogether in the present invention, 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)
The underscore part of wherein HAP1 is the aptamer of object parasiticin, the inscribe that underscore marks in HAP2
EnzymeNt.BbvCIIdentification cutting sequence.Lower stroke of boldface is the sequence rich in G in HAP2, and G is formed by the sequence rich in G
Tetrad, catalysis oxidation hydrogen peroxide and ABTS generate color change to quantify detection 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
Under the action of primer and template, holds to 3 ' ends and grow from 5 ' along template strand, polymerize and template strand base complete complementary
Series.Nt.BbvCI can realize in specific position and be cut to the specificity of DNA double chain that the cutting of specificity identifies sequence
It is：CC TCA GC, cleavage site are between front second and third base.
The detection of parasiticin is realized in homogeneous phase solution in the present invention, and letter is realized by way of two steps cycle
Number amplification, to realize the highly sensitive detection of parasiticin, and obtain lower Monitoring lower-cut.
The reaction occurred in homogeneous mainly has：HAP1 consists of two parts：Aptamer, and 3 with S2,Hold complementary series.
In the presence of having parasiticin, due between aptamer and object specific recognition and combination, HAP1 can be opened,
Make on HAP1 and 3 in S2,The complementary series at end is exposed to outside in the form of single-stranded.3 of S2 in then homogeneous,End can lead to
The HAP1 for crossing base pair complementarity and opening is hybridized to certain double-strand.
The 5 of S1 and S2,Base pair complementarity is held, certain double-strand is hybridized to.Under the action of phi29DNA polymerases, S2
3,End be template growth into the heteroduplex of complete complementary using open HAP1, and releases object parasiticin, free
Parasiticin out can continue to open other HAP1, then repeat the above process.This is first step cycle amplification（Object
The cycle of induction is amplified）.
Equally under the action of phi29DNA polymerases, the HAP1 of opening is template growth into the hybridization of complete complementary using S2
Double-strand, and S1 is released, free S1 can open more HAP2, then repeat the above process.This puts for second step cycle
Greatly.The amplification of this two step is carried out at the same time, and Phi29DNA polymerases act simultaneously.
In homogeneous reaction, reaction condition is 37 DEG C, and the reaction time is 4h.
Specific detecting step is as follows：
（1）DNA chain is pre-processed；
（2）It is mixed in homogeneous phase solution；
（3）By color developing agent ABTS with mixed solution is warm is detected.
The preparation method, concrete operation step are as follows：
（1）By the centrifuge tube equipped with DNA chain, it is put into 12000R/min in centrifuge, 10min is centrifuged, after centrifugation, presses
According to the ratio on centrifuge tube, suitable aqua sterilisa is added, then put and shake 30s in an oscillator, keeps chain warm uniformly；
（2）By aqua sterilisa, 10 × buffer buffer solutions, HAP1 and object to be measured object be added in centrifuge tube, shake 30s,
It is put into 37 DEG C of insulating box and is incubated 1h；
（3）By step（2）The centrifuge tube of middle incubation is taken out, then this is added in S1 and S2, phi29 archaeal dna polymerase, dNTPs
In a centrifuge tube, 30s is shaken, is put into 37 DEG C of insulating box and is incubated 1h；
（4）By step（3）The centrifuge tube of middle incubation, which is taken out, is added HAP2 and Nt.BbvCI restriction endonucleases in centrifuge tube, concussion
30s is put into 37 DEG C of insulating box and is incubated 1h；
（5）By step（4）The centrifuge tube of middle incubation is taken out, and iron chloride ferroheme is added in centrifuge tube, shakes 30s, puts
Enter and be incubated 1h in 37 DEG C of insulating box, H is added after incubation2O2It is detected with ABTS；
The detection mode of the invention is colorimetric detection, is detected using respective sample and the color change of blank sample
's.Using ultraviolet specrophotometer, quantitative detection can be realized to detection sample.It is fixed to be realized by the difference of absorbance value
Amount detection, the position of absorption peak is 415 or so.The present invention is based on the specific recognitions of aptamer and object, have chain
The phi29DNA polymerases of permutation function, endonuclease is in specific position to the dissection of nucleic acid chains, strand displacement isothermal
The redox characteristic of amplification and G tetrads constructs colorimetric bio sensor.The sensor has detection speed fast, detection
Limit low, the advantages that specificity is high, can make up the shortcomings and deficiencies of the existing detection method of parasiticin, realize it is quick to its, it is accurate
True quantitative detection.
Beneficial effects of the present invention：
1, the Idiotype identification of aptamer is utilized, is realized using the aptamer of parasiticin as identification substance
The high specific of object parasiticin is detected；
2, using the phi29 archaeal dna polymerases with strand displacement function, recycling for object and primer strand is realized,
It is exaggerated detection signal, improves the sensitivity of detection；
3, the identification using core restriction endonuclease to specific sequence and dissection, signal generate；
4, the reaction condition of the sensor is mild, and reaction speed is fast；
5, all processes of testing principle are realized in homogeneous, are improved reaction speed, are reduced answering for operation
Miscellaneous degree realizes the quick of object, simply, sensitive to detect；
6, preparation method is simple, and performance is stablized, the detection of parasiticin and biosensor production suitable for food security
The practical application of industry；
7, the process costs for making sensor are low, the inexpensive requirement suitable for industrialization.
Description of the drawings
Fig. 1 is the schematic diagram of the experiment；
Fig. 2 is 1 assist probes S1 concentration optimization testing result figures of embodiment；
Fig. 3 is 2 desired specificities testing result figure of embodiment；
Fig. 4 is the working curve of sensor detection.
Specific implementation mode
With reference to specific embodiment, invention is further explained.
The preparation method of the biosensor, includes the following steps：
（1）DNA chain is pre-processed；
（2）It is mixed in homogeneous phase solution；
（3）By color developing agent ABTS with mixed solution is warm is detected.
The preparation method, concrete operation step are as follows：
（1）By the centrifuge tube equipped with DNA chain, it is put into 12000R/min in centrifuge, 10min is centrifuged, after centrifugation, presses
According to the ratio on centrifuge tube, suitable aqua sterilisa is added, then put and shake 30s in an oscillator, keeps chain warm uniformly.
（2）By aqua sterilisa, 10 × buffer buffer solutions, HAP1 and object to be measured object be added in centrifuge tube, shake 30s,
It is put into 37 DEG C of insulating box and is incubated 1h；
（3）The centrifuge tube being incubated in step 2 is taken out, then this is added in S1 and S2, phi29 archaeal dna polymerase, dNTPs
In a centrifuge tube, 30s is shaken, is put into 37 DEG C of insulating box and is incubated 1h；
（4）The centrifuge tube being incubated in step 3 is taken out, HAP2 and Nt.BbvCI restriction endonucleases are added in centrifuge tube, concussion
30s is put into 37 DEG C of insulating box and is incubated 1h；
（5）The centrifuge tube being incubated in step 4 is taken out, iron chloride ferroheme is added in centrifuge tube, shakes 30s, is put into
It is incubated 1h in 37 DEG C of insulating box, H is added after incubation2O2It is detected with ABTS；
The detection mode of the invention is colorimetric detection, is detected using respective sample and the color change of blank sample
's.Using ultraviolet specrophotometer, quantitative detection can be realized to detection sample.It is fixed to be realized by the difference of absorbance value
Amount detection, the position of absorption peak is 415 or so.The present invention is based on the specific recognitions of aptamer and object, have chain
The phi29DNA polymerases of permutation function, endonuclease is in specific position to the dissection of nucleic acid chains, strand displacement isothermal
The redox characteristic of amplification and G tetrads constructs colorimetric bio sensor.The sensor has detection speed fast, detection
Limit low, the advantages that specificity is high, can make up the shortcomings and deficiencies of the existing detection method of parasiticin, realize it is quick to its, it is accurate
True quantitative detection.Schematic diagram is as shown in Figure 1.
Embodiment 1
The key step of specific operation process is as follows：
A, by the centrifuge tube equipped with DNA chain, it is put into 12000R/min in centrifuge, 10min is centrifuged, after centrifugation, presses
According to the ratio on centrifuge tube, suitable aqua sterilisa is added, then put and shake 30s in an oscillator, keeps chain warm uniformly.
B, by aqua sterilisa, 10 × buffer buffer solutions, HAP1（10 μM）, object to be measured object（10 μM）, centrifugation is added
Guan Zhong shakes 30s, is put into 37 DEG C of insulating box and is incubated 1h.
C, the centrifuge tube being incubated in b is taken out, then by the S1 of various concentration（2 μM, 5 μM, 10 μM, 15 μM）And S2（5μ
M）, phi29 archaeal dna polymerases（2 μL）, dNTPs（2 μL）It is added in this centrifuge tube, shakes 30s, be put into 37 DEG C of insulating box
Middle incubation 1h；
D, will（c）The centrifuge tube of middle incubation is taken out, and HAP2 is added（5μM）And Nt.BbvCI（2μL）Restriction endonuclease is in centrifuge tube
In, 30s is shaken, is put into 37 DEG C of insulating box and is incubated 1h；
E, the centrifuge tube being incubated in d is taken out, iron chloride ferroheme is added（1mM）In centrifuge tube, 30s is shaken, is put into
It is incubated 1h in 37 DEG C of insulating box, H is added after incubation2O2(27 μM) and ABTS (0.1 μM) are detected；
F, it is detected with ultraviolet specrophotometer, detection peak value is quantified 420 or so according to the variation of absorbance
Detect the content of object.
The buffer solution of 10X（buffer）It buys, 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 in high-pressure sterilizing pot at a temperature of 120 DEG C 20 min.
As a result Fig. 2 is seen, it can be seen from the figure that the blank detected and response signal are as the concentration of S1 is in 2-10 μM of area
It is interior increase and increase, after concentration is more than 10 μM, blank signal also enhances, and causes the signal absorption peak value of blank and response
Gap value becomes smaller.So the optium concentration of S1 is 10 μM.
Embodiment 2
The key step of experimentation is as follows：
A, by the centrifuge tube equipped with DNA chain, it is put into 12000R/min in centrifuge, 10min is centrifuged, after centrifugation, presses
According to the ratio on centrifuge tube, suitable aqua sterilisa is added, then put and shake 30s in an oscillator, keeps chain warm uniformly.
B, by aqua sterilisa, 10 × buffer buffer solutions, HAP1（10 μM）, object to be measured object parasiticin（10 μM）,
Streptomysin（10 μM）, terramycin（10 μM）, kanamycins（10 μM）It is added in centrifuge tube, shakes 30s, be put into 37 DEG C of perseverance
1h is incubated in incubator.
C, the centrifuge tube being incubated in b is taken out, then by the S1 of various concentration（10 μM）And S2（5μM）, phi29 DNA it is poly-
Synthase（2 μL）, dNTPs（2 μL）It is added in this centrifuge tube, shakes 30s, be put into 37 DEG C of insulating box and be incubated 1h；
D, will（c）The centrifuge tube of middle incubation is taken out, and HAP2 is added（5μM）And Nt.BbvCI（2μL）Restriction endonuclease is in centrifuge tube
In, 30s is shaken, is put into 37 DEG C of insulating box and is incubated 1h；
E, the centrifuge tube being incubated in d is taken out, iron chloride ferroheme is added（1mM）In centrifuge tube, 30s is shaken, is put into
It is incubated 1h in 37 DEG C of insulating box, H is added after incubation2O2(27 μM) and ABTS (0.1 μM) are detected；
F, it is detected with ultraviolet specrophotometer, detection peak value is quantified 420 or so according to the variation of absorbance
Detect the content of object.
As a result see Fig. 3, by image, it can be seen that, the sample that parasiticin only is added has apparent absorption peak,
Absorbance value and the blank sample gap that the sample of other antibiotic is added are little, so this sensor has specificity.
Embodiment 3
A kind of parasiticin detection method, key step are as follows：
A, by the centrifuge tube equipped with DNA chain, it is put into 12000R/min in centrifuge, 10min is centrifuged, after centrifugation, presses
According to the ratio on centrifuge tube, suitable aqua sterilisa is added, then put and shake 30s in an oscillator, keeps chain warm uniformly.
B, by aqua sterilisa, 10 × buffer buffer solutions, HAP1（10 μM）, the object to be measured object benzyl mould of various concentration
Element（100pM, 1nM, 10nM, 100nM, 1 μM, 10 μM）It is added in centrifuge tube, shakes 30s, be put into 37 DEG C of insulating box and be incubated
1h。
C, the centrifuge tube being incubated in b is taken out, then by the S1 of various concentration（10 μM）And S2（5μM）, phi29 DNA it is poly-
Synthase（2 μL）, dNTPs（2 μL）It is added in this centrifuge tube, shakes 30s, be put into 37 DEG C of insulating box and be incubated 1h；
D, will（c）The centrifuge tube of middle incubation is taken out, and HAP2 is added（5μM）And Nt.BbvCI（2μL）Restriction endonuclease is in centrifuge tube
In, 30s is shaken, is put into 37 DEG C of insulating box and is incubated 1h；
E, the centrifuge tube being incubated in d is taken out, iron chloride ferroheme is added（1mM）In centrifuge tube, 30s is shaken, is put into
It is incubated 1h in 37 DEG C of insulating box, H is added after incubation2O2(27 μM) and ABTS (0.1 μM) are detected；
Detected using ultraviolet specrophotometer, setting wave-length coverage from 300 to 500, go out peak position 415 to 420 it
Between, quantitative detection is carried out by the absorbance value detected.Testing result is as shown in figure 4, a to f is respectively 100pM, 1nM,
10nM, 100nM, 1 μM, 10 μM.In figure it will be seen that when the concentration of parasiticin is at 100pM to 10 μM, benzyl mould
The logarithm of plain concentration and absorbance size are proportional, meanwhile, we continue on the basis of the concentration of 100pM to lower dense
Degree detection, after testing when concentration is less than 100pM, the relationship of absorbance value and concentration is no longer complies with matched curve rule just,
Therefore this method can be obtained in the minimum point of absorbance i.e. in figure Monitoring lower-cut is 100pM.
。
<110>University Of Ji'nan
<120>A method of detection 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 of detection parasiticin, which is characterized in that include the following steps：
（1）By the centrifuge tube equipped with each DNA chain, it is put into 12000R/min in centrifuge, centrifuges 10min, aqua sterilisa is added, then put
30s is shaken in an oscillator, keeps chain warm uniformly；
（2）By aqua sterilisa, 10 × buffer buffer solutions, 10 μM of HAP1 and object to be measured object be added in centrifuge tube, shake 30s,
It is put into 37 DEG C of insulating box and is incubated 1h；
（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, shakes 30s, is put into 37 DEG C of insulating box and is incubated 1h；
（4）By step（3）The centrifuge tube of middle incubation, which is taken out, is added 5 μM of HAP2 and 2 μ L Nt.BbvCI restriction endonucleases in centrifuge tube,
30s is shaken, is put into 37 DEG C of insulating box and is incubated 1h；
（5）By step（4）The centrifuge tube of middle incubation is taken out, and iron chloride ferroheme is added, and shakes 30s, is put into 37 DEG C of insulating box
Middle incubation 1h, H is added after incubation2O2And ABTS, using UV spectrophotometer measuring；
Step（1）Each DNA chain, sequence are 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)；
Step（2）The nucleotide sequence of the HAP1 is as shown in SEQ ID NO.1；
Step（3）The nucleotide sequence of the S1 is as shown in SEQ ID NO.2；
Step（4）The nucleotide sequence of the HAP2 is as shown in SEQ ID NO.3；
Step（3）The nucleotide sequence of the S2 is as shown in SEQ ID NO.4.
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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 |
| CN109283147B (en) * | 2018-09-20 | 2021-03-16 | 湖北师范大学 | Homogeneous phase biosensing method for detecting kanamycin and application thereof |
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| KR100880516B1 (en) * | 2003-10-16 | 2009-01-28 | 써드 웨이브 테크놀로지스, 아이앤씨. | Direct Nucleic Acid Detection in Bodily Fluids |
| US20060199202A1 (en) * | 2005-02-09 | 2006-09-07 | Third Wave Technologies, Inc. | Detection of allelic expression imbalance |
| CN105238852B (en) * | 2015-08-10 | 2018-09-04 | 济南大学 | The biosensor and preparation method thereof of salmonella typhimurium is detected based on aptamer |
| CN105158320B (en) * | 2015-10-10 | 2017-12-26 | 济南大学 | Electrochemical sensor based on aptamer detection kanamycins and preparation method thereof |
| CN105925699B (en) * | 2016-06-07 | 2019-10-01 | 济南大学 | A method of detection kanamycins |
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