CN111751540B - SERS lateral flow test strip for nuclease detection and detection method - Google Patents
SERS lateral flow test strip for nuclease detection and detection method Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- 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
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
Abstract
The invention relates to a SERS lateral flow test strip for nuclease detection and a detection method. The SERS lateral flow test strip comprises a bottom plate, a sample pad, a combination pad, a reaction film and a water absorption pad, wherein the reaction film is provided with a detection line and a control line, the detection line is arranged at one end close to the combination pad, and the control line is arranged at one end close to the absorption pad; the combination pad is coated with Raman reporter molecules and nanoparticles for detecting DNA marks to serve as SERS nano probes, the detection lines are fixed with streptavidin, and the control lines are fixed with anti-single-chain DNA antibodies. The method is used for quantitative analysis by the SERS technology, the SERS signal is strong, no light drifting is caused, the fingerprint information is rich, the stability is strong, so the detection has higher sensitivity, the SERS lateral flow test strip is prepared by combining the immunochromatography technology, the sample demand is low, the operation is simple and convenient, the reaction is rapid, the result is accurate, the price is low, the detection limit is low, the selectivity is good, and the mass production is easy to realize.
Description
Technical Field
The invention relates to a SERS lateral flow test strip for nuclease detection and a detection method, belonging to the technical field of photochemical analysis.
Background
An enzyme capable of cleaving the phosphodiester bond of a polynucleotide chain is called a nuclease. Nucleases belong to the hydrolases and act on the P-O position of the phosphodiester bond. Nucleases are nucleic acids that act to hydrolyze phosphodiester bonds between nucleotides in the first step of nucleic acid degradation. The ester has an effect on phosphodiester bonds in higher animals and plants. The specificity and the mode of action of nucleases are different from one source to another. Some nucleases can only act on RNA, called ribonucleases (rnases), some can only act on DNA, called deoxyribonucleases (dnases), and some have low specificity and can act on both RNA and DNA, and are therefore collectively called nucleases (nucleases). Depending on the position of action of nucleases, nucleases can be further classified into exonucleases (exonuclease) and endonucleases (endonuclease).
Some nucleases hydrolyze single nucleotides one by one from one end of a DNA or RNA strand and are called exonucleases. Exonucleases that act only on DNA are called deoxyribonucleases, exonucleases that act only on RNA are called ribonucleases; there are also a number of exonucleases that can act on DNA or RNA. Exonuclease hydrolyzes nucleotides one by one from the 3 'end, called 3' → 5 'exonuclease, e.g., snake venom phosphodiesterase, a 3' → 5 'exonuclease, with the hydrolysis product being 5' nucleotides; exonucleases hydrolyze nucleotides one by one starting from the 5' end, called 5' → 3' exonucleases, for example: bovine splenic phosphodiesterase is a 5' → 3' exonuclease with the hydrolysis product being the 3' nucleotide.
With the progress of the times, the fields of modern medical biology and the like have proven that nucleases play a key role in biological processes involving replication, recombination, DNA repair, molecular cloning, genotyping and mapping, and therefore, it is of great significance to accurately analyze the activity of nucleases. The traditional detection methods of enzymes include gel electrophoresis, radiolabeling, high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA), although these methods generally have higher accuracy, the methods still have many critical problems to be solved, such as tedious operation, long period, introduction of radioactive substances, expensive instruments or complex modification processes, and difficulty in realizing high-throughput and automatic detection. Therefore, the current enzyme detection technology needs to be further improved and applied to the actual life production.
Disclosure of Invention
The invention aims to provide a simple, quick and sensitive SERS lateral flow test strip for nuclease detection and a detection method aiming at the defects.
In order to achieve the purpose, the invention adopts the technical scheme that:
a SERS lateral flow test strip for nuclease detection comprises a bottom plate, and a sample pad, a binding pad, a reaction film and a water absorption pad which are sequentially overlapped on the bottom plate from left to right, wherein the reaction film is provided with a detection line and a control line, the detection line is arranged at one end close to the binding pad, and the control line is arranged at one end close to the adsorption pad;
streptavidin is fixed on the detection line, and an anti-single-chain DNA antibody is fixed on the control line;
the combination pad is dropwise added with an SERS nano probe, and the SERS nano probe consists of a Raman reporter molecule and nano particles for detecting DNA marks;
dropwise adding a mixed solution of substrate DNA and nuclease to the sample pad;
the nano particles are one of gold nano particles, silver nano particles and gold/silver composite nano particles, the particle size of the nano particles is 20-100nm, and the nano particles are spherical, rod-shaped, sheet-shaped, triangular, nanoflower or dendritic;
the Raman reporter molecule is one of Malachite Green Isothiocyanate (MGITC), 4-mercaptopyridine, rhodamine B isothiocyanate and Raman reporter molecules containing sulfydryl or isothiocyanate;
the detection DNA is single-stranded DNA with one end modified with sulfydryl or amino;
coupling Raman reporter molecules and detection DNA to the nanoparticles in a covalent coupling mode to obtain an SERS nano probe;
diluting the SERS nano probe by using a cross-linking solution and then coating the diluted SERS nano probe on a bonding pad;
the nuclease is exonuclease I, exonuclease II I and S1 nuclease;
the SERS nano probe and substrate DNA are hybridized to form a double-chain structure, and biotin modified by the tail end of the substrate DNA is combined with streptavidin on a test line; the detection DNA modified on the surface of the SERS nano probe is combined with the anti-single-chain DNA antibody on the control line through the interaction of the antibody and the antigen.
Further, the sample pad is prepared by drying after being treated by the sample pad treatment liquid.
Further, the reaction membrane is one of a nitrocellulose membrane, a polyvinylidene fluoride cellulose membrane, a polyacetate cellulose membrane, a polysulfonamide membrane and a polyethyleneimine membrane;
streptavidin and anti-single-chain DNA antibodies are coated on the detection line and the control line of the reaction membrane respectively by using phosphate buffer.
The invention also provides a preparation method of the SERS lateral flow test strip for nuclease detection, which comprises the following steps:
(1) Treating the sample pad by using a sample solution, and drying;
(2) Coupling the Raman reporter molecule and the detection DNA to the nano-particles in a covalent coupling mode to obtain an SERS nano-probe; diluting the SERS nano probe by using a cross-linking solution and then coating the diluted SERS nano probe on a bonding pad;
(3) Respectively coating streptavidin and anti-single-chain DNA antibodies on a detection line and a control line of the reaction membrane by using a phosphate buffer solution;
(4) And sequentially attaching a sample pad, a combination pad, a reaction film and a water absorption pad from left to right on the bottom plate in a staggered manner to obtain the SERS lateral flow test strip.
The invention also provides a detection method of nuclease, which comprises the following steps: the method comprises the steps of dropwise adding a mixed solution of substrate DNA and nuclease into a test strip sample pad, combining with an SERS material dropwise added to a combination pad in advance, sequentially flowing through a test line and a control line by means of running buffer in a 96-well plate, and detecting the nuclease by observing the test line through a colorimetric method and detecting the test line through an SERS detection method.
During detection, a mixture of a nuclease sample to be detected and a substrate DNA modified by biotin with the same volume is dripped onto a sample pad, and a test strip carrying the nucleic acid sample is immersed into a buffer solution; substrate DNA on the sample pad is hydrolyzed into small fragments by nuclease, the hydrolyzed fragment DNA and unhydrolyzed substrate DNA reach the binding sheet through the sample sheet due to the siphoning effect principle, the unhydrolyzed substrate DNA and SERS nano-probes on the binding sheet are hybridized to form double-stranded DNA through base complementary pairing action, the double-stranded DNA diffuses towards the upper part of the reaction film and is subjected to coupling reaction with a detection line fixed on the reaction film, and redundant SERS nano-probes continuously diffuse towards the direction of a control line and react with the control line fixed on the reaction film; and (3) observing the color change of the detection line by naked eyes within 5-15 minutes at the temperature of 25 ℃, wherein the reaction results have the following two conditions:
a. when the detection line shows color, the detection result is negative, which indicates that the detected sample does not contain nuclease;
b. when the detection line does not display color, the detection result is positive, which indicates that the detected sample contains nuclease;
and (3) measuring SERS signals on the detection line by using a Raman instrument, and carrying out quantitative analysis on the content of nuclease.
Has the advantages that: the method is used for quantitative analysis by the SERS technology, the SERS signal is strong, no light drifting is caused, the fingerprint information is rich, the stability is strong, so the detection has higher sensitivity, the SERS lateral flow test strip is prepared by combining the immunochromatography technology, the sample demand is low, the operation is simple and convenient, the reaction is rapid, the result is accurate, the price is low, the detection limit is low, the selectivity is good, and the mass production is easy to realize.
Drawings
FIG. 1 is a schematic diagram of the detection of nuclease by colorimetric method of the present invention.
FIG. 2 is a representation, ultraviolet spectrum, scanning electron microscope and particle size characterization of the nano-gold material.
FIG. 3 is a representation of SERS nanoprobes, UV spectrogram, SEM image, and particle size characterization.
FIG. 4 is a graph of the test strip's Exo I standard curve and standard curve equation data.
FIG. 5 is a diagram of the specificity of the test strip.
FIG. 6 is a graph of the Exo I standard curve and standard curve equation data in human serum using test strips.
Detailed Description
The following examples further illustrate the working procedures and efficacy of the present invention, but the present invention is not limited thereto.
Reagent:
polyoxyethylene octyl phenyl ether (triton x-100), tris-HCl buffer, SSC buffer, bovine Serum Albumin (BSA), and Tris (2-carboxyethyl) phosphine (TCEP) were all purchased from Sigma-Aldrich, mercaptopyridine (4-MPY), sodium phosphate, and sucrose from Shanghai pharmaceutical group chemical Co., ltd. Streptavidin, PBS buffer, and DNA sequences were purchased from Biotechnology (Shanghai) Inc.
5-SH-TTT TTT TTT TTT TTT TTT TT CAC TCG AGC AGA-3
5-biotin-TCG AAC TCT GCT CGA GTG-3
Example 1
Using the present invention for the example of detecting Exo I nuclease, exo I nuclease can degrade the 3 '-end of single-stranded DNA to produce 3' -end single-stranded nucleotides or oligonucleotides. The preparation and detection steps are as follows:
(1) Preparation of the lateral flow test strip: the sample pad was soaked with 50 mM Tris-HCl containing 0.25% triton x-100 and 150 mM NaCl and dried in an oven at 37 ℃ for 2h. The test strip used consists of three parts, the sample pad and the absorbent pad are sequentially stuck to the nitrocellulose membrane with a PVC support, and firm contact must be ensured. Cutting the test strip into strips with the width of 4mm by using a numerical control strip cutting machine, and placing the cut test strips in a sterile environment for later use. And (3) dropwise adding 1 mu L of anti-single-chain DNA antibody to 1/5 of the test strip by using a liquid transfer gun, and dropwise adding 1 mu L of streptavidin of 0.5 mg/mL to form a control line and a detection line respectively at 2/5 of the test strip. And placing the prepared test strip of the control line and the test line in a sterile environment for waiting use.
(2) The SERS substrate gold nano material is synthesized by boiling 100mL (1 mM) of chloroauric acid solution, then quickly adding 10mL (38.8 mM) of sodium citrate solution (the color of the solution gradually changes from yellow to light gray and finally changes to wine red) into the solution, continuously boiling for 30min, stopping heating, stirring and cooling to room temperature, and finally storing in a refrigerator at 4 ℃ for later use.
(3) Constructing an SERS probe, namely, setting the concentration of 0.8 mu L to be 10 -4 Adding 4-MPY of M into 1mL of nano-gold solution, keeping out of the sun for 30min, adding Detection DNA into the nano-gold particle solution for modifying the Raman reporter molecule, uniformly mixing by vortex, and standing overnight. The next day, a mixed salt stable solution of ph7.0 containing 100 mM pbs, 1% SDS, 0.2M NaCl was added to the gold nanoparticles 5mL every 20 min for 22 times (after each addition, the solution was mixed well to prevent aggregation of the gold nanoparticle solution due to local concentration being too high), 12 h was placed in the dark. And concentrating and centrifuging the solution of the gold nanoparticles for modifying the Detection DNA, and removing the gold nanoparticles which are not combined with the Detection DNA. The method comprises the following specific steps: centrifuging at the rotating speed of 2500 r/min for 30min, discarding 900 μ L of supernatant, adding 1mL of 0.1 × pH7.0 PBS buffer solution into the precipitate, mixing by vortex, and repeating the above steps for 2 times. 100 μ L of a crosslinking solution (20 mM Na) was added to the precipitate 3 PO 4 ·12H 2 O, 5% BSA, 0.25% Tween 20, and 10% sucrose), and placing at 4 deg.C for refrigeration.
(4) The detection of Exo I enzyme by the test strip comprises the following specific steps: placing the prepared test strip with the control line and the test line in an aseptic environment as shown in fig. 1A, dropwise adding 5 μ L of modified SERS probes on the surface of the combination pad, waiting for natural air drying, mixing Exo i enzyme (0-0.01U/mL) with different concentrations and 10 μ L,4 μ M of Substrate DNA, and then dropwise adding the mixture into a sample plate of the test strip, vertically inserting the test strip into a 96-well plate of a Running buffer as shown in fig. 1B, allowing the Substrate DNA and the Exo i enzyme to sequentially flow through the test line and the control line from bottom to top along the test strip through capillary action, allowing the SERS probes on the surface of the combination pad to be combined with the test line and flow upwards when the Substrate DNA and the Exo i enzyme flow through the surface of the test strip, and performing SERS detection on the test line by means of color change of the surface of the test line. And (3) observing the color change of the detection line by naked eyes within 5-15 minutes at the temperature of 25 ℃, wherein the reaction results have the following two conditions:
a. when the detection line shows color, the detection result is negative, which indicates that the detected sample does not contain nuclease;
b. when the detection line does not show color, the detection result is positive, which indicates that the detected sample contains nuclease.
The SERS nano probe is hybridized with substrate DNA to form a double-chain structure, and biotin modified by the tail end of the substrate DNA is combined with streptavidin on a test line; the detection DNA modified on the surface of the SERS nano probe is combined with the anti-single-chain DNA antibody on the control line through the interaction of the antibody and the antigen.
FIG. 2 is a representation of a nanogold material, an ultraviolet spectrogram, a scanning electron micrograph and a particle size characterization chart. The color of the gold nano material synthesized as shown in FIG. 2A shows obvious wine red; the gold nano-material shown in FIG. 2B is in a regular circle shape, and has a good dispersion state, and the diameter is about 40 nm; the ultraviolet absorption wavelength of the gold nano-material shown in fig. 2C and 2D is 528nm, and the particle size is 38.98 +/-2 nm.
FIG. 3 is a graph of SERS nanoprobe phenomenon, UV spectrogram, SEM image, and particle size characterization. As shown in fig. 3A, the SERS probe has an obvious SERS signal, and the original AuNPs has no obvious SERS signal; as shown in FIG. 3B, the ultraviolet absorption peak of the SERS probe is at 529nm, and the absorption peak of AuNPs is at 528 nm; as shown in FIG. 3-C, the particle size of AuNPs is 38.95nm, and the particle size of SERS probe is 51.25nm, which fully indicates the success of SERS nanoprobe modification.
FIG. 4 is a diagram of a test strip sensor Exo I standard curve and a standard curve equation. As shown in FIG. 4A, when the concentration of detected Exo I enzyme is increased from 0 to 0.01U/mL in sequence, the color of the test line of the test strip is gradually lightened; when SERS detection is performed on the test line as shown in FIG. 4B, the obtained SERS signal value is gradually reduced along with the increase of the concentration of the detected Exo I enzyme; as shown in FIGS. 4C and 4D, the SERS signal peak (1100 cm) was determined -1 ) Drawing a standard curve and deducing a standard curve equation for the obtained data, andcan obtain R 2 =0.997。
Specific study: the specificity of the test strip is examined under the same experimental conditions, the concentrations of deoxyribonuclease I (DNaseI), thrombin (Thrombin) and Glucose Oxidase (Glucose Oxidase) are all 0.1U/mL, in addition, a control detection enzyme Exo I nuclease and a blank are set, and the concentration of the Exo I nuclease is 0.005U/mL. The overall operation steps are consistent as described above, and other used SERS probes, test strips, and the like are not changed except for the change of the detection object.
FIG. 5 is a chart of test strip specificity data. As shown in FIG. 5A, the test line of the test strip for detecting Exo I is nearly colorless, and the test lines of the other test strips are very similar in color; as shown in fig. 5B, SERS detection is performed on the test lines of all the test strips to obtain the highest SERS signal of the blank test line, and the lowest SERS signal of the test strip test line for detecting Exo i; proves that the sensor can specifically hydrolyze the Substrate DNA, and further proves that the sensor has strong specificity for detecting the ExoI enzyme and achieves the prior aim.
Application example
The application of the test strip in the serum actual sample comprises the following steps: a standard addition method is used, and serum diluted by 100 times is used as a solvent to prepare ExoI nuclease standard solutions (0-0.01U/mL) with different concentrations; the rest preparation and detection operation steps are unchanged from the steps; and (4) carrying out test strip detection on Exo I nuclease in serum, and carrying out SERS detection on a test line.
FIG. 6 is a graph of the Exo I standard curve and standard curve equation data in human serum using test strips. As shown in fig. 6A, the color of the test strip detection line obtained by performing test strip detection on Exo i enzyme in human serum gradually becomes lighter as the concentration of the Exo i enzyme increases; as shown in fig. 6B, SERS detection is sequentially performed on the test strip detection line, and the value of the obtained SERS signal is continuously reduced with the increase of the Exo i enzyme concentration; as shown in FIGS. 6C and 6D, the signal peak was fixed by 1100cm -1 Drawing a standard curve and deducing a standard curve equation for data obtained under different Exo I concentrations; the Raman signal and the Exo I concentration in serum are goodLinear relation (R) 2 = 0.998), detection limit is 3 × 10-5U/mL. The test strip can be used for actual detection.
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments of the invention are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and not by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (6)
1. A SERS lateral flow test strip for nuclease detection comprises a bottom plate, and a sample pad, a binding pad, a reaction film and a water absorption pad which are sequentially overlapped on the bottom plate from left to right, wherein the reaction film is provided with a detection line and a control line, the detection line is arranged at one end close to the binding pad, and the control line is arranged at one end close to the adsorption pad; the method is characterized in that:
streptavidin is fixed on the detection line, and an anti-single-chain DNA antibody is fixed on the control line;
the combination pad is dropwise added with an SERS nano probe, and the SERS nano probe consists of Raman reporter molecules and nano particles for detecting DNA marks;
the sample pad is dropwise added with a mixed solution of substrate DNA and nuclease;
the nano particles are one of gold nano particles, silver nano particles and gold/silver composite nano particles, the particle size of the nano particles is 20-100nm, and the nano particles are spherical, rod-shaped, flaky, triangular, nanoflower or dendritic;
the Raman reporter molecule is one of malachite green isothiocyanate MGITC, 4-mercaptopyridine, rhodamine B isothiocyanate and Raman reporter molecules containing sulfydryl or isothiocyanate radicals;
the detection DNA is single-stranded DNA with one end modified with sulfydryl or amino;
coupling Raman reporter molecules and detection DNA to the nanoparticles in a covalent coupling mode to obtain an SERS nano probe;
diluting the SERS nano probe by using a cross-linking solution and then coating the diluted SERS nano probe on a bonding pad;
the nuclease is exonuclease I, exonuclease II I, S1 nuclease;
the SERS nano probe is hybridized with substrate DNA to form a double-chain structure, and biotin modified by the tail end of the substrate DNA is combined with streptavidin on a test line; the detection DNA modified on the surface of the SERS nano probe is combined with the anti-single-chain DNA antibody on the control line through the interaction of the antibody and the antigen.
2. The SERS lateral flow test strip of claim 1, wherein: and the sample pad is prepared by drying after being treated by the sample pad treatment liquid.
3. The SERS lateral flow test strip of claim 1, wherein: the reaction membrane is one of a nitrocellulose membrane, a polyvinylidene fluoride cellulose membrane, a polyacetate cellulose membrane, a polysulfonamide membrane and a polyethyleneimine membrane;
streptavidin and anti-single-chain DNA antibodies are coated on the detection line and the control line of the reaction membrane respectively by using phosphate buffer.
4. A method of preparing a SERS lateral flow test strip according to any of claims 1 to 3, wherein: the method comprises the following steps:
(1) Treating the sample pad by using a sample solution, and drying;
(2) Coupling Raman reporter molecules and detection DNA to the nanoparticles in a covalent coupling mode to obtain an SERS nano probe; diluting the SERS nano probe by using a cross-linking solution and then coating the diluted SERS nano probe on a bonding pad;
(3) Respectively coating streptavidin and an anti-single-chain DNA antibody on a detection line and a control line of the reaction membrane by using a phosphate buffer solution;
(4) And sequentially attaching the sample pad, the combination pad, the reaction film and the water absorption pad from left to right in a staggered manner on the bottom plate to obtain the SERS transverse flow test strip.
5. A method for detecting a nuclease, comprising: the SERS lateral flow test strip of any one of claims 1-3 is used for detection, and comprises the following steps: the method comprises the steps of dropwise adding a mixed solution of substrate DNA and nuclease into a test strip sample pad, combining with an SERS material dropwise added to a combination pad in advance, sequentially flowing through a test line and a control line by means of running buffer in a 96-well plate, and detecting the nuclease by observing the test line through a colorimetric method and detecting the test line through an SERS detection method.
6. The detection method according to claim 5, characterized in that: during detection, a mixture of a nuclease sample to be detected and a substrate DNA modified by biotin with the same volume is dripped onto a sample pad, and a test strip carrying the nucleic acid sample is immersed into a buffer solution; substrate DNA on the sample pad is hydrolyzed into small fragments by nuclease, the hydrolyzed fragment DNA and unhydrolyzed substrate DNA reach the binding sheet through the sample sheet due to the siphoning effect principle, the unhydrolyzed substrate DNA and SERS nano probes on the binding sheet are hybridized through base complementary pairing to form double-stranded DNA, the double-stranded DNA diffuses towards the upper part of the reaction film and is subjected to coupling reaction with a detection line fixed on the reaction film, and redundant SERS nano probes continue to diffuse towards the direction of a control line and react with the control line fixed on the reaction film; and (3) observing the color change of the detection line by naked eyes within 5-15 minutes at the temperature of 25 ℃, and obtaining the following two reaction results:
a. when the detection line shows color, the detection result is negative, which indicates that the detected sample does not contain nuclease;
b. when the detection line does not display color, the detection result is positive, and the detected sample contains nuclease;
and (3) measuring SERS signals on the detection line by using a Raman instrument, and carrying out quantitative analysis on the content of nuclease.
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| ITTO20141040A1 (en) * | 2014-12-15 | 2016-06-15 | Fondazione St Italiano Tecnologia | PROCEDURE FOR THE COLORIMETRIC DETECTION OF NUCLEASE CONTAMINATION |
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