CN103667448A - Difunctional aptamer detection kit and detection method - Google Patents
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Abstract
The invention relates to a difunctional aptamer detection kit and a detection method capable of simultaneously detecting two metal ions of Hg<2+> and Ag<+1>. According to the difunctional aptamer detection method, in the simultaneous presence of the two metal ions of Hg<2+> and Ag<+1>, T-Hg<2+>-T and C-Ag<+1>-C structures are formed to respectively connect mismatch of thymine and mismatch of cytosine on the 3' end of a first template DNA and the 3' end of a second template DNA, so that the hybridized first template DNA and the second template DNA can be amplified, a first oligonucleotide and a second oligonucleotide formed by cutting of a first DNA nickase are respectively hybridized with a first probe sequence and a second probe sequence, so that the first probe sequence and the second probe sequence are respectively cut by a second DNA nickase and a third DNA nickase, a first fluorophore and a second fluorophore are exposed finally, and the two metal ions of Hg<2+> and Ag<+1> are simultaneously detected by fluorescence detection.
Description
Technical field
The present invention relates to environmental analysis and food safety field, particularly relate to and a kind ofly can carry out Hg simultaneously
2+and Ag
+difunctional aptamers detection kit and detection method that two metal ion species detect.
Background technology
Mercury and silver are two kinds of very common heavy metals, are widely used in pharmacy, the fields such as battery and photography industry.But when the by product of these products or using discarded after, part heavy metal, the most at last with ionic species entered environment, has caused serious pollution to water body and soil.Mercury ion all has a great impact animals and plants and HUMAN HEALTH, and long-term exposure is in the permanent injury containing causing central nervous system and other organ in the environment of mercury ion.The direct toxicity of silver ions is relatively little, but it can produce very strong inrichment in hydrobiont, thereby enters in human body by the mode of food chain, makes it to become one of heavy metal element having high harm.
Existing Hg
2+and Ag
+detection technique comprises atomic absorption spectrum, ICP-MS, organic fluorescence molecule and aptamers method etc.Wherein, atomic absorption spectrometry is traditional analytical procedure, during test, sample is dissolved by appropriate means, makes contained mercury all be converted into Hg
2+, then with reductive agent by Hg
2+be reduced into mercury vapour, import in mercury vapor analyzer and measure; Sample pretreatment process and the atomic absorption spectrum of ICP-MS method are similar, first need the mercury in sample to dissolve to change into ionic condition, after the effect of clearing up, sample are injected into mass spectrograph and are measured; Organic fluorescence molecule process is that the method by chemosynthesis produces a kind of specific groups, this group can react with heavy metal ion specifically, thereby cause structural change and strengthen or cancellation fluorescence, finally utilizing the variation of fluorescence intensity to reach the object of detection.A kind of novel method that aptamers method has been developed recently, has highly sensitive and specificity, its be mainly utilize thymus pyrimidine (T) and cytosine(Cyt) (C) respectively with Hg
2+and Ag
+form two kinds of special pairing structures, i.e. T-Hg
2+-T and C-Ag
+-C, carries out analysis heavy metal ion thereby design corresponding biosensor.
Aptamers method receives a lot of concerns with its sensitivity and specificity, has developed the analytical procedure of many kinds of metal ions by composite structure, as fluorescence, and electrochemistry, colorimetric and Raman method etc.But these methods can only be confined to detect a metal ion species (Hg
2+or Ag
+), can not carry out Hg simultaneously
2+and Ag
+the detection of two metal ion species.
Summary of the invention
Based on this, be necessary to provide a kind of and can carry out Hg simultaneously
2+and Ag
+difunctional aptamers detection kit and detection method that two metal ion species detect.
A difunctional aptamers detection kit, for carry out Hg simultaneously
2+and Ag
+two metal ion species detect, and comprising:
The first masterplate DNA and the second masterplate DNA, in described the first masterplate DNA, contain 15 bases with described the second masterplate DNA complementary pairing, and described the first masterplate DNA and described the second masterplate DNA form the 3 ' end of rear described the first masterplate DNA of hybridization by described 15 bases and the 3 ' end of described the second masterplate DNA forms respectively the mispairing of 3 thymus pyrimidines and the mispairing of 3 cytosine(Cyt)s, and the mispairing of described thymus pyrimidine is at Hg
2+existence under, can form T-Hg
2+the special pairing structure of-T, increases for masterplate thereby make the 3 ' end of described the first masterplate DNA after hybridization can take the 5 ' end of described the second masterplate DNA, and the mispairing of described cytosine(Cyt) is at Ag
+existence under, can form C-Ag
+the special pairing structure of-C, increases for masterplate thereby make the 3 ' end of described the second masterplate DNA after hybridization can take the 5 ' end of described the first masterplate DNA;
Archaeal dna polymerase and a DNA nickase, a described DNA nickase can be sheared and form the first oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification described the first masterplate DNA out, and a described DNA nickase can also be sheared and form the second oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification described the second masterplate DNA out;
First fluorophore cancellation group's probe and the second fluorophore cancellation group probe, described the first fluorophore cancellation group probe comprises the first probe sequence and is combined in the first fluorophore and the first cancellation group on described the first probe sequence, described the first probe sequence can with described the first oligonucleotide hybridization, there is cancellation reaction in described the first fluorophore and described the first cancellation group, described the second fluorophore cancellation group probe comprises the second probe sequence and is combined in the second fluorophore and the second cancellation group on described the second probe sequence, described the second probe sequence can with described the second oligonucleotide hybridization, there is cancellation reaction in described the second fluorophore and described the second cancellation group, described the first fluorophore is different with described the second fluorophore,
The 2nd DNA nickase, the 3rd DNA nickase and dNTPs, it is separated with described the first cancellation group that rear described the first fluorophore be sheared and be sheared to described the 2nd DNA nickase can to described the first probe sequence after hybridization, and it is separated with described the second cancellation group that rear described the second fluorophore be sheared and be sheared to described the 3rd DNA nickase can to described the second probe sequence after hybridization.
In one embodiment, described the first masterplate DNA is the sequence shown in SEQ ID No.1, and the second masterplate DNA is the sequence shown in SEQ ID No.2.
In one embodiment, described the first probe sequence is the sequence shown in SEQ ID No.3, and described the second probe sequence is the sequence shown in SEQ ID No.4.
In one embodiment, described the first fluorophore is Fluoresceincarboxylic acid, and described the second fluorophore is tetramethyl-rhodamine.
In one embodiment, described archaeal dna polymerase is archaeal dna polymerase Klenow fragment, a described DNA nickase is DNA nickase Nb.BbvCI, and described the 2nd DNA nickase is DNA nickase Nb.BtsI, and described the 3rd DNA nickase is DNA nickase Nt.AlwI.
A difunctional aptamers detection method, for carry out Hg simultaneously
2+and Ag
+two metal ion species detect, and comprise the steps:
After testing sample, the first masterplate DNA and the second masterplate DNA are mixed, there is hybridization and obtain hybridization solution, wherein, in described the first masterplate DNA, contain 15 bases with described the second masterplate DNA complementary pairing, and described the first masterplate DNA and described the second masterplate DNA form after hybridization the 3 ' end of described the first masterplate DNA by described 15 bases and the 3 ' end of described the second masterplate DNA forms respectively the mispairing of 3 thymus pyrimidines and the mispairing of 3 cytosine(Cyt)s;
In described hybridization solution, add dNTPS, archaeal dna polymerase and a DNA nickase generation oligonucleotide strand displacement amplified reaction, obtain oligonucleotide amplification liquid, wherein, the mispairing of thymus pyrimidine is at Hg
2+existence under, form T-Hg
2+the special pairing structure of-T, increases for masterplate thereby make the 3 ' end of the first masterplate DNA after hybridization take the 5 ' end of the second masterplate DNA, and the mispairing of cytosine(Cyt) is at Ag
+existence under, form C-Ag
+the special pairing structure of-C, thereby making the 3 ' end of the second masterplate DNA after hybridization take the 5 ' end of the first masterplate DNA increases for masterplate, a described DNA nickase is sheared and is formed the first oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification described the first masterplate DNA out, and a described DNA nickase is also sheared and formed the second oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification described the second masterplate DNA out;
In described oligonucleotide amplification liquid, add the first fluorophore cancellation group probe, the second fluorophore cancellation group probe, there is difunctional amplified fluorescence reaction in the 2nd DNA nickase and the 3rd DNA nickase, obtain fluoroscopic examination liquid, wherein, described the first fluorophore cancellation group probe comprises the first probe sequence and is combined in the first fluorophore and the first cancellation group on described the first probe sequence, described the first probe sequence can with described the first oligonucleotide hybridization, there is cancellation reaction in described the first fluorophore and described the first cancellation group, described the second fluorophore cancellation group probe comprises the second probe sequence and is combined in the second fluorophore and the second cancellation group on described the second probe sequence, described the second probe sequence can with described the second oligonucleotide hybridization, there is cancellation reaction in described the second fluorophore and described the second cancellation group, described the first fluorophore is different with described the second fluorophore, it is separated with described the first cancellation group that rear described the first fluorophore is sheared and sheared to described the 2nd DNA nickase to described the first probe sequence after hybridizing, it is separated with described the second cancellation group that rear described the second fluorophore is sheared and sheared to described the 3rd DNA nickase to described the second probe sequence after hybridizing,
Described fluoroscopic examination liquid is carried out to fluoroscopic examination, complete described difunctional aptamers detection method.
In one embodiment, described the first masterplate DNA is the sequence shown in SEQ ID No.1, and the second masterplate DNA is the sequence shown in SEQ ID No.2.
In one embodiment, described the first probe sequence is the sequence shown in SEQ ID No.3, and described the second probe sequence is the sequence shown in SEQ ID No.4.
In one embodiment, described archaeal dna polymerase is archaeal dna polymerase Klenow fragment, a described DNA nickase is DNA nickase Nb.BbvCI, and described the 2nd DNA nickase is DNA nickase Nb.BtsI, and described the 3rd DNA nickase is DNA nickase Nt.AlwI.
In one embodiment, in described oligonucleotide amplification liquid, add the first fluorophore cancellation group probe, the second fluorophore cancellation group probe, the 2nd DNA nickase and the 3rd DNA nickase to occur, in the operation of difunctional amplified fluorescence reaction, also to need to add wherein bovine serum albumin.
This difunctional aptamers detection method, at Hg
2+and Ag
+in the simultaneous situation of two metal ion species, can be due to T-Hg
2+-T and C-Ag
+the formation of-C-structure, make the 3 ' end of the first masterplate DNA and the 3 ' end of the second masterplate DNA form respectively the mispairing of 3 thymus pyrimidines and the mispairing of 3 cytosine(Cyt)s forms respectively connection, thereby hybridization the first masterplate DNA and the second masterplate DNA together can be amplified, then the cutting by a DNA nickase forms the first oligonucleotide and the second oligonucleotide, the first oligonucleotide and the second oligonucleotide are hybridized with the first probe sequence and the second probe sequence respectively, thereby cause the first probe sequence and the second probe sequence by the 2nd DNA nickase and the 3rd DNA nickase, to be cut respectively, final the first fluorophore and the second fluorophore are exposed, by detecting the fluorescence of fluoroscopic examination liquid, can be simultaneously at Hg
2+and Ag
+two metal ion species detect.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the difunctional aptamers detection method of an embodiment;
Fig. 2 is the schema of difunctional aptamers detection method as shown in Figure 1;
Fig. 3 is the native polyacrylamide gel electrophoresis result figure of the oligonucleotide amplification liquid that obtains of oligonucleotide strand displacement amplified reaction;
Fig. 4 A is different concns Hg
2+fluorescent spectrum curve;
Fig. 4 B is that fluorescent spectrum curve is as shown in Figure 4 A usingd the fluorescence intensity at 520nm place as ordinate zou, Hg
2+the X-coordinate matched curve of the logarithmic value of concentration;
Fig. 5 A is different concns Ag
+fluorescent spectrum curve;
Fig. 5 B is that fluorescent spectrum curve is as shown in Figure 5A usingd the fluorescence intensity at 582nm place as ordinate zou, Ag
+the X-coordinate matched curve of the logarithmic value of concentration;
Fig. 6 is the fluorometric analysis figure of the specificity experiment of difunctional aptamers detection method.
Embodiment
For above-mentioned purpose of the present invention, feature and advantage can be become apparent more, below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in detail.A lot of details have been set forth in the following description so that fully understand the present invention.But the present invention can implement to be much different from alternate manner described here, and those skilled in the art can do similar improvement without prejudice to intension of the present invention in the situation that, so the present invention is not subject to the restriction of following public concrete enforcement.
The difunctional aptamers detection kit of one embodiment, for carry out Hg simultaneously
2+and Ag
+two metal ion species detect, and comprising:
The first masterplate DNA, the second masterplate DNA, archaeal dna polymerase, a DNA nickase, the 2nd DNA nickase, the 3rd DNA nickase, dNTPs, first fluorophore cancellation group's probe and the second fluorophore cancellation group probe.
In the first masterplate DNA, contain 15 bases with the second masterplate DNA complementary pairing, and the first masterplate DNA and the second masterplate DNA form after hybridization the 3 ' end of the first masterplate DNA by these 15 bases and the 3 ' end of the second masterplate DNA forms respectively the mispairing of 3 thymus pyrimidines (T) and the mispairing of 3 cytosine(Cyt)s (C).
The mispairing of thymus pyrimidine (T) is at Hg
2+existence under, can form T-Hg
2+the special pairing structure of-T, increases for masterplate thereby make the 3 ' end of the first masterplate DNA after hybridization can take the 5 ' end of the second masterplate DNA.Work as Hg
2+while not existing, because the mispairing of 3 thymus pyrimidines (T) forms blocking effect, thereby make the 3 ' end of the first masterplate DNA after hybridization can not take the 5 ' end of the second masterplate DNA, increase for masterplate.
The mispairing of cytosine(Cyt) (C) is at Ag
+existence under, can form C-Ag
+the special pairing structure of-C, increases for masterplate thereby make the 3 ' end of the second masterplate DNA after hybridization can take the 5 ' end of the first masterplate DNA.Work as Ag
+while not existing, because the mispairing of 3 cytosine(Cyt)s (C) forms blocking effect, thereby make the 3 ' end of the second masterplate DNA after hybridization can not take the 5 ' end of the first masterplate DNA, increase for masterplate.
The one DNA nickase can be sheared and form the first oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification the first masterplate DNA out, and a DNA nickase can also be sheared and form the second oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification the second masterplate DNA out.
The first fluorophore cancellation group probe comprises the first probe sequence and is combined in the first fluorophore and the first cancellation group on the first probe sequence, the first probe sequence can with the first oligonucleotide hybridization, there is cancellation reaction in the first fluorophore and the first cancellation group.
The second fluorophore cancellation group probe comprises the second probe sequence and is combined in the second fluorophore and the second cancellation group on the second probe sequence, the second probe sequence can with the second oligonucleotide hybridization, there is cancellation reaction in the second fluorophore and the second cancellation group.
In present embodiment, first fluorophore cancellation group's probe and the second fluorophore cancellation group probe are made by probe Synesis Company " precious biotechnology (Dalian) ".
In present embodiment, the first fluorophore is FAM(Fluoresceincarboxylic acid), the second fluorophore is TAMRA(tetramethyl-rhodamine).The first cancellation group is Eclipse(Eclipse are trademarks of Epoch Biosciences, Inc.), the second cancellation group is Eclipse(Eclipse are trademarks of Epoch Biosciences, Inc.).
In present embodiment, the first fluorophore is different with the second fluorophore.
The 2nd DNA nickase identification double-stranded DNA, after can the first probe sequence after hybridization being sheared and be sheared, the first fluorophore be separated with the first cancellation group, thus the first fluorophore activation can be sent fluorescence.
The 3rd DNA nickase identification double-stranded DNA, after can the second probe sequence after hybridization being sheared and be sheared, the second fluorophore be separated with the second cancellation group, thus the second fluorophore activation can be sent fluorescence.
This difunctional aptamers detection kit, at Hg
2+and Ag
+in the simultaneous situation of two metal ion species, can be due to T-Hg
2+-T and C-Ag
+the formation of-C-structure, make the 3 ' end of the first masterplate DNA and the 3 ' end of the second masterplate DNA form respectively the mispairing of 3 thymus pyrimidines and the mispairing of 3 cytosine(Cyt)s forms respectively connection, thereby hybridization the first masterplate DNA and the second masterplate DNA together can be amplified, then the cutting by a DNA nickase forms the first oligonucleotide and the second oligonucleotide, the first oligonucleotide and the second oligonucleotide are hybridized with the first probe sequence and the second probe sequence respectively, thereby cause the first probe sequence and the second probe sequence by the 2nd DNA nickase and the 3rd DNA nickase, to be cut respectively, final the first fluorophore and the second fluorophore are exposed, by fluoroscopic examination, can be simultaneously at Hg
2+and Ag
+two metal ion species detect.
In present embodiment, the first masterplate DNA is the sequence shown in SEQ ID No.1, and the second masterplate DNA is the sequence shown in SEQ ID No.2.
In present embodiment, the first probe sequence is the sequence shown in SEQ ID No.3, and the second probe sequence is the sequence shown in SEQ ID No.4.
Archaeal dna polymerase can be archaeal dna polymerase Klenow fragment, and a DNA nickase can be DNA nickase Nb.BbvCI, and the 2nd DNA nickase can be DNA nickase Nb.BtsI, and the 3rd DNA nickase can be DNA nickase Nt.AlwI.
Below in conjunction with Fig. 1 and Fig. 2, provide a kind of difunctional aptamers detection method of the above-mentioned difunctional aptamers detection kit of employing of an embodiment, for carry out Hg simultaneously
2+and Ag
+two metal ion species detect, and comprise the steps:
S10, there is hybridization after testing sample, the first masterplate DNA and the second masterplate DNA are mixed and obtain hybridization solution.
The damping fluid using in S10 is the first reaction buffer, wherein, and 10 * the first reaction buffers (the Trisaminomethane acetic acid of 0.2 mole every liter, pH7.9, the magnesium acetate of 0.1 mole every liter, the Potassium ethanoate of 0.5 mole every liter, the dithiothreitol (DTT) of 0.01 mole every liter).
In the first masterplate DNA, contain 15 bases with the second masterplate DNA complementary pairing, and the first masterplate DNA and the second masterplate DNA form after hybridization the 3 ' end of the first masterplate DNA by 15 bases and the 3 ' end of the second masterplate DNA forms respectively the mispairing of 3 thymus pyrimidines and the mispairing of 3 cytosine(Cyt)s.
In present embodiment, the first masterplate DNA is the sequence shown in SEQ ID No.1, and the second masterplate DNA is the sequence shown in SEQ ID No.2.
In S20, the hybridization solution that obtains to S10, add dNTPS, archaeal dna polymerase and a DNA nickase generation oligonucleotide strand displacement amplified reaction, obtain the oligonucleotide amplification liquid that contains the first oligonucleotide and the second oligonucleotide.
The mispairing of thymus pyrimidine (T) is at Hg
2+existence under, form T-Hg
2+the special pairing structure of-T, increases for masterplate thereby make the 3 ' end of the first masterplate DNA after hybridization take the 5 ' end of the second masterplate DNA.Work as Hg
2+while not existing, because the mispairing of 3 thymus pyrimidines (T) forms blocking effect, thereby make the 3 ' end of the first masterplate DNA after hybridization can not take the 5 ' end of the second masterplate DNA, increase for masterplate.
The mispairing of cytosine(Cyt) (C) is at Ag
+existence under, form C-Ag
+the special pairing structure of-C, increases for masterplate thereby make the 3 ' end of the second masterplate DNA after hybridization take the 5 ' end of the first masterplate DNA.Work as Ag
+while not existing, because the mispairing of 3 cytosine(Cyt)s (C) forms blocking effect, thereby make the 3 ' end of the second masterplate DNA after hybridization can not take the 5 ' end of the first masterplate DNA, increase for masterplate.
The one DNA nickase is sheared and is formed the first oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification the first masterplate DNA out, and a DNA nickase is also sheared and formed the second oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification the second masterplate DNA out.
In S30, the oligonucleotide amplification liquid that obtains to S20, add the first fluorophore cancellation group probe, the second fluorophore cancellation group probe, the 2nd DNA nickase and the 3rd DNA nickase that difunctional amplified fluorescence reaction occurs, obtain fluoroscopic examination liquid.
The damping fluid using in S30 is the second reaction buffer, wherein, and 10 * the second reaction buffers (the Trisaminomethane hydrochloric acid of 0.1 mole every liter, pH7.9, the magnesium chloride of 0.1 mole every liter, the sodium-chlor of 0.5 mole every liter, the dithiothreitol (DTT) of 0.01 mole every liter).
Generally speaking, while carrying out difunctional amplified fluorescence reaction in S30, also need to add bovine serum albumin.
The effect of bovine serum albumin is for auxiliary the 2nd DNA nickase.
The first fluorophore cancellation group probe comprises the first probe sequence and is combined in the first fluorophore and the first cancellation group on the first probe sequence, the first probe sequence can with the first oligonucleotide hybridization, there is cancellation reaction in the first fluorophore and the first cancellation group.
The second fluorophore cancellation group probe comprises the second probe sequence and is combined in the second fluorophore and the second cancellation group on the second probe sequence, the second probe sequence can with the second oligonucleotide hybridization, there is cancellation reaction in the second fluorophore and the second cancellation group.
In present embodiment, the first fluorophore is different with the second fluorophore.
In present embodiment, first fluorophore cancellation group's probe and the second fluorophore cancellation group probe are made by probe Synesis Company " precious biotechnology (Dalian) ".
In present embodiment, the first fluorophore is FAM(Fluoresceincarboxylic acid), the second fluorophore is TAMRA(tetramethyl-rhodamine).The first cancellation group is Eclipse(Eclipse are trademarks of Epoch Biosciences, Inc.), the second cancellation group is Eclipse(Eclipse are trademarks of Epoch Biosciences, Inc.).
The 2nd DNA nickase identification double-stranded DNA, after can the first probe sequence after hybridization being sheared and be sheared, the first fluorophore be separated with the first cancellation group, thus the first fluorophore activation can be sent fluorescence.
The 3rd DNA nickase identification double-stranded DNA, after can the second probe sequence after hybridization being sheared and be sheared, the second fluorophore be separated with the second cancellation group, thus the second fluorophore activation can be sent fluorescence.
In present embodiment, the first probe sequence is the sequence shown in SEQ ID No.3, and the second probe sequence is the sequence shown in SEQ ID No.4.
Archaeal dna polymerase can be archaeal dna polymerase Klenow fragment, and a DNA nickase can be DNA nickase Nb.BbvCI, and the 2nd DNA nickase can be DNA nickase Nb.BtsI, and the 3rd DNA nickase can be DNA nickase Nt.AlwI.
S40, the fluoroscopic examination liquid that S30 is obtained carry out fluoroscopic examination, complete difunctional aptamers detection method.
Because the first fluorophore is different with the second fluorophore, the fluorescence sending after the first fluorophore and the activation of the second fluorophore is also different.
By fluoroscopic examination liquid is carried out to fluoroscopic examination, analyze concrete wavelength of fluorescence and the fluorescence intensity obtaining that detect, can be to Hg
2+and Ag
+two metal ion species carry out qualitative and quantitative analysis.
This difunctional aptamers detection method, at Hg
2+and Ag
+in the simultaneous situation of two metal ion species, can be due to T-Hg
2+-T and C-Ag
+the formation of-C-structure, make the 3 ' end of the first masterplate DNA and the 3 ' end of the second masterplate DNA form respectively the mispairing of 3 thymus pyrimidines and the mispairing of 3 cytosine(Cyt)s forms respectively connection, thereby hybridization the first masterplate DNA and the second masterplate DNA together can be amplified, then the cutting by a DNA nickase forms the first oligonucleotide and the second oligonucleotide, the first oligonucleotide and the second oligonucleotide are hybridized with the first probe sequence and the second probe sequence respectively, thereby cause the first probe sequence and the second probe sequence by the 2nd DNA nickase and the 3rd DNA nickase, to be cut respectively, final the first fluorophore and the second fluorophore are exposed, by detecting the fluorescence of fluoroscopic examination liquid, can be simultaneously at Hg
2+and Ag
+two metal ion species detect.
This difunctional aptamers detection method also tool has the following advantages:
Amplification efficiency is high, by the difunctional amplification mode of two steps is together in series, has greatly improved amplification efficiency, has greatly improved detection sensitivity;
High specificity, passes through T-Hg
2+-T and C-Ag
+-C-structure, has strengthened the specificity detecting greatly;
Be applicable to actual sample and detect, when actual sample detects, do not need complicated pre-treatment step, and not finding any matrix interference effect in water sample detection from the beginning, in actual measurement, have a good application prospect.
Be specific embodiment below.In embodiment, the first masterplate DNA is the sequence shown in SEQ ID No.1, the second masterplate DNA is the sequence shown in SEQ ID No.2, the first probe sequence is the sequence shown in SEQ ID No.3, the second probe sequence is the sequence shown in SEQ ID No.4, archaeal dna polymerase is archaeal dna polymerase Klenow fragment, the one DNA nickase is DNA nickase Nb.BbvCI, the 2nd DNA nickase is DNA nickase Nb.BtsI, the 3rd DNA nickase is DNA nickase Nt.AlwI, first fluorophore cancellation group's probe and the second fluorophore cancellation group probe are made by probe Synesis Company " precious biotechnology (Dalian) ", the first fluorophore is FAM, the second fluorophore is TAMRA.The first cancellation group is Eclipse, and the second cancellation group is Eclipse.
Reagent is prepared: the reagent of preparing respectively following concentration: the first masterplate DNA of 1 every liter of micromole, the second masterplate DNA of 1 every liter of micromole, the mixture of tetra-kinds of deoxyribonucleotides of dNTPs(of 2.5 every liter of mmole), 10 * the first reaction buffers (the Trisaminomethane acetic acid of 0.2 mole every liter, pH7.9, the magnesium acetate of 0.1 mole every liter, the Potassium ethanoate of 0.5 mole every liter, the dithiothreitol (DTT) of 0.01 mole every liter), the Trisaminomethane hydrochloric acid of 10 * reaction buffer 2(0.1 mole every liter, pH7.9, the magnesium chloride of 0.1 mole every liter, the sodium-chlor of 0.5 mole every liter, the dithiothreitol (DTT) of 0.01 mole every liter), the DNA nickase Nb.BbvCI of the 10 every microlitres of unit, Nb.BtsI and Nt.AlwI, the 5 every microlitre archaeal dna polymerase Klenow fragment of unit (3 '-5 ' exo-), 100 * BSA (bovine serum albumin, 10mg/mL), first fluorophore cancellation group's probe (QF probe-1) of 100 every liter of micromole and the second fluorophore cancellation group probe (QF probe-2), the mercury ion of sterilized water and different concns and silver ions.
Difunctional amplified reaction: reaction soln is divided into A liquid, B liquid, C liquid and D liquid, and cumulative volume is 50 microlitres.The mixture that wherein A liquid comprises 0.25 microlitre the first masterplate DNA, 0.25 microlitre the second masterplate DNA, tetra-kinds of deoxyribonucleotides of 1 microlitre dNTPs(), 1 microlitre 10 * the first reaction buffers, 1 microlitre metal target ion (Hg
2+or Ag
+) and 6 microlitre sterilized waters; B liquid comprises 0.3 microlitre DNA nickase Nb.BbvCI, 0.2 microlitre archaeal dna polymerase Klenow fragment (3 '-5 ' exo-); The sterilized water that C liquid comprises 0.125 microlitre QF probe-1 and 0.3 microlitre QF probe-2,5 microlitre 10 * the second reaction buffers, 33.875 microlitres (or 33.7 microlitres); D liquid comprise 0.5 microlitre DNA nickase Nb.BtsI and 1 microlitre DNA nickase Nt.AlwI and 0.5 microlitre BSA (bovine serum albumin, 10mg/mL).First A liquid hatch 3 minutes at 95 ℃, is then cooled to 37 ℃, then add B liquid, the two is reacted 60 minutes at 37 ℃, after mixing with C liquid again, at 37 ℃, react 15 minutes, then add D liquid at 37 ℃, to react 45 minutes, finally can collect signal by luminoscope.
Amplification principle is in conjunction with Fig. 1: the first step is that hybridization occurs for the first masterplate DNA and the second masterplate DNA, and there are 15 base complementrities between the two, but respectively have three base mispairings at 3 end places, be respectively three thymus pyrimidines and three cytosine(Cyt)s.Second step is oligonucleotide strand displacement amplified reaction, when adding Hg2
+and Ag
+time, the base mismatch generation combination of template strand 3 ends, forms T-Hg
2+-T and C-Ag
+the composite structure of-C, this is just for amplified reaction provides condition.If there is no Hg
2+and Ag
+, can due to the mispairing of 3 ends, cause amplified reaction not start.After this, mixture at archaeal dna polymerase Klenow fragment (3 '-5 ' exo-) and tetra-kinds of deoxyribonucleotides of dNTPs() under effect, take the first masterplate DNA and the second masterplate DNA extends as amplification template, thereby forms complete duplex structure.The nicking enzyme recognition site (this nicking enzyme is only identified duplex structure) of the duplex structure of DNA nickase Nb.BbvCI identification subsequently, and at the base place, the 2nd, downstream in this site, new synthetic chain is carried out to nicking, this has just produced new polysaccharase replication site.This site can be extended again by polysaccharase Klenow fragment (3 '-5 ' exo-), its strand displacement vigor can cement out the downstream oligonucleotide fragment after nicking simultaneously, and sequence after again extending can be by nicking enzyme Nb.BbvCI nicking, then being aggregated enzyme Klenow fragment (3 '-5 ' exo-) extends again, this extension and nicking circulation repeatedly formed strand displacement amplification reaction, and it can increase and produce a large amount of short oligonucleotide fragments.And this kind of difunctional template can produce two kinds of not homotactic first oligonucleotide and the second oligonucleotide simultaneously.The 3rd step is difunctional amplified fluorescence reaction, and the oligonucleotide fragment of generation can be respectively with QF probe-1 and the combination of QF probe-2 probe hybridization.Because the two strands forming contains corresponding recognition site, can by nicking enzyme Nb.BtsI and Nt.AlwI, be cut respectively, cause QF probe-1 and QF probe-2 probe to be cut into two sections, fluorophore and cancellation group are occurred separated, thereby produce strong fluorescent signal.Now oligonucleotide fragment can be separated again from duplex structure, is combined with new not cut QF probe probe, has so just formed in the circulation of a cutting-separation-hybridization, and fluorescent signal is strengthened significantly.And QF probe-1 and QF probe-2 probe have different fluorophores, thus the fluorescence of different emission can be produced, and then reach the object of difunctional amplified fluorescence.
Oligonucleotide strand displacement amplified reaction confirmatory experiment..
Adopt native polyacrylamide gel electrophoresis analysis to verify oligonucleotide strand displacement amplified reaction.
As shown in Figure 3, not there is not Hg in its result
2+and Ag
+situation under, control tube electrophoresis road (control road) do not find the band of any amplification; Only there is Hg
2+or Ag
+situation under, the 20bp in its electrophoresis road down locates to find obvious oligonucleotide target stripe, this explanation chain replaces amplified reaction successfully to be realized.There is at the same time Hg
2+and Ag
+situation under, oligonucleotide target stripe also can significantly be observed, and its band is bright during obviously than Individual existence one metal ion species, this explanation two metal ion species simultaneously in the presence of time, successfully amplify two kinds of different oligonucleotide.In a word, whole difunctional chain replaces amplified reaction can distinguish echo signal and control signal well, and feasibility is very good.
Sensitivity experiment.
By the Hg of different concns
2+and Ag
+carried out detecting and analyzed, the sensitivity of validation test method, result is as shown in Fig. 4 A, Fig. 4 B, Fig. 5 A and Fig. 5 B.Fig. 4 A has shown different concns Hg
2+fluorescent spectrum curve, show the Hg of this technical scheme to different concns
2+there is good discrimination.In order to assess its quantitative analysis ability, we adopt the fluorescence intensity at 520nm place as ordinate zou, Hg
2+the logarithmic value of concentration is X-coordinate matched curve, obtains Fig. 4 B.Fig. 4 B presents good linear relationship, and its linear equation is F=-43.32+110.88log
10c(relation conefficient is 0.9958), through calculating, the detectability of this technical scheme can reach every liter of 1.93 picomole.
That Fig. 5 A shows is different concns Ag
+fluorescent spectrum curve, as can be seen from the figure the fluorescence curve under different concns has discrimination clearly.For Quantitative Study, the fluorescence intensity that we adopt 582nm place is ordinate zou, Ag
+the logarithmic value of concentration is X-coordinate matched curve, obtains Fig. 5 B.Fig. 5 B shows good linear relationship, and its linear equation is F=-63.56+91.33log
10c(relation conefficient is 0.9967), through calculating, the detectability of this technical scheme can reach every liter of 15.80 picomole.
Therefore this testing method is at Hg
2+and Ag
+aspect all has very high detection sensitivity.
Specificity experiment and actual water sample analysis.
In order to assess Hg
2+and Ag
+specificity, selected 7 kinds of other metal ions (Cu
2+, Ca
2+, Cd
2+, Fe
3+, Pb
2+, Mn
2+, Zn
2+)carried out detecting and analyzed, detected result as shown in Figure 6.
As seen from Figure 6, at Hg
2+and Ag
+place has strong fluorescent signal, and other seven metal ion species only have faint fluorescence intensity, show that the specificity of this technical scheme is very good.
In addition, adopt this detection method to be applied to the detection of actual water sample, this actual sample is tap water.Result is as shown in table 1.
Selected respectively the Hg of every liter of 0.5 and 1 nmole
2+or Ag
+join in tap water, through calculating, find that its rate of recovery is between 93%-106%, relative standard deviation is all less than 6%.And can find out in table, in reality detects, do not find any matrix interference effect, the ionic concn adding is 0 o'clock, what detect is also 0.Result shows that this technical scheme can analyze with sensitivity to actual sample.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a difunctional aptamers detection kit, for carry out Hg simultaneously
2+and Ag
+two metal ion species detect, and it is characterized in that, comprising:
The first masterplate DNA and the second masterplate DNA, in described the first masterplate DNA, contain 15 bases with described the second masterplate DNA complementary pairing, and described the first masterplate DNA and described the second masterplate DNA form the 3 ' end of rear described the first masterplate DNA of hybridization by described 15 bases and the 3 ' end of described the second masterplate DNA forms respectively the mispairing of 3 thymus pyrimidines and the mispairing of 3 cytosine(Cyt)s, and the mispairing of described thymus pyrimidine is at Hg
2+existence under, can form T-Hg
2+the special pairing structure of-T, increases for masterplate thereby make the 3 ' end of described the first masterplate DNA after hybridization can take the 5 ' end of described the second masterplate DNA, and the mispairing of described cytosine(Cyt) is at Ag
+existence under, can form C-Ag
+the special pairing structure of-C, increases for masterplate thereby make the 3 ' end of described the second masterplate DNA after hybridization can take the 5 ' end of described the first masterplate DNA;
Archaeal dna polymerase and a DNA nickase, a described DNA nickase can be sheared and form the first oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification described the first masterplate DNA out, and a described DNA nickase can also be sheared and form the second oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification described the second masterplate DNA out;
First fluorophore cancellation group's probe and the second fluorophore cancellation group probe, described the first fluorophore cancellation group probe comprises the first probe sequence and is combined in the first fluorophore and the first cancellation group on described the first probe sequence, described the first probe sequence can with described the first oligonucleotide hybridization, there is cancellation reaction in described the first fluorophore and described the first cancellation group, described the second fluorophore cancellation group probe comprises the second probe sequence and is combined in the second fluorophore and the second cancellation group on described the second probe sequence, described the second probe sequence can with described the second oligonucleotide hybridization, there is cancellation reaction in described the second fluorophore and described the second cancellation group, described the first fluorophore is different with described the second fluorophore,
The 2nd DNA nickase, the 3rd DNA nickase and dNTPs, it is separated with described the first cancellation group that rear described the first fluorophore be sheared and be sheared to described the 2nd DNA nickase can to described the first probe sequence after hybridization, and it is separated with described the second cancellation group that rear described the second fluorophore be sheared and be sheared to described the 3rd DNA nickase can to described the second probe sequence after hybridization.
2. difunctional aptamers detection kit according to claim 1, is characterized in that, described the first masterplate DNA is the sequence shown in SEQ ID No.1, and the second masterplate DNA is the sequence shown in SEQ ID No.2.
3. difunctional aptamers detection kit according to claim 1, is characterized in that, described the first probe sequence is the sequence shown in SEQ ID No.3, and described the second probe sequence is the sequence shown in SEQ ID No.4.
4. difunctional aptamers detection kit according to claim 1, is characterized in that, described the first fluorophore is Fluoresceincarboxylic acid, and described the second fluorophore is tetramethyl-rhodamine.
5. difunctional aptamers detection kit according to claim 1, it is characterized in that, described archaeal dna polymerase is archaeal dna polymerase Klenow fragment, a described DNA nickase is DNA nickase Nb.BbvCI, described the 2nd DNA nickase is DNA nickase Nb.BtsI, and described the 3rd DNA nickase is DNA nickase Nt.AlwI.
6. a difunctional aptamers detection method, for carry out Hg simultaneously
2+and Ag
+two metal ion species detect, and it is characterized in that, comprise the steps:
After testing sample, the first masterplate DNA and the second masterplate DNA are mixed, there is hybridization and obtain hybridization solution, wherein, in described the first masterplate DNA, contain 15 bases with described the second masterplate DNA complementary pairing, and described the first masterplate DNA and described the second masterplate DNA form after hybridization the 3 ' end of described the first masterplate DNA by described 15 bases and the 3 ' end of described the second masterplate DNA forms respectively the mispairing of 3 thymus pyrimidines and the mispairing of 3 cytosine(Cyt)s;
In described hybridization solution, add dNTPS, archaeal dna polymerase and a DNA nickase generation oligonucleotide strand displacement amplified reaction, obtain oligonucleotide amplification liquid, wherein, the mispairing of thymus pyrimidine is at Hg
2+existence under, form T-Hg
2+the special pairing structure of-T, increases for masterplate thereby make the 3 ' end of the first masterplate DNA after hybridization take the 5 ' end of the second masterplate DNA, and the mispairing of cytosine(Cyt) is at Ag
+existence under, form C-Ag
+the special pairing structure of-C, thereby making the 3 ' end of the second masterplate DNA after hybridization take the 5 ' end of the first masterplate DNA increases for masterplate, a described DNA nickase is sheared and is formed the first oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification described the first masterplate DNA out, and a described DNA nickase is also sheared and formed the second oligonucleotide the complementary sequence of the 5 ' terminal sequence of amplification described the second masterplate DNA out;
In described oligonucleotide amplification liquid, add the first fluorophore cancellation group probe, the second fluorophore cancellation group probe, there is difunctional amplified fluorescence reaction in the 2nd DNA nickase and the 3rd DNA nickase, obtain fluoroscopic examination liquid, wherein, described the first fluorophore cancellation group probe comprises the first probe sequence and is combined in the first fluorophore and the first cancellation group on described the first probe sequence, described the first probe sequence can with described the first oligonucleotide hybridization, there is cancellation reaction in described the first fluorophore and described the first cancellation group, described the second fluorophore cancellation group probe comprises the second probe sequence and is combined in the second fluorophore and the second cancellation group on described the second probe sequence, described the second probe sequence can with described the second oligonucleotide hybridization, there is cancellation reaction in described the second fluorophore and described the second cancellation group, described the first fluorophore is different with described the second fluorophore, it is separated with described the first cancellation group that rear described the first fluorophore is sheared and sheared to described the 2nd DNA nickase to described the first probe sequence after hybridizing, it is separated with described the second cancellation group that rear described the second fluorophore is sheared and sheared to described the 3rd DNA nickase to described the second probe sequence after hybridizing,
Described fluoroscopic examination liquid is carried out to fluoroscopic examination, complete described difunctional aptamers detection method.
7. difunctional aptamers detection method according to claim 6, is characterized in that, described the first masterplate DNA is the sequence shown in SEQ ID No.1, and the second masterplate DNA is the sequence shown in SEQ ID No.2.
8. difunctional aptamers detection method according to claim 6, is characterized in that, described the first probe sequence is the sequence shown in SEQ ID No.3, and described the second probe sequence is the sequence shown in SEQ ID No.4.
9. difunctional adaptive health check-up detection method according to claim 6, it is characterized in that, described archaeal dna polymerase is archaeal dna polymerase Klenow fragment, a described DNA nickase is DNA nickase Nb.BbvCI, described the 2nd DNA nickase is DNA nickase Nb.BtsI, and described the 3rd DNA nickase is DNA nickase Nt.AlwI.
10. difunctional adaptive health check-up detection method according to claim 6, it is characterized in that, in described oligonucleotide amplification liquid, add the first fluorophore cancellation group probe, the second fluorophore cancellation group probe, the 2nd DNA nickase and the 3rd DNA nickase to occur, in the operation of difunctional amplified fluorescence reaction, also to need to add wherein bovine serum albumin.
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| CN107677661A (en) * | 2017-08-16 | 2018-02-09 | 樊之雄 | A kind of detection method of the chemiluminescence sensor based on adaptation body measurement silver ion |
| CN108342456A (en) * | 2018-02-08 | 2018-07-31 | 中国农业大学 | A kind of visualization of dual heavy metal ion quantifies new detecting method |
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