CN116179654B - Rolling circle amplification detection system for detecting cadmium ions in water and application thereof - Google Patents
Rolling circle amplification detection system for detecting cadmium ions in water and application thereof Download PDFInfo
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Abstract
The invention provides a rolling circle amplification detection system for detecting cadmium ions in water and application thereof, belonging to the technical field of cadmium ion detection. The invention provides a rolling circle amplification detection system for detecting cadmium ions in water, which comprises a DNA template and an allosteric transcription factor CadC. The invention specifically recognizes Cd 2+ The allosteric transcription factor CadC of (2) is taken as a recognition element, the triggering of rolling circle amplification is regulated and controlled, and the Cd can be realized 2+ Is an ultra-sensitive detection of (a). The detection limit of the rolling circle transcription amplification system controlled by CadC can reach 0.133nM.
Description
Technical Field
The invention belongs to the technical field of cadmium ion detection, and particularly relates to a rolling circle amplification detection system for detecting cadmium ions in water and application thereof.
Background
Heavy metal ions in the environment are difficult to degrade and can be led toThe biological enrichment effect causes serious harm to human health. Cadmium has wide application in the fields of electronics, military industry, metallurgy and the like, and once the cadmium enters a human body, cadmium ions (Cd) 2+ ) Accumulation in the body can lead to dysfunction of the kidneys and central nervous system, and may also lead to renal and prostate tumors. The highest limit value of cadmium specified in the drinking water quality standard and class III surface water quality standard in China is 44.48nmol/L (0.005 mg/L). At present, the conventional methods commonly used for detecting heavy metals, such as inductively coupled plasma mass spectrometry (ICP-MS) and Atomic Absorption Spectrometry (AAS), have higher sensitivity and accuracy, but are realized by expensive instruments and experienced technicians, so that the method is unfavorable for field detection. While the emerging biosensor is simple to operate, but has low sensitivity, and is difficult to meet the national standard requirement, and a signal amplification method is needed to further reduce the detection limit. Rolling circle amplification is a recently developed isothermal nucleic acid amplification method, and amplification of nucleic acid signals can be achieved under isothermal conditions. Allosteric transcription factors (allosteric transcription factor, aTF) are a class of regulatory proteins that are widely distributed in bacteria, typically comprising two domains, an effector sensing domain (EBD) and a DNA Binding Domain (DBD). The small molecule effector alters the conformation of aTF by binding to EBD to enhance or attenuate the binding capacity of aTF to DNA, thereby regulating transcription and expression of the gene. Nothing is disclosed in the prior art about the detection of cadmium ions in water by means of the modulation of rolling circle amplification by allosteric transcription factors.
Disclosure of Invention
In view of the above, the invention aims to provide a rolling circle amplification detection system for detecting cadmium ions in water, which adopts an allosteric transcription factor CadC for specifically recognizing the cadmium ions as a recognition element, and combines the rolling circle amplification in vitro transcription system to detect the cadmium ions in water, so that the detection limit of the cadmium ions can be greatly improved, and the detection limit of the cadmium ions can reach 0.133nM.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a rolling circle amplification detection system for detecting cadmium ions in water, which comprises a DNA template and an allosteric transcription factor CadC.
Preferably, the rolling circle amplification detection system further comprises 5 Xtranscription buffer, NTP, pyrophosphatase TIPP, thiothioflavin ThT, T7RNA polymerase and double distilled water.
Preferably, the DNA template is prepared from a short chain with a nucleotide sequence shown as SEQ ID NO.1 and a long chain with a nucleotide sequence shown as SEQ ID NO. 2.
Preferably, the amino acid sequence of the allosteric transcription factor CadC is shown as SEQ ID NO. 3.
The invention also provides a method for preparing the rolling circle amplification detection system, which comprises the following steps: mixing the DNA template and the allosteric transcription factor CadC, incubating at 25-42 ℃ for 15-30min, and adding the rest substances.
The invention also provides application of the rolling circle amplification detection system in detection of cadmium ions in water.
The invention also provides a method for detecting cadmium ions in water, which comprises the following steps: dropwise adding a water sample to be detected into the rolling circle amplification detection system, incubating at 25-42 ℃ for more than 2 hours, measuring the fluorescence intensity by using an enzyme-labeled instrument, and calculating according to a quantitative formula Y=182.2×X+462.9 to obtain the content of cadmium ions in the water sample; in the quantitative formula, Y is a fluorescence value, and X is the logarithm of the concentration of cadmium ions.
Preferably, the volume ratio of the water sample to be detected to the rolling circle amplification detection system is 1:9.
Preferably, the excitation wavelength of the enzyme-labeled instrument is 425nm, and the emission wavelength is 490nm.
The invention has the beneficial effects that:
the invention specifically recognizes Cd 2+ The allosteric transcription factor CadC of (2) is taken as a recognition element, the triggering of rolling circle amplification is regulated and controlled, and the Cd can be realized 2+ Is an ultra-sensitive detection of (a). The detection limit of the rolling circle transcription amplification system controlled by CadC can reach 0.133nM.
Compared with the existing method, the detection method has the advantages of simplicity in operation, high sensitivity and strong specificity. Specifically, the method of the invention is carried out by a DNA template andafter the preparation of the allosteric transcription factor CadC, the detection system can be produced in batch, and one centrifuge tube is the detection system. In the practical application of detection, the detection of cadmium ions can be realized by only adding the water sample to be detected into the centrifuge tube, and complex operation is not required. In addition, the detection method of the invention specifically recognizes Cd 2+ The allosteric transcription factor CadC of (2) is taken as a recognition element, and the small molecule effector is combined with the EBD to change the conformation of the allosteric transcription factor so as to enhance or weaken the binding capacity of the allosteric transcription factor and DNA, thereby regulating the transcription and expression of genes. CadC can specifically identify cadmium ions, and trigger the rolling circle amplification to be carried out through an allosteric effect, so that the detection specificity is very strong.
Drawings
FIG. 1 is a graph showing the statistics of fluorescence values generated by each group of reaction systems;
FIG. 2 shows agarose gel electrophoresis of each group of reaction systems, wherein 4 lanes are 1 lane 2000bp marker from left to right in sequence; lane 2 DNA; lane 3 dna+cadc; lane 4 DNA+CadC+Cd 2+ ；
FIG. 3 is a graph showing the result of obtaining the quantitative formula for detecting cadmium ions according to the present invention;
FIG. 4 shows the result of the method for detecting cadmium ions according to the present invention having high specificity.
Detailed Description
The invention provides a rolling circle amplification detection system for detecting cadmium ions in water, which comprises a DNA template and an allosteric transcription factor CadC.
In the present invention, the DNA template is preferably composed of a nucleotide sequence as shown in SEQ ID NO.1:5'-TAATACGACTCACTATAGGGAGACTCAAATAAATATTTGAATGAA-3' the short chain and nucleotide sequence is shown in SEQ ID NO.2: 5'-GTCGTATTACCCACCCAACCCACCCTTCCCACCCAACCCACCCTTCCCACCCAACCCACCCTTCATTCAAATATTTATTTGAGTCTCCCTATAGTGA-3'. In the present invention, the preparation method of the DNA template preferably comprises the steps of: the short-chain DNA (SEQ ID NO. 1) and the long-chain DNA (SEQ ID NO. 2) with the final concentration of 1.1 mu mol/L are mixed, and the two single chains are subjected to base complementary pairing to form partial double chains in a water bath at 95 ℃ for 10 min. Then the temperature is slowly reduced to room temperature after 30min, so that the double-chain structure is stable. And then, carrying out overnight treatment on the DNA by using T4 DNA ligase at the temperature of 4 ℃ to ensure that long-chain DNA with a phosphate group marked at the 5' end is connected end to form a local double-chain annular DNA template.
In the present invention, the amino acid sequence of the allosteric transcription factor CadC is preferably as shown in SEQ ID NO.3:
MKKKDTCEIFCYDEEKVNRIQGDLQTVDISGVSQILKAIADENRAKITYALCQDEELCVCDIANILGVTIANASHHLRTLYKQGVVNFRKEGKLALYSLGDEHIRQIMMIALAHKKEVKVNV. The specific source of the allosteric transcription factor CadC is not particularly limited, and can be synthesized by using the conventional protein synthesis procedure in the art.
In the present invention, the rolling circle amplification detection system preferably further comprises 5 Xtranscription buffer, NTP, pyrophosphatase TIPP, thiothiothioflavin ThT, T7RNA polymerase and double distilled water. The NTP preferably consists of ATP, UTP, CTP and GTP. The specific sources of the substances in the rolling circle amplification detection system are not particularly limited, and the products which are conventionally and commercially available in the field can be adopted. In the invention, the volume and dosage proportion relation of each substance in the rolling circle amplification detection system is preferably (based on the total volume of the rolling circle amplification detection system of 18 mu l): 1.5. Mu.l (1. Mu. Mol/L), 2. Mu.l (10. Mu. Mol/L) CadC, 4. Mu.l of 5 Xtranscription buffer, 0.6. Mu.l (100 mmol/L) ATP, 0.6. Mu.l (100 mmol/L) UTP, 0.6. Mu.l (100 mmol/L) CTP, 0.6. Mu.l (100 mmol/L) GTP, 0.15. Mu.l (0.3U/L) pyrophosphatase TIPP, 1. Mu.l (10 mmol/L) thiothiothioflavin ThT, 2. Mu.l (200U/mu.l) T7RNA polymerase, and 4.95. Mu.l double distilled water. The rolling circle amplification detection system has the advantages that all substances are necessary substances, no any substance is absent, normal reaction can not be carried out, and the highest reaction efficiency is achieved under the volume and dosage ratio of all substances defined by the rolling circle amplification detection system.
The invention also provides a method for preparing the rolling circle amplification detection system, which comprises the following steps: mixing the DNA template and the allosteric transcription factor CadC, incubating at 25-42 ℃ for 15-30min, and adding the rest substances.
In the method for preparing the rolling circle amplification detection system, the incubation mode is preferably water bath, the incubation temperature is preferably 30-40 ℃, more preferably 35-38 ℃, the incubation time is preferably 16-25min, more preferably 19-22min, the DNA template and the allosteric transcription factor CadC are mixed and incubated for a period of time, and then the rest substances are added, so that the DNA template and the CadC can be fully combined. In the present invention, the surplus substances are substances required for performing the rolling circle amplification reaction, and the surplus substances of the present invention preferably include 5X transcription buffer, NTP, pyrophosphatase TIPP, thiothiothioflavin ThT, T7RNA polymerase and double distilled water.
The invention also provides application of the rolling circle amplification detection system in detection of cadmium ions in water.
The invention also provides a method for detecting cadmium ions in water, which comprises the following steps: dropwise adding a water sample to be detected into the rolling circle amplification detection system, incubating at 25-42 ℃ for more than 2 hours, measuring the fluorescence intensity by using an enzyme-labeled instrument, and calculating according to a quantitative formula Y=182.2×X+462.9 to obtain the content of cadmium ions in the water sample; in the quantitative formula, Y is a fluorescence value, and X is the logarithm of the concentration of cadmium ions.
In the invention, the volume ratio of the water sample to be detected to the rolling circle amplification detection system is preferably 1:9. After the water sample to be detected is dripped into the rolling circle amplification detection system, the incubation temperature is preferably 30-40 ℃, more preferably 35-38 ℃, and the incubation time is preferably 3h. The invention has no special limitation on the specific model and source of the enzyme-labeled instrument, and can adopt the enzyme-labeled instrument which is commonly sold in the field. In the invention, the excitation wavelength of the enzyme label instrument is preferably 425nm, and the emission wavelength is preferably 490nm.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the following examples, conventional methods are used unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
(a) Preparation of DNA template:
the DNA template is prepared from two single strands, the short chain length is 45bp, and the sequence is 5'-TAATACGACTCACTATAGGGAGACTCAAATAAATATTTGAATGAA-3' (SEQ ID NO. 1); the long chain length is 97bp, and the sequence is 5'-GTCGTATTACCCACCCAACCCACCCTTCCCACCCAACCCACCCTTCCC ACCCAACCCACCCTTCATTCAAATATTTATTTGAGTCTCCCTATAGTGA-3' (SEQ ID NO. 2). The single strands constituting the DNA template were synthesized by the division of biological engineering (Shanghai).
Mixing short-chain DNA with the final concentration of 1.1 mu mol/L and long-chain DNA with the final concentration of 1 mu mol/L, and carrying out base complementary pairing on the two single chains to form partial double chains in a water bath at 95 ℃ for 10 min. Then the temperature is slowly reduced to room temperature after 30min, so that the double-chain structure is stable. And then, carrying out overnight treatment on the DNA by using T4 DNA ligase at the temperature of 4 ℃ to ensure that long-chain DNA with a phosphate group marked at the 5' end is connected end to form a local double-chain annular DNA template, thus obtaining the DNA template of the method.
(b) Preparation of the allosteric transcription factor CadC: an allosteric transcription factor CadC which specifically recognizes cadmium ions is 122aa, and the amino acid sequence is (SEQ ID NO. 3): MKKKDTCEIFCYDEEKVNRIQGDLQTVDISGVSQILKAIADENRAKITYAL CQDEELCVCDIANILGVTIANASHHLRTLYKQGVVNFRKEGKLALYSLGD EHIRQIMMIALAHKKEVKVNV. The synthesis work was done by the company Kirsry Biotech Co.
(c) Reagent source: 5 Xtranscription buffer, T7RNA polymerase, ATP, UTP, CTP, GTP, available from Sieimer, USA; TIPP is available from NEB company in the united states; thT is purchased from shanghai alaa Ding Shenghua technologies.
(d) Establishment of a rolling circle amplification detection system: 1.5. Mu.l (1. Mu. Mol/L) of the DNA template and 2. Mu.l (10. Mu. Mol/L) of CadC were sequentially added to a 200. Mu.l centrifuge tube, and the mixture was subjected to a water bath at 37℃for 15 minutes. Then, 4. Mu.l of 5 Xtranscription buffer, 0.6. Mu.l (100 mmol/L) of ATP, 0.6. Mu.l (100 mmol/L) of UTP, 0.6. Mu.l (100 mmol/L) of CTP, 0.6. Mu.l (100 mmol/L) of GTP, 0.15. Mu.l (0.3U/L) of pyrophosphatase TIPP, 1. Mu.l (10 mmol/L) of thiothiothioflavin ThT, 2. Mu.l (200U/mu.l) of T7RNA polymerase and 4.95. Mu.l of double distilled water were sequentially added to constitute an 18. Mu.l rolling circle amplification detection system.
(e) Detecting cadmium ions in a water sample: 2 μl of the water sample to be detected is dripped into the rolling circle amplification detection system established in the step (d), after incubation is carried out for 3 hours at 37 ℃, 20 μl of the reaction system is transferred into a 384-well plate (transparent bottom), and the generated fluorescence intensity is measured by using an enzyme-labeled instrument. Selecting a microplate reader FL mode, and setting a microplate reader program: the excitation wavelength is 425nm, the emission wavelength is 490nm, the fluorescence intensity is measured by selecting the area where the sample adding hole is located, and the content of cadmium ions in the water sample is calculated according to a quantitative formula.
(f) Obtaining a cadmium ion detection quantitative formula: detecting the cadmium ion solution with the concentration of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500 and 1000nmol/L by using the rolling circle amplification detection system established in (d). Recording and analyzing the fluorescence value measured by the enzyme label instrument, wherein the fluorescence value has a linear relation with the logarithm of the concentration of cadmium ions, the linear equation of the fluorescence value is Y=182.2×X+462.9 between 0.1 nmol/L and 500nmol/L, and R is as follows 2 = 0.9911, where Y is the fluorescence value and X is the logarithm of the cadmium ion concentration.
Example 2
Setting 3 groups of reaction systems, and carrying out three parallel tests on each group of reaction systems, wherein the three parallel tests are as follows: (1) The rolling circle amplification system of only DNA template (i.e., compared with the rolling circle amplification detection system of example 1, except that CadC is not contained, the other materials and the amount of each material are the same as those of the rolling circle amplification detection system of example 1); (2) Adding CadC with a final concentration of 1 mu mol/L on the basis of the step (1); (3) Cadmium ions with a final concentration of 1 mu mol/L are added on the basis of (2). The fluorescence values of the 3-group systems were measured after 3 hours of reaction (the specific procedure is the same as in example 1), and the results are shown in Table 1 and FIG. 1. 15. Mu.L of the reaction system was subjected to 1% agarose gel electrophoresis for 40min, and the results are shown in FIG. 2.
TABLE 1 fluorescence values generated by the reaction systems of the respective groups
Experimental results show that the fluorescence value is obviously reduced after CadC is added, and the fluorescence value is increased again with the addition of cadmium ions. As can also be seen from agarose gel electrophoresis, there is a clear RNA formation in the rolling circle amplification reaction of only the DNA template, almost no RNA formation occurs after CadC is added, and the molecular weight of the DNA is increased due to the combination of the DNA and CadC, so that the phenomenon of migration lag occurs; and with the addition of cadmium ions, the DNA is separated from CadC, and the rolling circle amplification is triggered again to generate RNA. The fluorescence result corresponds to the result of a gel electrophoresis experiment, and proves that the allosteric transcription factor mediated rolling circle amplification detection system of the invention is successfully constructed.
Example 3
The detection of 0.1, 0.5, 1, 5, 10, 50, 100, 500nmol/L of cadmium ion solution was performed by using the rolling circle amplification detection system constructed in example 1, and the specific detection procedure is the same as that of example 1, and 3 parallel tests were performed on each cadmium ion solution, and the results are shown in Table 2 and FIG. 3.
TABLE 2 fluorescence values generated for the detection of cadmium ions at different concentrations
Experimental results show that the detection method has great application potential in the aspect of actual quantitative detection of cadmium ions in water. Calculation of detection limit: the detection limit is also called detection limit, which refers to the average value of the background signal generated by the matrix blank plus 3 times of average standard deviation. The rolling circle amplification detection system established in example 1 (d) was used for 10 times to obtain fluorescence values y=x±3sd=257.4+3× 15.33753566 = 303.412607 corresponding to the detection limit, taking ultrapure water containing no cadmium ions as a blank sample, and bringing the fluorescence values into a quantitative linear formula: y=182.2×x+462.9, x= -0.875, and the lowest detection limit concentration after logarithmic conversion is 0.133nmol/L. Namely, the detection limit of the detection method is as low as 0.133nmol/L, and ultrasensitive detection of cadmium ions in water can be realized.
Example 4
In order to verify the detection specificity of the detection system for detecting the rolling circle amplification mediated by the allosteric transcription factor, the detection system of the embodiment 1 is used for detecting cadmium, zinc, copper, mercury, chromium, nickel and arsenic with the final concentration of 1 mu mol/L and the mixed heavy metal ion solution, and the specific detection steps are the same as those of the embodiment 1, and the detection result is shown in fig. 4. The result shows that other heavy metal ions have no influence on the detection result of the detection method, and the detection of cadmium ions is not influenced under the condition that mixed heavy metal ions exist, so that the detection method has high specificity for the detection of cadmium ions.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (6)
1. The rolling circle amplification detection system for detecting cadmium ions in water is characterized by comprising a DNA template and an allosteric transcription factor CadC;
the rolling circle amplification detection system also comprises 5 Xtranscription buffer solution, NTP, pyrophosphatase TIPP, thiothiothioflavin ThT, T7RNA polymerase and double distilled water;
the DNA template is prepared from a short chain with a nucleotide sequence shown as SEQ ID NO.1 and a long chain with a nucleotide sequence shown as SEQ ID NO. 2;
the amino acid sequence of the allosteric transcription factor CadC is shown as SEQ ID NO. 3.
2. A method of preparing the rolling circle amplification detection system of claim 1, comprising the steps of: after mixing the DNA template and the allosteric transcription factor CadC, incubation is carried out for 15min at 37℃and 5 Xtranscription buffer, NTP, pyrophosphatase TIPP, thiothiothioflavin ThT, T7RNA polymerase and double distilled water are added.
3. The use of the rolling circle amplification detection system of claim 1 in the detection of cadmium ions in water.
4. A method for detecting cadmium ions in water, comprising the steps of: dropwise adding a water sample to be detected into the rolling circle amplification detection system of claim 1, incubating for 3 hours at 37 ℃, measuring the fluorescence intensity by using an enzyme-labeled instrument, and calculating according to a quantitative formula Y=182.2×X+462.9 to obtain the content of cadmium ions in the water sample; in the quantitative formula, Y is a fluorescence value, and X is the logarithm of the concentration of cadmium ions.
5. The method of claim 4, wherein the volume ratio of the water sample to be tested to the rolling circle amplification detection system is 1:9.
6. The method of claim 4, wherein the excitation wavelength of the microplate reader is 425nm and the emission wavelength is 490nm.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946681A (en) * | 2014-12-24 | 2015-09-30 | 温州医科大学 | Microbiological method for detecting heavy-metal cadmium in water body |
| KR20160052328A (en) * | 2014-10-29 | 2016-05-12 | 한국생명공학연구원 | Protein Expression System Induced by Heavy Metals And Biosensor for Detecting Heavy Metals |
| CN110684789A (en) * | 2019-10-24 | 2020-01-14 | 南京林业大学 | Fusion gene, recombinant vector and preparation method thereof, cadmium ion whole-cell biosensor and preparation method and application thereof |
| CN112011597A (en) * | 2020-07-24 | 2020-12-01 | 南京师范大学 | Cadmium ion sensing method combining induced allosteric probe with rolling circle amplification |
| CN113347993A (en) * | 2018-10-22 | 2021-09-03 | 艾赛澳克莱克斯疗法有限责任公司 | Mutant vaccinia virus and uses thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2018379996A1 (en) * | 2017-12-05 | 2020-06-25 | BioPlx, Inc. | Methods and compositions to prevent microbial infection |
| WO2021145889A1 (en) * | 2020-01-17 | 2021-07-22 | Zepto Life Technology, LLC | Systems and methods for sensing analytes in gmr-based detection of biomarkers |
| CN113355400B (en) * | 2021-04-06 | 2023-08-22 | 南京师范大学 | Cadmium ion detection method based on T3 DNA ligase |
| CN116179654B (en) * | 2022-12-30 | 2023-09-15 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Rolling circle amplification detection system for detecting cadmium ions in water and application thereof |
-
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-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160052328A (en) * | 2014-10-29 | 2016-05-12 | 한국생명공학연구원 | Protein Expression System Induced by Heavy Metals And Biosensor for Detecting Heavy Metals |
| CN104946681A (en) * | 2014-12-24 | 2015-09-30 | 温州医科大学 | Microbiological method for detecting heavy-metal cadmium in water body |
| CN113347993A (en) * | 2018-10-22 | 2021-09-03 | 艾赛澳克莱克斯疗法有限责任公司 | Mutant vaccinia virus and uses thereof |
| CN110684789A (en) * | 2019-10-24 | 2020-01-14 | 南京林业大学 | Fusion gene, recombinant vector and preparation method thereof, cadmium ion whole-cell biosensor and preparation method and application thereof |
| CN112011597A (en) * | 2020-07-24 | 2020-12-01 | 南京师范大学 | Cadmium ion sensing method combining induced allosteric probe with rolling circle amplification |
Non-Patent Citations (3)
| Title |
|---|
| 3D-printed rolling circle amplification chip for on-site colorimetric detection of inorganic mercury in drinking water;Ji Won Lim等;Food Chemistry;第300卷;125177 * |
| 基于核酸适配体的镉离子可视化检测方法;赵旭;吴世嘉;乐琳;王周平;;食品与机械(第06期);35-38 * |
| 滚环扩增(RCA)技术放大信号实现17β-雌二醇(E2)的超灵敏检测;王伟亚等;解放军预防医学杂志;第38卷(第12期);78-83 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL2035508B1 (en) * | 2022-12-30 | 2024-07-09 | Tianjin Inst Of Environmental And Operational Medicine | Rolling circle amplification detection system for detecting cadmium ions in water and application thereof |
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