JP4675671B2 - Novel short-time detection method for nucleic acid amplification reaction - Google Patents

Description

  The present invention relates to a simple method for detecting nucleic acid and pyrophosphate which are polymerization reaction products of deoxynucleoside tri-phosphate (hereinafter referred to as dNTP) in nucleic acid amplification methods such as PCR.

  Nucleic acid polymerization (Polymerase Chain Reaction; PCR) and ligase chain reaction (LCR) methods have been used as diagnostic methods using genes. In order to carry out these, not only was an expensive thermal cycler (temperature cycling amplification method) necessary, but it was also possible to react only with a specific thermal cycler, which could not be a simple and inexpensive rapid diagnosis. . As a method that can replace this temperature cycling amplification method, an isothermal amplification method capable of amplifying DNA and RNA at a constant temperature at a high frequency has been developed. As a method using DNA, the Standard Displacement Amplification (SDA) method, the Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids (ICAN) method, and the modified UCAN method, which is a modified method such as the UCAN method, the MPI method, and the modified method are the UCAN method. As a method using RNA, a transcription reverse transcription (TRC) method, a nucleic acid sequence-based amplification (NASBA) method, a transcription produced amplification There is a method (TMA) method. All of these amplification methods are dNTP polymerization reactions.

As a method for confirming the amplification reaction, there is a method for detecting directly amplified DNA or RNA. For example, there are various methods such as staining with ethidium bromide after electrophoresis, luminescence method using biotinylated probe, excimer-forming fluorescent sensor, electrochemical sensor, etc., all of which require expensive equipment and reagents, It has a drawback that the operation becomes complicated.
Moreover, although the method of detecting the pyrophosphoric acid in dNTP polymerization reaction liquid by a phosphomolybten reaction is seen in patent document 1 or 2, all are couple | bonded with dNTP, DNA, and RNA which exist in dNTP polymerization reaction liquid. Not only does it contain the danger of detecting phosphoric acid, but it has not been made into a simple kit.

The detection method of DNA and RNA by the isothermal amplification method is almost the same as the conventional PCR detection method except for a part, and no improvement regarding the detection method is recognized. The only loop-mediated thermal amplification (LAMP) method developed by Eiken Chemical does not require an expensive thermal cycler, and DNA amplification (isothermal amplification method) is frequently performed at a constant temperature of about 60 ° C. Confirmation of DNA amplification can be performed by a simple method in which the pyrophosphate-magnesium precipitated by the reaction between pyrophosphate produced during DNA amplification and magnesium added in excess is visually observed. However, a reaction time for securing a production amount until the pyrophosphate-magnesium can be visually confirmed is required, and generally about 1 hour or more is required. Furthermore, the primers used in the LAMP method are more specific and complex than PCR primers, and have the disadvantage that specific primers that are very limited are required to carry out sufficient reactions until they are visible.
JP 7-59600 A JP 2004-205298 A

  The object of the present invention is to visually observe the results of frequently used dNTP polymerization reactions within 5 minutes without using expensive equipment and reagents such as conventional electrophoresis and fluorescence methods. The method that can be determined by is provided as a kit.

  In order to achieve the above object, the present inventors paid attention to nucleic acids, free pyrophosphoric acid and pyrophosphoric acid metal salts, which are products produced during dNTP polymerization, and phosphoric acid and phosphoric acid metal salts, which are pyrophosphoric acid degradation products. In general, in a dNTP polymerization reaction solution, a divalent metal ion such as magnesium, manganese, or zinc plays a coenzyme role for an enzyme necessary for the dNTP polymerization reaction. Although pyrophosphoric acid is produced in proportion to the amount of dNTP polymerized, this pyrophosphoric acid forms a chelate with the divalent metal ion contained in the reaction solution, and is insoluble pyrophosphoric acid-divalent metal salt At the same time, phosphoric acid-divalent metal salt is formed even with phosphoric acid decomposed by pyrophosphoric acid. On the other hand, dNTP remaining as an unreacted substance coexists in the dNTP polymerization reaction solution, and these all bind phosphoric acid. Since phosphoric acid bound to dNTP is also detected by the molybden method, which is a total phosphoric acid measurement method, it is specifically detected in the reaction solution containing these in the pyrophosphate-2valent metal or phosphate-2valent metal. It was considered difficult to detect pyrophosphate and phosphate easily in a short time. However, as a result of intensive studies, the present inventor has found that there are conditions under which organic phosphoric acid bound in unreacted dNTP cannot be colored, and specifically pyrophosphate-2valent metal or phosphate-2valent. We succeeded in detecting pyrophosphoric acid and phosphoric acid in metals easily in a short time. Furthermore, it has been found that the nucleic acid produced by the dNTP polymerization reaction is precipitated under strongly acidic conditions in the presence of betaine, and that the progress of the dNTP polymerization reaction can be confirmed also by precipitation of the nucleic acid.

That is, the present invention is as follows.
According to a first aspect of the present invention, by making a polymerization reaction solution of deoxynucleoside tri-phosphate (dNTP) in an acidic condition in the presence of betaine, nucleic acid is detected as a precipitate in the reaction solution. This is a dNTP polymerization reaction result determination method for determining the result of the polymerization reaction as an index.
In the second aspect of the invention, the dNTP polymerization reaction solution is made acidic under conditions where the organic phosphoric acid bonded to the unreacted dNTP cannot be colored, and molybdate or molybdate and a reducing agent are added. This is a method for determining the result of a dNTP polymerization reaction in which free pyrophosphate and metal pyrophosphate produced by dNTP polymerization reaction and coloration of free phosphoric acid and metal phosphate are used as indicators.
According to a third aspect of the present invention, dNTP polymerization reaction solution is made acidic in the presence of betaine under conditions where organic phosphoric acid bonded to unreacted dNTP cannot be colored, and reduced with molybdate or molybdate. By adding a reagent, nucleic acid is detected as a precipitate in the reaction solution and / or free pyrophosphate and metal pyrophosphate produced by polymerization reaction of dNTP and free phosphate and metal phosphate This is a method for determining the result of a dNTP polymerization reaction that is determined using coloration as an index.
4th invention is a kit used for the result determination method of said dNTP polymerization reaction which consists of at least molybthenic acid or molybdate, acidic water, and a reducing agent.

According to the method of the first invention, the dNTP polymerization reaction proceeds by precipitation of nucleic acid as a dNTP polymerization reaction product in the presence of betaine, which is usually used to destabilize double-stranded DNA and dissolve nucleic acid. Can be detected.
The method of the second invention is a pyrophosphate metal salt and a phosphate metal salt formed with pyrophosphate and its decomposition product phosphoric acid produced in proportion to the dNTP polymerization reaction and a metal contained in the reaction solution. Is a method of detecting the progress of the dNTP polymerization reaction by coloring by addition of molybridenic acid (salt) and a reducing agent under acidic conditions, and has a specificity that is not colored in a reaction solution in which the dNTP polymerization reaction does not proceed.
The method of the third invention is a metal pyrophosphate salt in which a salt is formed with pyrophosphoric acid produced in proportion to the dNTP polymerization reaction, phosphoric acid that is a decomposition product thereof, and a metal contained in the reaction solution along with precipitation of nucleic acid. In addition, in the reaction solution in which dNTP polymerization reaction does not proceed, the metal phosphate is detected by coloration by addition of molyribdenic acid (salt) and a reducing agent in the presence of betaine and acidic conditions. Specificity not to be colored. According to this method, it is possible to confirm both the nucleic acid and pyrophosphate produced by the dNTP polymerization reaction, and the progress of the dNTP polymerization reaction can be confirmed more reliably.

  According to the above-described method of the present invention, the determination of the result can be greatly shortened particularly in the method of producing a large amount of dNTP polymerized substance (DNA or RNA) used in the LAMP method or the like. Furthermore, since the detection sensitivity is excellent, an incomplete primer can be used if a certain amount or more of dNTP is polymerized. The “colorimetric detection method for pyrophosphate using a filter” disclosed in Patent Document 2 also uses the phosphomolybten reaction. However, this is a method for colorimetric detection by collecting colored substances on the filter after the molybden reaction in the dNTP polymerization reaction completion solution, and has the inconvenience that cannot be detected in the same container. . Further, “Target Nucleic Acid Detection Method and Diagnostic Test Kit” of Patent Document 1 is a detection method of inorganic orthophosphate, and there is no description regarding detection of free pyrophosphate, its metal salt, and metal phosphate.

  The molybdenum method itself used in this detection method is a known method. However, there is no example in which a means for detecting pyrophosphoric acid or phosphoric acid in divalent metal pyrophosphate or divalent metal phosphate is distinguished from phosphoric acid in dNTP coexisting in the reaction solution. Moreover, there is no example which reports that precipitation and the amount of precipitation of the nucleic acid of a specific dNTP polymerization reaction product increase under the acidic conditions in the presence of betaine or betaine and molybdate or molybdate. This detection method can detect not only the organic phosphate in dNTP coexisting in the dNTP polymerization reaction solution but also the inorganic pyrophosphate, which is a reactive organism, as well as the produced nucleic acid. Can do. In other words, by adjusting the concentration of the acidic substance to be added, molybdate or molybdate and / or the reducing agent, it was possible to intentionally produce a nucleic acid precipitate, and the specific coloration of pyrophosphate was also achieved. Not only has it become possible, but also the detection sensitivity has increased.

  The principle of the present invention is to specifically deposit nucleic acid, which is a dNTP polymerization reaction product, and at the same time, use pyrophosphoric acid, which is inorganic phosphoric acid, and organic phosphoric acid in unreacted dNTP by utilizing the difference in sensitivity of the phosphomolybten reaction. It is to detect only pyrophosphate with high sensitivity. Furthermore, the detection sensitivity in visual judgment can also be improved by adjusting the acidic substance concentration and the concentration of molybdate or molybdate and the reducing agent. In particular, when the reducing agent is stannous chloride, the precipitate is dyed blue, so visual observation in the precipitated state is extremely easy to determine. Therefore, not only can an error in determination by an individual be reduced, but also there is an advantage that detection sensitivity is increased. The time required for determination is about 5 seconds to 5 minutes, which is significantly shorter than the conventional method.

The present invention is described in detail below.
The method of the present invention can be used for determination of all dNTP polymerization reaction results without using a phosphate-containing buffer. For example, PCR method, reverse transcription (RT) -PCR method, Arbitrary Primed (AP) -PCR method, Single Strain Counting (SSC) -PCR method, ligation tactication (LCR) method, isothermal amplification method LAMP method, Standard Displacement Amplification (SDA method), Ligase chain reaction (LCR method), Single Nucleotide Polymerism (SNP method), Isotherm-immersed-Camp ification of Nucleic acids (ICAN method) can be directed to a polymerization reaction liquid such.

In the first and third inventions, the nucleic acid is precipitated by acidification in the presence of betaine. A typical example of betaine is trimethylglycine, but other betaines such as quaternary ammonium salts, sulfonium salts, and phosphonium salts are also used. The amount of betaine varies depending on the reaction product to be produced, but it is usually 10 to 5000 times the amount of dNTP added. Note that, for example, betaine is used in the amplification method itself as in the LAMP method, and the reaction solution may already contain sufficient betaine, but in that case, it is not necessary to add betaine again.
In addition, when the amount of nucleic acid produced in the polymerization reaction solution is large, precipitation may be seen only by acidification even in the absence of betaine. However, if betaine is present, the amount of precipitation increases and the determination is easy. Become.

  In the present invention, the acidic condition for precipitating nucleic acid in the dNTP polymerization reaction solution is preferably pH 3 or less, more preferably pH 1 or less. The acidifying reagent can be used without particular limitation as long as it is an acidic substance that does not inhibit the phosphomolybtenic acid reaction. For example, hydrochloric acid, sulfuric acid, nitric acid and the like can be exemplified. The acid concentration is preferably 0.25 to 3N. If the acidic condition is too strong, for example, if it exceeds 3N with sulfuric acid or hydrochloric acid, the precipitation of nucleic acid is reduced.

In the second and third inventions, the color reaction is performed using the phosphomolybdenum reaction. Commonly used molybdates (salts) are commonly used molybdate, ammonium molybdate, aluminum molybdate, cadmium molybdate, calcium molybdate, barium molybdate, bismuth molybdate, lithium molybdate, potassium molybdate , Sodium molybdate, zinc molybdate and alkyl ammonium molybdate (having alkyl having 1 to 6 carbon atoms). Further, as a reducing agent, an SH-containing reducing agent such as ascorbic acid or mercaptoethanol, aminonaphtholsulfonic acid, p-methylaminophenol sulfate, stannous chloride, phenylhydrazine, hydroquinone, ferrous sulfate, 2,4-diaminophenol N-phenyl-p-phenylenediamine and a silver halide compound are preferable, but stannous chloride or 2-mercaptoethanol which can be colored in a short time is preferable. If necessary to increase sensitivity, ammonium amidosulfate, potassium tartrate antimonate or potassium antimornyl tartrate can be used.
The substance may be a commercially available reagent or a synthetic product. Further, it may be liquid or solid. Furthermore, it may be an individual reagent itself or a mixed state. Moreover, you may take the form of the salt which can be created.

  In the second and third inventions, free pyrophosphoric acid and pyrophosphoric acid metal salt produced by the polymerization reaction of dNTP and free metal phosphate under the condition that the organic phosphoric acid bound in unreacted dNTP cannot be colored. The phosphoric acid and metal phosphate are colored by the addition of molybdate or molybdate and a reducing agent. The conditions under which the organic phosphoric acid bound in the unreacted dNTP cannot be colored are mainly the concentration and pH of the reducing agent in the reaction solution. These conditions can be determined by experiment. The concentration of the reducing agent varies depending on the reducing agent to be used, but a sufficient amount to reduce the formed phosphomolybthenic acid compound to give a blue color is desirable. Generally, the final concentration is 0.01 to 10% by weight. The amount added is preferred. In the case of stannous chloride, 0.15 to 2% by weight is particularly preferred in the reaction solution, and in the case of 2-mercaptoethanol, 0.01 to 0.5M is particularly preferred. When stannous chloride is used as the reducing agent, if less than 0.01%, the color becomes too weak, and if it exceeds 2%, a black precipitate is formed in the reaction solution, making it difficult to determine. In the case of 2-mercaptoethanol, if the amount is less than 0.01M, the color becomes too weak, and if it exceeds 0.5M, the color of the mercaptoethanol itself (orange) becomes too strong, making it difficult to determine. The pH of the reaction solution is preferably 3 or less, more preferably 1 or less.

  The addition amount of the phosphomolybten reagent in the second and third inventions is not limited because it varies depending on the amount of dNTP used in the dNTP polymerization reaction and the amount of the solution. It is desirable to contain molybthenic acid (salt), and a final concentration of 0.01 to 10% by weight of molybthenic acid (salt) is preferable, and more preferably 0.125 to 1% by weight.

The acidic substance, molybden compound, and reducing agent used in the present invention can be made into an arbitrary dosage form by a conventional method separately or in combination. For example, it can be a solidified preparation or a liquid preparation. In addition, the carrier may take a form containing individual or plural reagents. For example, a filter paper soaked with a reagent and dried can be used.
As the reagent, an acidic substance, a molybdenum compound, and a reducing agent can be provided as a kit. Each of these can be provided separately or in combination in a container. Preferably, a kit in which individual reagents containing acidic substance-containing water, molybdate compound-containing water, and reducing agent-containing water are separated is preferable in terms of stability. It is preferable to use a container in which the contents can be dropped and the amount of dropping liquid per drop is constant and clear. By using such a container, the weighing operation can be simplified, and excessive contact between the reducing agent and air can be avoided, so that the stability of the reagent can be maintained. In addition, a stabilizer may be mix | blended with a reagent.

For example, the present invention is performed as follows.
In the case of 1st invention, an acidic substance and betaine if necessary are added to these polymerization reaction liquids, and it is made to react at 5-40 degreeC (room temperature) for 5 to 20 seconds. When polymerization proceeds and a polymerization reaction product is generated, nucleic acid is detected as a precipitate.
In the case of the second and third inventions, molybthenic acid (salt), a reducing agent, if necessary betaine and an acidic substance are added to these polymerization reaction solutions, and reacted at 5 to 40 ° C. (room temperature) for 5 seconds to 10 minutes. . When the polymerization proceeds, nucleic acid is detected as a precipitate and pyrophosphate (metal salt) or free phosphoric acid (metal salt) is colored. Since phosphoric acid bonded to dNTP does not react, it does not color when polymerization does not proceed.

Hereinafter, the present invention will be described by way of examples.
Example 1
The primers for detection of S. aureus (femA) used in the LAMP method are SEQ ID NO: 1 (f-1), SEQ ID NO: 2 (f-2), SEQ ID NO: 3 (f-3), and SEQ ID NO: 4 (f-4). It was used.
In the LAMP reaction solution, 2.0 μL of the extracted DNA (sterilized distilled water was added as a control), 100 pmol / μL of each primer, f-1 and f-2 were each 0.8 μL, f-3 and f-4 were each A nucleic acid amplification reaction was performed by adding 2.0 μL of 0.1 μL and 8.0 U / μL of Bst DNA Polymerase, respectively. In addition to the above primers and enzyme, the reaction solution was 20 mM Tris HCl (pH 8.8), 10 mM KCl, 8 mM MgSO ₄ , 10 mM SO ₄ (NH ₄ ) ₂ , 0.1% Tween 20, 0.8 M Betaine, 1.4 mM etch. of dNTPs is contained in a total of 50 μL of the reaction solution. The reaction solution was heated at 63 ° C. for 60 minutes, 10 μL of 25% HCl (final concentration 2.5%, pH 1 or less) was added, and a photograph was taken, as shown in FIG. For comparison, photographs of the LAMP unreacted solution and the LAMP unreacted solution before HCl addition are also shown. In FIG. 1, 1 is a photograph of a LAMP reaction end solution + 0.25N sulfuric acid, 2 is a LAMP reaction end solution, and 3 is a LAMP unreacted solution.
In the case of 1 in which the LAMP reaction end solution is acidified, clear cloudiness is confirmed in comparison with 2 that remains neutral. Moreover, the unreacted liquid 3 remained transparent.

Example 2
15 μL of 1.0% ammonium molybdate (final concentration 0.15%) and 25 μL of 1.0% stannous chloride (final concentration 0.25%) were sequentially mixed with the reaction solution to which HCl of Example 1 was added. Then, after standing for 5 minutes, a photograph was taken. At the same time, a photo was taken by centrifuging at 7,000 g for 1 minute. At the same time, phosphomolybthenic acid reaction using stannous chloride was performed with each solution obtained by diluting the LAMP reaction-terminated solution with LAMP unreacted solution to 1/64 times by two-step dilution, and compared with electrophoresis. A photograph of the result of the phosphomolybthenic acid reaction is shown in FIG. 2, and a photograph of the result of the electrophoresis is shown in FIG. The electrophoresis method followed the following procedure. 4.0 μL of 0.1% bromophenol blue was added to 8.0 μL of the LAMP reaction solution and the two-fold diluted solution, and prepared with Tris (hydroxyl-methyl) aminomethane-acetic acid-Disodium EDTA (pH 8.0) buffer. 8 μL of each sample was added to the prepared 2% agarose gel. As a control, 3 μL of DNA ladder was added, followed by electrophoresis for 45 minutes in Tris (hydroxymethyl) aminomethane-acetic acid-disodium EDTA (pH 8.0) buffer with a voltage of 100V. After completion of electrophoresis, the agarose gel was immersed in 1.0 μg / mL ethidium bromide for 20 minutes and then immersed in purified water twice for 10 minutes. The band was confirmed with a UV lamp.

Similarly, to 50 μL of the LAMP reaction solution, 20 μL of 7.5 N sulfuric acid (final concentration 1.5 N), 20 μL of 3.75% ammonium molybdate (final concentration 0.75%), 10 μL of 1M 2-mercaptoethanol (final concentration) 0.1M) was sequentially mixed (pH 1 or less), allowed to stand for 5 minutes, and then centrifuged at 7,000 g for 1 minute. The photographed results before and after centrifugation 5 minutes after the start of the reaction are shown in FIG.
The phosphomolybthenic acid reaction using stannous chloride as the reducing agent was visually confirmed up to a 32-fold dilution, and after centrifugation was confirmed up to a 64-fold dilution. In electrophoresis, it was confirmed that the dilution was 64 times or more, and thus this detection method is considered to have the same sensitivity as the electrophoresis method. Moreover, although the time point at which white turbidity can be visually confirmed by the LAMP reaction is at most about 4-fold dilution, this detection method has a strong sensitivity of about 16-fold. That is, it has been found that detection by this detection method is possible even when no cloudiness is confirmed by the LAMP reaction. Moreover, the phosphomolybtenic acid reaction using 2-mercaptoethanol as a reducing agent was visually confirmed from 32- to 64-fold dilution, and was the same even after centrifugation. However, the coloration time was slightly faster when stannous chloride was used, and the precipitates were colored blue.

Example 3
Examination of PCR reaction completion solution using lympholybten reaction reagent PCR is a region of SEQ ID NO: 5 and SEQ ID NO: 6 (PCR products 306 bp), which is a region specifically retained only by S. aureus, among genes possessed by staphylococci. Detected with primers. The PCR reaction solution was 0.2 μL of S. aureus extract DNA (sterilized distilled water was added as a control), 0.2 μL of each 100 pmol / μL primer, and 5.0 U / μL of AmpliTaq Gold (Applied Biosystems). 0.4 μL, 1.0 μL of 2 mM dTNPmix (GeneAmp), 3.0 μL of 25 mM MgCl ₂ , 5.0 μL of 10 × PCR buffer (Applied Biosystems), and 40.0 μL of distilled water were added. Cycling time and temperature setting for femA was heated at 95 ° C for 12 minutes, then (94 ° C, 30sec. → 52 ° C, 1min. → 70 ° C, 1min.) X 40 cycles and treated at 70 ° C for 10 minutes did. As the thermal cycler, GeneAmp PCR System 9700 (Applied Biosystems) was used. The reaction results are shown in FIG. The left side is a photograph after addition of betaine, the center is 0.5M betaine addition, and the right side is a photograph after 0.5M betaine addition centrifugation. Like the LAMP reaction solution, it was detectable with a phosphomolybthenic acid reaction reagent (reducing agent: stannous chloride), and precipitation of nucleic acid was also confirmed in the same manner as in Example 1 by adding betaine.

It is a photograph of the LAMP reaction completion liquid of Example 1 + 0.25N sulfuric acid (1), the LAMP reaction completion liquid (2), and the LAMP unreacted liquid (3). 2 is a photograph of the results of a phosphomolybthenic acid reaction using stannous chloride of Example 2. 4 is a photograph of electrophoresis as a comparison with Example 2. 2 is a photograph of the results of a phosphomolybdate reaction using 2-mercaptoethanol in Example 2. FIG. 4 is a photograph of the results of a phosphomolybthenic acid reaction using stannous chloride in the PCR reaction completion solution of Example 3.

Claims (11)

  By setting the polymerization reaction solution of deoxynucleoside tri-phosphate (dNTP) to acidic conditions in the presence of betaine, the polymerization reaction is performed using nucleic acid as a precipitate in the reaction solution as an index. DNTP polymerization reaction result determination method for determining the result of   The dNTP polymerization reaction solution was brought to acidic conditions of pH 3 or less in the presence of betaine, and 0.01 to 10% by weight of a reducing agent was added to the reaction solution in the reaction solution by adding molybdic acid or molybdate and the reaction solution. DNTP polymerization is judged using as indicators the detection of nucleic acids as precipitates and the coloration of free pyrophosphate and metal pyrophosphate and free phosphate and metal phosphate produced by the polymerization reaction of dNTP Method for determining the result of the reaction.   The method according to claim 1 or 2, wherein the amount of betaine is 10 to 5000 times the amount of dNTP added during the polymerization reaction.   The determination method according to claim 2, wherein the reducing agent is stannous chloride or 2-mercaptoethanol.   The determination method according to claim 4, wherein the stannous chloride concentration in the reaction solution is 0.01 to 2% by weight or the concentration of 2-mercaptoethanol is 0.01 to 0.5M.   The determination method according to claim 2, wherein the concentration of molybdic acid or molybdate in the reaction solution is 0.01 to 10% by weight.   The determination method according to claim 2, wherein the concentration of molybdic acid or molybdate is 0.05 to 2.5% by weight and the acid substance concentration is 0.1 to 5N.   The determination method according to claim 2, wherein the reaction solution is further concentrated after the coloration.   The kit used for the result determination method of the dNTP polymerization reaction according to claim 2, comprising at least molybdic acid or molybdate, acidic water, and a reducing agent.   The kit according to claim 9, wherein molybdic acid or molybdate-containing water, acidic water, and reducing agent-containing water are individually or combined and placed in a container with a clear amount of dropping liquid.   The kit according to claim 9, wherein molybdic acid or molybdate and / or a reducing agent is supported on a carrier.

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