Reagents for PCR and methods of making the same, kits and PCR methods

Abstract

The application provides a reagent for PCR, a preparation method, a kit and a PCR method, and relates to the technical field of nucleic acid amplification. The reagent for PCR contains acid orange G, acid yellow 23, brilliant green or ethyl violet, and the concentrations of the acid orange G, the acid yellow 23, the brilliant green or the ethyl violet in a PCR reaction system are respectively 0.00001-0.0001 g / mL of acid orange G, 0.00003-0.0003 g / mL of acid yellow, 0.00001-0.0001 g / mL of brilliant green or 0.00001-0.0001 g / mL of ethyl violet. The coloring agents can give different colors to different PCR components, do not interfere with the amplification reaction, do not interfere with the freeze-drying and reconstitution of the reagent, and enhance the amplification signal in the fluorescent PCR amplification program, thereby solving the technical problem that different PCR reagents of different systems are not easy to distinguish.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid amplification technology, and in particular to a reagent for PCR, its preparation method, kit, and PCR method. Background Technology

[0002] Real-time fluorescence PCR (PCR) is a technique for amplifying DNA or RNA by adding fluorescent groups to the PCR reaction and using the accumulation of fluorescence signals to detect the entire PCR process in real time, making each cycle "visible." Currently, commercially available nucleic acid detection kits mainly consist of specific primers and fluorescent probes, enzymes, metal cations, and buffer systems, with a large proportion of biologically active reagents. These components are thermally unstable and easily deformed and inactivated, and large protein molecules may react with other substances. If the kit is in liquid form, it requires cold chain transportation and low-temperature storage throughout the process. Due to the presence of water, the components in the reaction reagents are relatively active, and active molecular ions are easily denatured and inactivated. Therefore, to solve the problems of stable storage and transportation of diagnostic reagents, lyophilization technology has been widely used in the biomedical diagnostic reagent industry.

[0003] Freeze-drying technology, also known as vacuum freeze-drying technology, involves freezing wet materials or solutions into a solid state at a low temperature, then sublimating the water in the solution directly into a gaseous state under vacuum, ultimately dehydrating the material. Freeze-dried reagents retain essentially the same bioactive components, maximizing the protection of sensitive components and allowing for long-term storage at room temperature or in a regular refrigerator. Currently, freeze-dried reagents for nucleic acid products are typically in white powder or cake form. With the increasing number of nucleic acid products for detecting different pathogens, labeling is usually done on the outer packaging or test tubes containing the freeze-dried reagents to facilitate differentiation between different products or different components of the same product. However, once the outer packaging is removed, it is still difficult to identify the different components. Labeling on the test tubes is also problematic because PCR test tubes are usually small, resulting in very small markings that are difficult to see with the naked eye. Furthermore, coloring or marking on the test tubes can interfere with the reading of fluorescence signals during PCR amplification. Therefore, improving PCR reagents to facilitate differentiation between different products is a pressing issue that needs to be addressed.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The first objective of this invention is to provide a reagent for PCR, wherein a coloring agent is premixed, thereby alleviating the technical problem that existing PCR reagents with different components are not easily distinguishable.

[0006] A second objective of this invention is to provide a PCR kit comprising individually packaged colorants or PCR reagents premixed with colorants, facilitating the differentiation of different components.

[0007] A third objective of this invention is to provide a method for preparing PCR reagents.

[0008] The fourth objective of this invention is to provide a PCR method.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] According to one aspect of the present invention, a reagent for PCR is provided, the reagent containing a colorant, said colorant including Acid Orange G, Acid Yellow 23, Brilliant Green or Ethyl Violet;

[0011] The concentration of the colorant in the PCR reaction system is:

[0012] Acid Orange G: 0.00001 g / mL to 0.0001 g / mL;

[0013] Acid Yellow 23: 0.00003 g / mL~0.0003 g / mL;

[0014] Brilliant Green: 0.00001 g / mL~0.0001 g / mL;

[0015] Alternatively, ethyl violet 0.00001 g / mL to 0.0001 g / mL.

[0016] Preferably, the concentration of Acid Orange G in the PCR reaction system is 0.00001 g / mL, 0.00005 g / mL, or 0.0001 g / mL;

[0017] Preferably, the concentration of Acid Yellow 23 in the PCR reaction system is 0.00003 g / mL, 0.00015 g / mL, or 0.0003 g / mL;

[0018] Preferably, the concentration of Brilliant Green in the PCR reaction system is 0.00001 g / mL, 0.00005 g / mL, or 0.0001 g / mL;

[0019] Preferably, the concentration of ethyl violet in the PCR reaction system is 0.00001 g / mL, 0.00005 g / mL, or 0.0001 g / mL.

[0020] Preferably, the reagents include one or more of the following: PCR reaction system, primer reagents, probe reagents, enzyme reagents, buffer system reagents, and lyophilization protectants;

[0021] Preferably, the reagent used for PCR is a lyophilized reagent;

[0022] Preferably, the reagent used for PCR is a reagent used for fluorescent PCR;

[0023] Preferably, the reagent used for PCR is a lyophilized reagent for fluorescent PCR.

[0024] Preferably, the reagent is a lyophilized reaction system for fluorescent PCR, which is obtained by lyophilizing the staining agent, PCR premix, lyophilization protectant, and primers; as well as probes and / or fluorescent dyes.

[0025] Preferably, the PCR premix is ​​selected from 5×HyperstartPremix-UNG or 5×SuperstartPremix-UNG;

[0026] Preferably, the probe has a CY5 fluorescent reporter group or a FAM fluorescent reporter group;

[0027] Preferably, the fluorescent dye includes CY5 fluorescent dye and / or FAM fluorescent dye.

[0028] According to another aspect of the invention, the invention also provides a kit for PCR, the kit comprising (A) or (B):

[0029] (A) The above-mentioned reagents for PCR containing coloring agents;

[0030] (B) Contains separately packaged colorants and PCR reagents, wherein the colorants include one or more of Acid Orange G, Acid Yellow 23, Brilliant Green and Ethyl Violet;

[0031] The concentration of the colorant in the PCR reaction system is:

[0032] Acid Orange G: 0.00001 g / mL to 0.0001 g / mL;

[0033] Acid Yellow 23: 0.00003 g / mL~0.0003 g / mL;

[0034] Brilliant Green: 0.00001 g / mL~0.0001 g / mL;

[0035] And, one or more of ethyl violet from 0.00001 g / mL to 0.0001 g / mL.

[0036] Preferably, the concentration of Acid Orange G in the PCR reaction system is 0.00001 g / mL, 0.00005 g / mL, or 0.0001 g / mL;

[0037] Preferably, the concentration of Acid Yellow 23 in the PCR reaction system is 0.00003 g / mL, 0.00015 g / mL, or 0.0003 g / mL;

[0038] Preferably, the concentration of Brilliant Green in the PCR reaction system is 0.00001 g / mL, 0.00005 g / mL, or 0.0001 g / mL;

[0039] Preferably, the concentration of ethyl violet in the PCR reaction system is 0.00001 g / mL, 0.00005 g / mL, or 0.0001 g / mL.

[0040] Preferably, the kit further includes one or more of primer reagents, probe reagents, enzyme reagents, buffer system reagents, fluorescent dyes, and lyophilization protectants;

[0041] Preferably, the probe reagent contains a probe with a CY5 fluorescent reporter group and / or a probe with a FAM fluorescent reporter group;

[0042] Preferably, the fluorescent dye includes CY5 fluorescent dye and / or FAM fluorescent dye.

[0043] According to another aspect of the present invention, the present invention also provides a method for preparing PCR reagents, comprising mixing a colorant into the PCR reagents; said colorant includes Acid Orange G, Acid Yellow 23, Brilliant Green, or Ethyl Violet;

[0044] The concentration of the colorant in the PCR reaction system is:

[0045] Acid Orange G: 0.00001 g / mL to 0.0001 g / mL;

[0046] Acid Yellow 23: 0.00003 g / mL~0.0003 g / mL;

[0047] Brilliant Green: 0.00001 g / mL~0.0001 g / mL;

[0048] Alternatively, ethyl violet 0.00001 g / mL to 0.0001 g / mL.

[0049] Preferably, the concentration of Acid Orange G in the PCR reaction system is 0.00001 g / mL, 0.00005 g / mL, or 0.0001 g / mL;

[0050] Preferably, the concentration of Acid Yellow 23 in the PCR reaction system is 0.00003 g / mL, 0.00015 g / mL, or 0.0003 g / mL;

[0051] Preferably, the concentration of Brilliant Green in the PCR reaction system is 0.00001 g / mL, 0.00015 g / mL, or 0.0001 g / mL;

[0052] Preferably, the concentration of ethyl violet in the PCR reaction system is 0.00001 g / mL or 0.0001 g / mL.

[0053] Preferably, the colorant is mixed in one or more of the following: PCR reaction system, primer reagent, probe reagent, enzyme reagent, buffer system reagent, and lyophilization protectant;

[0054] Preferably, the preparation method further includes lyophilizing the PCR reagent mixed with the colorant to obtain lyophilized reagent.

[0055] According to another aspect of the present invention, the present invention also provides a PCR method comprising preparing a PCR reaction system using the above-described reagents or the above-described kit.

[0056] Preferably, the PCR method is fluorescent PCR, which includes collecting fluorescence signals in the FAM channel and / or CY5 channel.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The PCR reagent provided by this invention premixes a coloring agent, including Acid Orange G (0.00001 g / mL to 0.0001 g / mL), Acid Yellow 23 (0.00003 g / mL to 0.0003 g / mL), Brilliant Green (0.00001 g / mL to 0.0001 g / mL), or Ethyl Violet (0.00001 g / mL to 0.0001 g / mL). Premixing the above coloring agents in the PCR reagent has the following beneficial effects:

[0059] (1) The PCR reaction system can effectively develop color and can distinguish different PCR reaction systems by different colors.

[0060] (2) Different intermediate reagents used to prepare the same PCR reaction system contain different coloring agents, which can distinguish different components of the same product.

[0061] (3) It has no adverse effect on the PCR reaction. The Ct value of the PCR reaction system with the above concentration was verified by fluorescent PCR method. The difference between the Ct value of the PCR reaction system with the above concentration and the Ct value of the control group was less than 0.5.

[0062] (4) The lyophilized reagents of the PCR reaction system premixed with the above coloring agents have no adverse effect on the PCR reaction after reconstitution, and are lyophilized in different colors, which makes them easy to distinguish.

[0063] (5) Premixed with the above concentration range of colorant, which enhances the amplification signal of the fluorescent PCR amplification results.

[0064] Based on the above-described inventive concept, the PCR kit and the application of the colorant in the preparation of PCR reagents provided by this invention also have the aforementioned beneficial effects. Especially when the PCR reagent is lyophilized, the colorant imparts color to the lyophilized reagent, allowing for effective differentiation between lyophilized reagents in different PCR reaction systems, effectively avoiding confusion between different PCR reaction systems. Therefore, using the above-described colorant-based PCR kit for PCR detection provides convenience for experimenters, reducing the possibility of confusing different PCR reaction systems or different components used to prepare the same PCR reaction system during the experiment. Simultaneously, because the above-described colorant enhances the amplification signal in the fluorescent PCR process, it further enhances the detection effect. Attached Figure Description

[0065] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0066] Figure 1 The control group and the PCR reaction solution containing Acid Orange G were used.

[0067] Figure 2 The control group and the PCR reaction solution containing Acid Yellow 23 were used.

[0068] Figure 3 The control group and the PCR reaction solution containing methylene blue were used.

[0069] Figure 4 The control group and the PCR reaction solution containing Acid Red 27 were used.

[0070] Figure 5 The control group and the PCR reaction solution containing Sunset Yellow FCF were used.

[0071] Figure 6 The control group and the PCR reaction solution containing bromophenol blue were used.

[0072] Figure 7 Control group and PCR reaction solution containing brilliant green;

[0073] Figure 8 Control group and PCR reaction solution containing ethyl violet. Detailed Implementation

[0074] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0075] According to one aspect of the present invention, the present invention also provides a reagent for PCR, the reagent containing a coloring agent, said coloring agent comprising Acid Orange G (CAS No.: 1936-96-5, molecular formula: C6H9NS) and Acid Yellow 23 (CAS No.: 1934-21-0, molecular formula C6H9NS). 16 H9N4Na3O9S2), Brilliant Green (CAS No.: 633-03-4, molecular formula C 27 H 34 N2O4S) or ethyl violet (CAS No.: 2390-59-2, molecular formula C 31 H 42 ClN3); the concentrations of the colorant in the PCR reaction system are as follows:

[0076] The concentration of Acid Orange G is 0.00001 g / mL to 0.0001 g / mL, for example, but not limited to, 0.00001 g / mL, 0.00002 g / mL, 0.00003 g / mL, 0.00004 g / mL, 0.00005 g / mL, 0.00006 g / mL, 0.00007 g / mL, 0.00008 g / mL, 0.00009 g / mL or 0.0001 g / mL, preferably 0.00001 g / mL or 0.0001 g / mL.

[0077] Acid Yellow 23 is present in concentrations of 0.00003 g / mL to 0.0003 g / mL, for example, but not limited to, 0.00003 g / mL, 0.00005 g / mL, 0.0001 g / mL, 0.00015 g / mL, 0.0002 g / mL, 0.00025 g / mL, or 0.0003 g / mL, preferably 0.00003 g / mL or 0.0003 g / mL.

[0078] The concentration of brilliant green is 0.00001 g / mL to 0.0001 g / mL, for example, but not limited to 0.00001 g / mL, 0.00003 g / mL, 0.00005 g / mL, 0.00008 g / mL, 0.0001 g / mL, 0.000015 g / mL, 0.00002 g / mL or 0.00004 g / mL, preferably 0.00001 g / mL or 0.0001 g / mL.

[0079] Ethyl violet is used at a concentration of 0.00001 g / mL to 0.0001 g / mL, for example, but not limited to, 0.00001 g / mL, 0.00003 g / mL, 0.00005 g / mL, 0.00008 g / mL, 0.0001 g / mL, 0.000015 g / mL, 0.00002 g / mL, or 0.00004 g / mL, preferably 0.00001 g / mL or 0.0001 g / mL.

[0080] This invention has found through experiments that premixing the above-mentioned coloring agent in PCR reagents has the following beneficial effects: (1) The PCR reaction system can effectively develop color, and different PCR reaction systems can be distinguished by different colors. (2) Different intermediate reagents used to prepare the same PCR reaction system contain different coloring agents, which can distinguish different components of the same product. (3) It has no adverse effect on the PCR reaction. The Ct value of the PCR reaction system premixed with the above concentration is less than 0.5 compared with the Ct value of the control group. (4) The lyophilized reagent of the PCR reaction system premixed with the above-mentioned coloring agent has no adverse effect on the PCR reaction after reconstitution, and it exhibits different colors after lyophilization, which is easy to distinguish. (5) Premixing the coloring agent within the above concentration range enhances the amplification signal of the amplification results of fluorescent PCR.

[0081] The reagents provided by this invention can optionally be PCR reaction systems used directly in PCR reactions, or can optionally be intermediate reagents used to prepare PCR reaction systems, such as, but not limited to, primer reagents, probe reagents, enzyme reagents, buffer system reagents, or lyophilization protectants. It is understood that when the reagent is a stock solution of a PCR system concentration multiple, the concentration of the colorant in the diluted PCR reaction system conforms to the above-mentioned concentration range; when the reagent is an intermediate reagent used to prepare PCR reaction systems, or a stock solution of an intermediate reagent concentration multiple, the concentration of the colorant in the PCR reaction system prepared by the reagent conforms to the above-mentioned concentration.

[0082] In some preferred embodiments, the lyophilized reagents retain essentially the same bioactive components, thus maximizing the protection of sensitive components and allowing for long-term storage at room temperature or in a regular refrigerator; and the lyophilized PCR reagents containing colorants exhibit different colors to distinguish different PCR systems, wherein the reagents used for PCR are lyophilized reagents.

[0083] In some preferred embodiments, the PCR reaction system with added colorant enhances the amplification signal of the amplification result when amplified using the fluorescent PCR method, wherein the reagent used for PCR is a reagent used for fluorescent PCR.

[0084] In some preferred embodiments, based on the synergistic effect of the colorant in both the lyophilized reagent and the fluorescent PCR reaction, the reagent for PCR is a lyophilized reagent for fluorescent PCR, more preferably a lyophilized reaction system for fluorescent PCR, obtained by lyophilizing the colorant, PCR premix, lyophilization protectant, and primers; and probes and / or fluorescent dyes. The PCR premix is ​​preferably 5×HyperstartPremix-UNG or 5×SuperstartPremix-UNG; the probe preferably carries a CY5 fluorescent reporter group or a FAM fluorescent reporter group; the fluorescent dye preferably includes a CY5 fluorescent dye and / or a FAM fluorescent dye.

[0085] According to another aspect of the invention, the invention also provides a kit for PCR.

[0086] In some optional embodiments, the kit includes the reagents described above for PCR. The kit contains PCR reagents premixed with at least one coloring agent; optionally, it includes multiple reagents containing different coloring agents for different PCR reaction systems, and optionally, it includes different intermediate reagents containing different coloring agents for preparing the same PCR reaction system, such as, but not limited to, premixed Acid Orange G primer lyophilized reagents, premixed ethyl violet probe lyophilized reagents, or buffer stock solutions premixed with a working solution concentration multiple of Brilliant Green.

[0087] In some alternative embodiments, this kit includes separately packaged colorants and other reagents for PCR, which are mixed with the colorants and other reagents to form the PCR reaction system. The colorants include one or more of Acid Orange G, Acid Yellow 23, Brilliant Green, and Ethyl Violet. The working concentrations of each colorant in the PCR reaction system after preparation are as follows:

[0088] Acid Orange G is 0.00001 g / mL to 0.0001 g / mL, for example, but not limited to, 0.00001 g / mL, 0.00002 g / mL, 0.00003 g / mL, 0.00004 g / mL, 0.00005 g / mL, 0.00006 g / mL, 0.00007 g / mL, 0.00008 g / mL, 0.00009 g / mL or 0.0001 g / mL, preferably 0.00001 g / mL or 0.0001 g / mL.

[0089] Acid Yellow 23 is present in concentrations of 0.00003 g / mL to 0.0003 g / mL, for example, but not limited to, 0.00003 g / mL, 0.00005 g / mL, 0.0001 g / mL, 0.00015 g / mL, 0.0002 g / mL, 0.00025 g / mL, or 0.0003 g / mL, preferably 0.00003 g / mL or 0.0003 g / mL.

[0090] The concentration of Brilliant Green is 0.00001 g / mL to 0.0001 g / mL, for example, but not limited to 0.00001 g / mL, 0.00003 g / mL, 0.00005 g / mL, 0.00008 g / mL, 0.0001 g / mL, 0.000015 g / mL, 0.00002 g / mL, or 0.00004 g / mL, preferably 0.00001 g / mL or 0.0001 g / mL.

[0091] Ethyl violet is used at a concentration of 0.00001 g / mL to 0.0001 g / mL, for example, but not limited to, 0.00001 g / mL, 0.00003 g / mL, 0.00005 g / mL, 0.00008 g / mL, 0.0001 g / mL, 0.000015 g / mL, 0.00002 g / mL, or 0.00004 g / mL, preferably 0.00001 g / mL or 0.0001 g / mL.

[0092] The kit provided by the present invention may also optionally include any reagents and / or consumables acceptable in the art for PCR reactions or for preparing PCR reaction systems. Specific embodiments may include, but are not limited to, one or more of enzymes, buffers, dNTPs, primers, probes, fluorescent dyes, salts or salt solutions, negative controls, positive controls, blank controls, calibrators, and PCR reaction containers.

[0093] In some optional embodiments, the kit is used for fluorescent PCR, and the kit further includes probes with a CY5 fluorescent reporter group and / or probes with a FAM fluorescent reporter group; or, the kit further includes CY5 fluorescent dye and / or FAM fluorescent dye.

[0094] According to another aspect of the present invention, the present invention also provides a method for preparing PCR reagents, the method comprising mixing a colorant into the PCR reagents; said colorant includes Acid Orange G, Acid Yellow 23, Brilliant Green, or Ethyl Violet;

[0095] The working concentrations of the colorant in the PCR reaction system are as follows:

[0096] The concentration of Acid Orange G is 0.00001 g / mL to 0.0001 g / mL, for example, but not limited to, 0.00001 g / mL, 0.00002 g / mL, 0.00003 g / mL, 0.00004 g / mL, 0.00005 g / mL, 0.00006 g / mL, 0.00007 g / mL, 0.00008 g / mL, 0.00009 g / mL or 0.0001 g / mL, preferably 0.00001 g / mL or 0.0001 g / mL.

[0097] Acid Yellow 23 is present in concentrations of 0.00003 g / mL to 0.0003 g / mL, for example, but not limited to, 0.00003 g / mL, 0.00005 g / mL, 0.0001 g / mL, 0.00015 g / mL, 0.0002 g / mL, 0.00025 g / mL, or 0.0003 g / mL, preferably 0.00003 g / mL or 0.0003 g / mL.

[0098] The concentration of brilliant green is 0.00001 g / mL to 0.0001 g / mL, for example, but not limited to 0.00001 g / mL, 0.00003 g / mL, 0.00005 g / mL, 0.00008 g / mL, 0.0001 g / mL, 0.000015 g / mL, 0.00002 g / mL or 0.00004 g / mL, preferably 0.00001 g / mL or 0.0001 g / mL.

[0099] Ethyl violet is used at a concentration of 0.00001 g / mL to 0.0001 g / mL, for example, but not limited to, 0.00001 g / mL, 0.00003 g / mL, 0.00005 g / mL, 0.00008 g / mL, 0.0001 g / mL, 0.000015 g / mL, 0.00002 g / mL, or 0.00004 g / mL, preferably 0.00001 g / mL or 0.0001 g / mL.

[0100] The above-mentioned coloring agent can be applied to the preparation of PCR reaction reagents, for example, but not limited to, in the following manner:

[0101] In some optional embodiments, the above-mentioned colorant is directly applied to the preparation of the PCR reaction system; alternatively, the PCR reaction system containing the colorant in the above concentration range is further prepared to obtain lyophilized reagent.

[0102] In some optional embodiments, the aforementioned colorant is applied to prepare intermediate reagents for formulating PCR reaction systems. These intermediate reagents may include, but are not limited to, primer reagents, probe reagents, enzyme reagents, buffer system reagents, or lyophilization protectants, all of which are optionally lyophilized reagents. Optionally, colorants exhibiting different colors can be premixed in different intermediate reagents used to prepare the same PCR reaction system. This allows for the differentiation of different components within the same product. For example, when preparing a kit, the primer lyophilized reagent may be premixed with Acid Orange G, and the probe lyophilized reagent may be premixed with Ethyl Violet. During use, the different reagents can be directly distinguished by the color presented in the packaging.

[0103] In some preferred embodiments, based on the beneficial effect that the colorant can distinguish different lyophilized reagents, has no effect on the amplification reaction, and enhances the amplification signal of the amplification result, the above preparation method further includes lyophilizing the PCR reagent mixed with the colorant to obtain the lyophilized reagent.

[0104] The concentrations of the colorants mentioned above refer to the working concentrations of each colorant in the PCR reaction system, that is, the concentrations of each colorant in the PCR reaction system when it actually participates in the reaction. It can be understood that when these colorants are used to prepare PCR reaction reagents, if a stock solution of a concentration multiple of the PCR reaction system is being prepared, the concentration of the colorant in the diluted PCR reaction system will conform to the above concentration range. Similarly, if the colorant is being prepared as an intermediate reagent for PCR reaction systems, or as a stock solution of a concentration multiple of the intermediate reagent, the concentration of each colorant in the PCR reaction system obtained after mixing with other PCR reagents will conform to the above concentrations.

[0105] According to another aspect of the present invention, the present invention also provides a PCR detection method, comprising preparing a PCR reaction system using the above-described PCR reagents or kits containing colorants. PCR reaction systems prepared using the above-described PCR reagents or kits can be distinguished by different colors, thereby reducing the possibility of confusion between different PCR systems or between different intermediate reagents used to prepare the PCR reaction system during the experiment, thus avoiding experimental failure. When the reagents containing colorants used are lyophilized reagents, since lyophilized reagents are more conducive to reagent preservation, well-preserved PCR reagents further ensure the accuracy and sensitivity of the PCR reaction, and different colors distinguish different lyophilized reagents, reducing the possibility of PCR lyophilized reagent confusion.

[0106] The PCR detection methods include, but are not limited to, conventional PCR detection methods or fluorescent PCR detection methods. The fluorescent detection method can be selectively used for qualitative, semi-quantitative, or quantitative detection of target samples. When using fluorescent PCR detection, the PCR reaction system with added colorant also enhances the amplification signal. When using fluorescent PCR detection, it is preferable to collect the fluorescence signal in the FAM channel and / or CY5 channel.

[0107] The technical solution and beneficial effects of the present invention will be further explained below with reference to preferred embodiments.

[0108] Example 1

[0109] A PCR reaction system was prepared, comprising paired primers and probes, a commercially available PCR premix, and a self-produced lyophilized protective solution. The target channels for this system are the FAM and CY5 channels. The final concentrations of each component in the system are as follows:

[0110] Table 1

[0111]

[0112] The colorants designed for comparison in this experiment and their final concentrations in the system are shown in Table 2 below:

[0113] Table 2. High, medium, and low concentrations of different colorants in the PCR system.

[0114] Colorant Name High concentration w / v medium concentration w / v low concentration w / v Acid Orange G 0.001g / mL 0.0001g / mL 0.00001g / mL Acid Yellow 23 0.003g / mL 0.0003g / mL 0.00003g / mL Methylene blue 0.0001g / mL 0.00001g / mL 0.000001g / mL Acid Red 27 0.0001g / mL 0.00001g / mL 0.000001g / mL Sunset Yellow FCF 0.0004g / mL 0.00004g / mL 0.000004g / mL Bromophenol blue 0.0002g / mL 0.00002g / mL 0.000002g / mL brilliant green 0.001g / mL 0.0001g / mL 0.00001g / mL Ethyl Violet 0.001g / mL 0.0001g / mL 0.00001g / mL

[0115] The PCR reaction solutions containing different coloring agents were prepared as follows. Simultaneously, a control group of reaction solutions without coloring agents was also prepared. The coloring results of the reaction solutions are shown below. Figures 1 to 8 , Figures 1 to 8 From left to right, tubes 1 and 2 are the control group, tubes 3 and 4 are the low-concentration group, tubes 5 and 6 are the medium-concentration group, and tubes 7 and 8 are the high-concentration group. Figures 1 to 8 As can be seen, the three reaction solutions containing Acid Orange G, Bromophenol Blue, and Brilliant Green are lighter in color when the colorant concentration is low. This may be due to issues with image clarity and lighting; however, they are still clearly distinguishable from the control (reaction solution without colorant) to the naked eye. The remaining reaction solutions containing colorants are clearly distinguishable from the control (reaction solution without colorant) in the images. In this embodiment, all components in Table 1 contain colorants. Different PCR reaction solutions were prepared according to the different types and concentrations of colorants listed in Table 2.

[0116] Example 2

[0117] The control group and 24 reaction solutions containing colorant from Example 1 were lyophilized. After lyophilization, the lyophilized reagents were reconstituted with template and detected. Samples with template amounts of 1000 copies per reaction in the FAM channel and 100,000 copies per reaction in the CY5 channel were tested in replicates for each group. The amplification results of the lyophilized reagent without colorant were used as controls for each group. Ct and Rn values ​​were compared. The result judgment criteria were: for samples of the same concentration, the mean Ct and Rn values ​​were taken; compared with the control, a difference of ≤0.5 in the mean Ct value or a decrease in Rn value of less than 10% was considered as no significant difference. The results are shown in Tables 3-6.

[0118] Table 3 Summary of FAM Channel Results (100,000 copies / T)

[0119]

[0120]

[0121] Table 4 Summary of FAM Channel 1000 copies / T Results

[0122]

[0123]

[0124] Table 5 Summary of CY5 channel results with 100,000 copies / T

[0125]

[0126]

[0127] Table 6 Summary of CY5 channel 1000 copies / T results

[0128]

[0129]

[0130] In Tables 3 to 6, the difference in Ct values ​​is obtained by subtracting the mean Ct value of the group containing colorant from the mean Ct value of the control group, and the difference in Rn is obtained by subtracting the mean Rn value of the group containing colorant from the mean Rn value of the control group / the mean Rn value of the control group × 100%.

[0131] The data in Tables 3 to 6 show that when the PCR lyophilized reagents contain colorants such as methylene blue, acid red 27, sunset yellow FCF, and bromophenol blue, the detection of both targets in the FAM and CY5 channels results in delayed Ct values ​​and reduced Rn values ​​exceeding the required range. In particular, when the concentrations of methylene blue, sunset yellow FCF, and bromophenol blue are high, some targets may even become undetectable.

[0132] The data in Tables 3-6 show that Acid Orange G and Acid Yellow 23 also exhibited delayed Ct values ​​and Rn values ​​exceeding the required range for the FAM channel at final concentrations of 0.00001 g / mL and 0.00003 g / mL, respectively. Acid Orange G showed the best amplification results at final concentrations of 0.00001 g / mL to 0.0001 g / mL; Acid Yellow 23 showed the best amplification results at final concentrations of 0.00003 g / mL to 0.0003 g / mL; Brilliant Green showed relatively good amplification results at final concentrations of 0.00001 g / mL to 0.00001 g / mL; and Ethyl Violet showed relatively good amplification results at final concentrations of 0.00001 g / mL to 0.00001 g / mL, with significant increases in amplification signals in both the FAM and CY5 channels.

[0133] Example 3

[0134] A PCR reaction system was prepared, comprising two pairs of paired primers and probes, commercially available PCR premix, and a self-produced lyophilization buffer. The target channels for this system were the FAM and CY5 channels. Unlike Example 2, the paired primer and probe sequences and the hot-start DNA polymerase were replaced. The final concentrations of each component in the system are as follows:

[0135] Table 7

[0136]

[0137] The colorants designed and added in this experiment, and their final concentrations in the system, are shown in Table 8 below.

[0138] Table 8

[0139]

[0140] PCR reaction solutions containing different colorants were prepared, and a control group without colorants was also prepared and lyophilized together. Template addition and reconstitution were performed on the control group and eight groups of lyophilized reagents containing colorants. Samples with 10 copies / reaction and 1000 copies / reaction template were used for the FAM channel, and samples with 100 copies / reaction and 10000 copies / reaction template were used for the CY5 channel. Two replicates were performed for each FAM channel sample, and four replicates were performed for the CY5 channel sample. The amplification results of the lyophilized reagent without colorants were used as controls for each group. Ct and Rn values ​​were compared. The result interpretation criteria were: the mean Ct and Rn values ​​for samples of the same concentration were taken, and the difference in mean Ct value compared to the control was ≤0.5.

[0141] Table 9 Summary of FAM Channel 10 copies / T Results

[0142]

[0143]

[0144] Table 10 Summary of FAM Channel 1000 copies / T Results

[0145]

[0146]

[0147] Table 11 Summary of CY5 channel 100 copies / T results

[0148]

[0149]

[0150] Table 12 Summary of Results for CY5 Channel with 10000 Copies / T

[0151]

[0152] In Tables 9 to 12, the difference in Ct values ​​is obtained by subtracting the mean Ct value of the group containing colorant from the mean Ct value of the control group, and the difference in Rn is obtained by subtracting the mean Rn value of the group containing colorant from the mean Rn value of the control group / the mean Rn value of the control group × 100%.

[0153] The experimental data in Tables 9 and 10 show that Acid Orange G achieved the best amplification results at a final concentration of 0.00001 g / mL to 0.0001 g / mL; Acid Yellow 23 achieved the best amplification results at a final concentration of 0.00003 g / mL to 0.0003 g / mL; Brilliant Green achieved relatively good amplification results at a final concentration of 0.00001 g / mL to 0.0001 g / mL; and Ethyl Violet achieved relatively good amplification results at a final concentration of 0.00001 g / mL to 0.0001 g / mL. The mean Ct value of the FAM channel was less than 0.5 different from that of the control group. All groups of lyophilized reagents with added colorant showed a significant increase in the amplification signal of the FAM channel. The results of detecting the Ct and Rn values ​​of the FAM channel using 0.000005 g / mL Acid Orange G, 0.0000015 g / mL Acid Yellow 23, 0.000005 g / mL Brilliant Green, and 0.000005 g / mL Ethyl Violet were within the required range, but had no beneficial effect on the amplification signal.

[0154] The experimental data in Tables 11 and 12 show that Acid Orange G achieved the best amplification results at a final concentration of 0.00001 g / mL to 0.0001 g / mL; Acid Yellow 23 achieved the best amplification results at a final concentration of 0.00003 g / mL to 0.0003 g / mL; Brilliant Green achieved relatively good amplification results at a final concentration of 0.00001 g / mL to 0.0001 g / mL; and Ethyl Violet achieved relatively good amplification results at a final concentration of 0.00001 g / mL to 0.0001 g / mL. The mean Ct value of the CY5 channel was less than 0.5 different from that of the control group. All groups of lyophilized reagents with added colorant showed a significant increase in the amplification signal of the CY5 channel. The Ct and Rn values ​​of the CY5 channel detected by 0.000005 g / mL Acid Orange G, 0.000005 g / mL Acid Yellow 23, 0.000005 g / mL Brilliant Green and 0.000005 g / mL Ethyl Violet were within the required range, but had no beneficial effect on the amplification signal.

[0155] The results show that the colorants in the table below can be used for labeling different systems and have the effect of enhancing FAM and CY5 amplification signals.

[0156] Table 13

[0157]

[0158]

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescent PCR method, characterized in that, A PCR reaction system was prepared using fluorescent PCR reagents for performing fluorescent PCR reactions; the fluorescent PCR reagents contained probe reagents and fluorescently enhanced staining agents that enhanced the amplification signal of fluorescent PCR. The probe reagent contains probes with CY5 fluorescent reporter groups and probes with FAM fluorescent reporter groups; The enhanced colorant includes at least one of Acid Orange G, Acid Yellow 23, Brilliant Green, or Ethyl Violet; The concentration of the enhanced staining agent in the PCR reaction system is as follows: Acid Orange G: 0.00005 g / mL ~ 0.0001 g / mL; Acid Yellow 23 0.00003 g / mL or 0.0003 g / mL; Brilliant Green: 0.00001 g / mL ~ 0.00005 g / mL; Alternatively, ethyl violet 0.00005 g / mL; The method described is not intended for disease diagnosis or treatment.

2. The fluorescent PCR method according to claim 1, characterized in that, The concentration of Acid Orange G in the PCR reaction system is 0.00005 g / mL or 0.0001 g / mL.

3. The fluorescent PCR method according to claim 1, characterized in that, The concentration of Brilliant Green in the PCR reaction system is 0.00001 g / mL or 0.00005 g / mL.

4. The fluorescent PCR method according to claim 1, characterized in that, The fluorescent PCR reagent also includes one or more of the following: primer reagents, enzyme reagents, buffer system reagents, and lyophilization protectants.

5. The fluorescent PCR method according to any one of claims 1 to 3, characterized in that, The fluorescent PCR reagent is a lyophilized reagent.

6. The fluorescent PCR method according to claim 5, characterized in that, The lyophilized reagent is obtained by lyophilizing the probe reagent as described in claim 1, the fluorescence-enhancing staining agent as described in claim 1, the PCR premix, the lyophilization protectant, and the primers.

7. The fluorescent PCR method according to claim 6, characterized in that, The PCR premix solution is selected from 5×HyperstartPremix-UNG or 5×SuperstartPremix-UNG.

Images