RT-qPCR (real-time quantitative polymerase chain reaction) full premix reaction reagent as well as application and product thereof

Abstract

The invention provides an RT-qPCR (real-time quantitative polymerase chain reaction) full premix reaction reagent as well as application and a product thereof, and belongs to the technical field of biochemical reagents. The RT-qPCR full premix reaction reagent provided by the invention comprises a PCR reaction additive, a DNA polymerase, a reverse transcriptase, a thermosensitive uracil DNA glycosidase, an RNA enzyme inhibitor, soluble cations and dNTPs (deoxyribonucleoside triphosphates). The PCR reaction additive is composed of 50 to 1000 mM of trehalose, 0.5 to 10.0% v / v of DMSO, 50 to 500 mM of betaine, 50 to 500 ng / [mu] L of SSB single-chain binding protein, and a stabilizing agent; the stabilizer is prepared from polyoxyethylene lauryl ether, glycerol and BSA (Bovine Serum Albumin). The RT-qPCR full premix reaction reagent provided by the invention is not only good in universality, high in specificity and sensitivity, but also relatively good in stability, and can be stably stored for one year in a refrigerator at-20 DEG C.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical reagents, and particularly relates to an RT-qPCR full-premix reaction reagent, its application and product. Background Art

[0002] Polymerase chain reaction (PCR) is a method for rapidly amplifying specific DNA fragments in vitro using single-stranded oligonucleotide primers, which can specifically amplify extremely trace amounts of target DNA fragments by millions of times in a short time. RT-qPCR is a method that adds reverse transcriptase on the basis of PCR to amplify DNA using RNA as the original template. RT-qPCR monitors each cycle of amplification products in the PCR reaction in real time according to the change in the fluorescence signal intensity accumulated by the fluorescent group added to the reaction system.

[0003] The currently commonly used RT-qPCR reaction system is composed of nucleic acid amplification buffer, enzyme mixture, primer-probe reaction solution, etc. and needs to be stored separately. When in use, each component needs to be dissolved at room temperature and fully mixed before use. When in use, each component is added in a fixed ratio, mixed well and then aliquoted into tubes. The whole process requires calculation and careful operation, and mistakes are often made. Moreover, the reaction system needs to be prepared and used immediately. If the nucleic acid amplification buffer, enzyme mixture, and primer-probe reaction solution are premixed in advance, stability problems will occur after freezing or refrigeration, specifically manifested as a decrease in sensitivity and non-specific amplification in negative samples.

[0004] The prior art CN116004776A discloses a one-tube RT-qPCR full-premix reaction reagent, application and RT-qPCR method. The reagent includes a Taq enzyme monoclonal antibody with double-blocking ability, nucleic acid aptamer, stabilizer and enhancer. The full-premix RT-qPCR reaction system can be stored stably at -20°C or below for 12 - 15 months without affecting the detection ability after being pressurized at 37°C for 7 days at high temperature, but the applicability and sensitivity of the premix system cannot be determined. Summary of the Invention

[0005] To solve the above problems, the present invention provides an RT-qPCR all-in-one reaction reagent and its applications and products. The RT-qPCR all-in-one reaction reagent provided by the present invention comprises a PCR reaction additive, a DNA polymerase, a reverse transcriptase, a thermophilic uracil DNA glycosylase, an RNase inhibitor, a soluble cation, and dNTPs; the PCR reaction additive is composed of 50-1000 mM trehalose, 0.5-10.0% v / v DMSO, 50-500 mM betaine, 50-500 ng / μL SSB single-strand binding protein, and a stabilizer; the stabilizer is composed of polyoxyethylene lauryl ether, glycerol, and BSA. The RT-qPCR all-in-one reaction reagent provided by the present invention not only has good versatility, high specificity and sensitivity, but also has good stability and can be stably stored in a -20°C refrigerator for 1 year.

[0006] The technical solution of the present invention includes:

[0007] In the first aspect, the present invention provides an RT-qPCR all-in-one reaction reagent, and the RT-qPCR all-in-one reaction reagent comprises a PCR reaction additive; the PCR reaction additive is composed of 50-1000 mM trehalose, 0.5-10.0% v / v DMSO, 50-500 mM betaine, 50-500 ng / μL SSB single-strand binding protein, and a stabilizer; the stabilizer is composed of polyoxyethylene lauryl ether, glycerol, and BSA.

[0008] Preferably, the PCR reaction additive is composed of 300-1000 mM trehalose, 2-10% v / v DMSO, 200-500 mM betaine, 100-500 ng / μL SSB single-strand binding protein, and a stabilizer.

[0009] More preferably, the PCR reaction additive is composed of 500 mM trehalose, 3.5% v / v DMSO, 200 mM betaine, 200 ng / μL SSB single-strand binding protein, and a stabilizer.

[0010] Specifically, the stabilizer is composed of 0.05-0.3% v / v polyoxyethylene lauryl ether, 0.5-5.0% v / v glycerol, and 20-400 ng / μL BSA.

[0011] Preferably, the stabilizer is composed of 0.15-0.3% v / v polyoxyethylene lauryl ether, 2.0-5.0% v / v glycerol, and 100-400 ng / μL BSA.

[0012] More preferably, the stabilizer is composed of 0.15% v / v polyoxyethylene lauryl ether, 2.4% v / v glycerol, and 200 ng / μL BSA.

[0013] Specifically, the RT-qPCR all-in-one reaction reagent further includes DNA polymerase, reverse transcriptase, thermophilic uracil DNA glycosylase, RNase inhibitor, soluble cations, and dNTPs.

[0014] More specifically, the addition amount of the DNA polymerase is 0.03 - 0.6 U / μL, the addition amount of the reverse transcriptase is 0.5 - 4 U / μL, the addition amount of the thermophilic uracil DNA glycosylase is 0.005 - 0.05 U / μL, and the addition amount of the RNase inhibitor is 0.1 - 3 U / μL.

[0015] Preferably, the addition amount of the DNA polymerase is 0.5 - 0.6 U / μL, the addition amount of the reverse transcriptase is 1 - 4 U / μL, the addition amount of the thermophilic uracil DNA glycosylase is 0.01 - 0.05 U / μL, and the addition amount of the RNase inhibitor is 0.1 - 3 U / μL.

[0016] More specifically, the soluble cations consist of Mg 2+ , K + , and NH 4+ .

[0017] Preferably, the soluble cations include: 2 - 12 mM Mg 2+ , 40 - 200 mM K + , and 10 - 100 mM NH 4+ .

[0018] More preferably, the soluble cations include: 4 - 12 mM Mg 2+ , 50 - 200 mM K + , and 10 - 100 mM NH 4+ .

[0019] More specifically, the dNTPs include: 0.02 - 1.2 mM dATP, 0.02 - 1.2 mM dCTP, 0.02 - 1.2 mM dGTP, 0.02 - 1.2 mM dTTP, and 0.04 - 2.4 mM dUTP.

[0020] Preferably, the dNTPs include: 0.5 - 1 mM dATP, 0.5 - 1 mM dCTP, 0.5 - 1 mM dGTP, 0.5 - 1 mM dTTP, and 1 mM dUTP.

[0021] In a second aspect, the present invention provides the application of the above RT-qPCR all-in-one reaction reagent in the preparation of an RT-qPCR kit.

[0022] In a third aspect, the present invention provides an RT-qPCR kit, which includes the aforementioned RT-qPCR all-in-one reaction reagent.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) After being mixed with primers, the RT-qPCR all-in-one reaction reagent of the present invention can be stored at -20°C for 1 year and at 37°C for 7 days;

[0025] (2) The RT-qPCR all-in-one reaction reagent of the present invention can be compatible with a variety of primer-probes, with strong versatility;

[0026] (3) The RT-qPCR all-in-one reaction reagent of the present invention can stably detect nucleic acid samples with an abundance of 500 copies / mL, with high sensitivity;

[0027] (4) The RT-qPCR all-in-one reaction reagent of the present invention has high specificity, strong anti-RNase inhibition ability, and can also digest aerosol contamination of 2×10 7 copies. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the detection result of the amplification ability of the mixed system in Example 1; in the figure, A is the freshly prepared mixed system in Example 1; B is the mixed system in Example 1 stored at -20°C for 1 year; C is the mixed system in Example 1 stored at 37°C for 7 days.

[0029] Figure 2 It is the detection result of the stability of the freshly prepared mixed system in Example 1; in the figure, A is the detection result of the amplification ability; B is the detection result of the specificity; C is the detection result of the sensitivity.

[0030] Figure 3 It is the detection result of the stability of the mixed system in Example 1 stored at -20°C for 1 year; in the figure, A is the detection result of the amplification ability; B is the detection result of the specificity; C is the detection result of the sensitivity.

[0031] Figure 4 It is the detection result of the stability of the mixed system in Example 1 stored at 37°C for 7 days; in the figure, A is the detection result of the amplification ability; B is the detection result of the specificity; C is the detection result of the sensitivity.

[0032] Figure 5 It is the detection result of the stability of the freshly prepared mixed system in Example 2; in the figure, A is the detection result of the amplification ability; B is the detection result of the specificity; C is the detection result of the sensitivity.

[0033] Figure 6Stability test results of the mixed system of Example 2 stored at -20°C for 1 year; in the figure, A is the amplification ability test result; B is the specificity test result; C is the sensitivity test result.

[0034] Figure 7 Stability test results of the mixed system of Example 2 stored at 37°C for 7 days; in the figure, A is the amplification ability test result; B is the specificity test result; C is the sensitivity test result.

[0035] Figure 8 Stability test results of the freshly prepared mixed system of Example 3; in the figure, A is the amplification ability test result; B is the specificity test result; C is the sensitivity test result.

[0036] Figure 9 Stability test results of the mixed system of Example 3 stored at -20°C for 1 year; in the figure, A is the amplification ability test result; B is the specificity test result; C is the sensitivity test result.

[0037] Figure 10 Stability test results of the mixed system of Example 3 stored at 37°C for 7 days; in the figure, A is the amplification ability test result; B is the specificity test result; C is the sensitivity test result.

[0038] Figure 11 Generalization test results of Mixed System 1 - Mixed System 4 of Example 1 and Mixed System 1 - Mixed System 4 of Comparative Example 1; in the figure, A is Mixed System 1 of Example 1 and Mixed System 1 of Comparative Example 1; B is Mixed System 2 of Example 1 and Mixed System 2 of Comparative Example 1; C is Mixed System 3 of Example 1 and Mixed System 3 of Comparative Example 1; D is Mixed System 4 of Example 1 and Mixed System 4 of Comparative Example 1.

[0039] Figure 12 Specificity test results of Mixed System 1 - Mixed System 4 of Example 1; in the figure, A is Mixed System 1 of Example 1; B is Mixed System 2 of Example 1; C is Mixed System 3 of Example 1; D is Mixed System 4 of Example 1.

[0040] Figure 13 Specificity test results of Mixed System 1 - Mixed System 4 of Comparative Example 1; in the figure, A is Mixed System 1 of Comparative Example 1; B is Mixed System 2 of Comparative Example 1; C is Mixed System 3 of Comparative Example 1; D is Mixed System 4 of Comparative Example 1.

[0041] Figure 14 Sensitivity test results of Mixed System 1 of Example 1; in the figure, A is the amplification effect of target 1; B is the amplification effect of target 2; C is the amplification effect of target 3; D is the amplification effect of target 4.

[0042] Figure 15 Digestion ability test results of the mixed system 4 in Example 1.

[0043] Figure 16 Anti-RNase inhibition ability test results in Example 1; A in the figure is the experimental group; B is the control group 1; C is the control group 2. Detailed implementation manners

[0044] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are conventional conditions unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0045] Preparation of primers and probes 1-4 in Basic Example 1

[0046] The present invention prepares primers and probes 1-4 for verifying the effects of the RT-qPCR all-premixed reaction reagents of the present invention. The compositions of primers and probes 1-4 are shown in Table 1 - Table 4.

[0047] Table 1 Primer and probe 1

[0048]

[0049] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; "P" represents the probe.

[0050] Table 2 Primer and probe 2

[0051]

[0052]

[0053] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; "P" represents the probe.

[0054] Table 3 Primer and probe 3

[0055]

[0056]

[0057] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; "P" represents the probe.

[0058] Table 4 Primer and probe 4

[0059]

[0060] Example 1 Preparation of RT-qPCR All-in-One Reaction Reagent and Its Mixing System

[0061] 1. Preparation of RT-qPCR All-in-One Reaction Reagent (2×ZCHS RT-qPCR Mix-1)

[0062] Refer to Table 5 to prepare the RT-qPCR all-in-one reaction reagent of Example 1, denoted as 2×ZCHS RT-qPCR Mix-1. The final concentrations in the table are the final concentrations of each component in the RT-qPCR all-in-one reaction reagent.

[0063] Table 5 Components and Concentrations of RT-qPCR All-in-One Reaction Reagent

[0064]

[0065]

[0066] 2. Preparation of the Mixing System of Example 1

[0067] The RT-qPCR all-in-one reaction reagent 2×ZCHS RT-qPCR Mix-1 of Example 1 paired with Primer Probe 1 is called the mixing system of Example 1. Prepare the mixing system of Example 1 according to Table 6.

[0068] Table 6 Mixing System of Example 1

[0069] Component Dosage 2×ZCHS RT-qPCR Mix-1 12.5 μL 10× Primer Probe 1 2.5 μL Sterile and DNase / RNase-free Water 5 μL

[0070] 3. Effect Verification

[0071] The mixing system of Example 1 is subjected to two treatments: a. Store at -20°C for 1 year; b. Store at 37°C for 7 days. Use the freshly prepared mixing system of Example 1 as a control to detect the amplification ability of 2×ZCHS RT-qPCR Mix-1 after the treatment is completed.

[0072] qPCR detection of the amplification ability of the RT-qPCR all-in-one reaction reagent (2×ZCHS RT-qPCR Mix-1), and the results are as Figure 1 shown. It can be seen that the RT-qPCR all-in-one reaction reagent of Example 1 has strong stability, and the amplification ability is still not affected after storing at -20°C for 1 year.

[0073] Detect the amplification ability, specificity, and sensitivity of the mixing system of Example 1 freshly prepared, stored at -20°C for 1 year, or stored at 37°C for 7 days. The experimental results are as Figures 2 - 4 shown. It can be seen that the RT-qPCR all-in-one reaction reagent (2×ZCHS RT-qPCR Mix-1) of Example 1 can be stored stably for a long time, and the amplification ability, specificity, and sensitivity are not affected by time.

[0074] Example 2 Preparation of RT-qPCR Premixed Reaction Reagent and Its Mixing System

[0075] 1. Preparation of RT-qPCR Premixed Reaction Reagent (2×ZCHS RT-qPCR Mix-2)

[0076] Refer to Table 7 to prepare the RT-qPCR premixed reaction reagent of Example 2, denoted as 2×ZCHS RT-qPCR Mix-2. The final concentrations in the table are the final concentrations of each component in the RT-qPCR premixed reaction reagent.

[0077] Table 7 Components and Concentrations of RT-qPCR Premixed Reaction Reagent

[0078]

[0079] 2. Preparation of the Mixing System of Example 2

[0080] The RT-qPCR premixed reaction reagent 2×ZCHS RT-qPCR Mix-2 of Example 2 paired with Primer Probe 2 is called the mixing system of Example 2. Prepare the mixing system of Example 2 according to Table 8.

[0081] Table 8 Mixing System of Example 1

[0082] Component Dosage 2×ZCHS RT-qPCR Mix-2 12.5 μL 10× Primer Probe 2 2.5 μL Sterile and DNase / RNase-free Water 5 μL

[0083] 3. Effect Verification

[0084] Detect the amplification ability, specificity, and sensitivity of the mixing system of Example 2 freshly prepared, stored at -20°C for 1 year, or stored at 37°C for 7 days. The experimental results are as Figures 5 - 7 shown. It can be seen that the RT-qPCR premixed reaction reagent (2×ZCHS RT-qPCR Mix-2) of Example 2 can be stored stably for a long time, and its amplification ability, specificity, and sensitivity are not affected by time.

[0085] Example 3 Preparation of RT-qPCR Premixed Reaction Reagent and Its Mixing System

[0086] 1. Preparation of RT-qPCR Premixed Reaction Reagent (2×ZCHS RT-qPCR Mix-3)

[0087] Refer to Table 9 to prepare the RT-qPCR premixed reaction reagent of Example 3, denoted as 2×ZCHS RT-qPCR Mix-3. The final concentrations in the table are the final concentrations of each component in the RT-qPCR premixed reaction reagent.

[0088] Table 9 Components and Concentrations of RT-qPCR Premixed Reaction Reagent

[0089]

[0090] 2. Preparation of the mixed system in Example 3

[0091] The RT-qPCR full premix reaction reagent 2×ZCHS RT-qPCR Mix-3 in Example 3 paired with primer probe 3 is called the mixed system in Example 3. Prepare the mixed system in Example 3 according to Table 10.

[0092] Table 10 Mixed system in Example 1

[0093]

[0094]

[0095] 3. Effect verification of the mixed system in Example 3

[0096] Detect the amplification ability, specificity, and sensitivity of the mixed system in Example 3 freshly prepared, stored at -20°C for 1 year, or stored at 37°C for 7 days. The experimental results are as Figures 8 - 10 shown. It can be seen that the RT-qPCR full premix reaction reagent (2×ZCHS RT-qPCR Mix-3) in Example 3 can be stored stably for a long time, and its amplification ability, specificity, and sensitivity are not affected by time.

[0097] Preparation of the RT-qPCR full premix reaction reagent in Comparative Example 1

[0098] The difference between the RT-qPCR full premix reaction reagent in Comparative Example 1 and the RT-qPCR full premix reaction reagent (2×ZCHS RT-qPCR Mix-1) in Example 1 is only that: the PCR reaction additives are different.

[0099] The composition and concentration of the PCR reaction additives in Comparative Example 1 are shown in Table 11. The final concentrations in the table are the final concentrations of each component in the RT-qPCR full premix reaction reagent. The RT-qPCR full premix reaction reagent in Comparative Example 1 is called 2×RT-qPCRMix-Comparative Example 1.

[0100] Table 11 Components and concentrations of the PCR reaction additives in 2×RT-qPCR Mix-Comparative Example 1

[0101]

[0102] Preparation of the RT-qPCR full premix reaction reagent in Comparative Example 2

[0103] The difference between the RT-qPCR all-in-one reaction reagent of Comparative Example 2 and the RT-qPCR all-in-one reaction reagent (2×ZCHS RT-qPCR Mix-1) of Example 1 lies only in that the PCR reaction additives are different.

[0104] The composition and concentration of the PCR reaction additives in Comparative Example 2 are shown in Table 12. The final concentrations in the table are the final concentrations of each component in the RT-qPCR all-in-one reaction reagent. The RT-qPCR all-in-one reaction reagent of Comparative Example 2 is designated as 2×RT-qPCR Mix-Comparative Example 2.

[0105] Table 12 Components and Concentrations of PCR Reaction Additives in 2×RT-qPCR Mix-Comparative Example 2

[0106]

[0107]

[0108] Experimental Example 1 Detection of the Versatility of RT-qPCR All-in-one Reaction Reagent

[0109] The above Examples 1 to 3 show that the RT-qPCR all-in-one reaction reagents 2×ZCHS RT-qPCR Mix-1, 2×ZCHS RT-qPCR Mix-2, and 2×ZCHS RT-qPCR Mix-3 provided by the present invention can be stored stably for a long time, and their amplification ability, specificity, and sensitivity are not affected by time. In this experimental example, the RT-qPCR all-in-one reaction reagent 2×ZCHS RT-qPCR Mix-1 of Example 1 was selected to prepare the corresponding mixed system to verify the versatility of the RT-qPCR all-in-one reaction reagent of the present invention.

[0110] The RT-qPCR all-in-one reaction reagent 2×ZCHS RT-qPCR Mix-1 of Example 1 paired with Primer Probe 1 is designated as Example 1 Mixed System 1. Take the RT-qPCR all-in-one reaction reagent 2×ZCHS RT-qPCR Mix-1 in Example 1 and prepare Example 1 Mixed Systems 1-4 according to Table 13.

[0111] Table 13 Compositions of Example 1 Mixed Systems 1-4

[0112]

[0113] Replace 2×ZCHS RT-qPCR Mix-1 in Table 13 with an equal volume of 2×RT-qPCR Mix-Comparative Example 1 to obtain Comparative Example 1 Mixed Systems 1-Comparative Example 1 Mixed System 4 as a control.

[0114] The general detection results of Example 1 Hybrid System 1 - Example 1 Hybrid System 4 and Comparative Example 1 Hybrid System 1 - Comparative Example 1 Hybrid System 4 are as follows Figure 11 shown; the results show that the fully premixed reaction reagent of Example 1 can adapt to a variety of primer probes and has strong versatility.

[0115] Experimental Example 2 Specificity Detection of RT-qPCR Fully Premixed Reaction Reagent

[0116] The specificity detection results of Example 1 Hybrid System 1 - Example 1 Hybrid System 4 are as follows Figure 12 shown, and the specificity detection results of Comparative Example 1 Hybrid System 1 - Comparative Example 1 Hybrid System 4 are as follows Figure 13 shown.

[0117] The results show that the RT-qPCR fully premixed reaction reagent (2×ZCHS RT-qPCR Mix-1) provided in Example 1 of the present invention has good specificity when paired with primer probes 1 - 4 respectively. It has a significant advantage in terms of specificity compared with Comparative Example 1.

[0118] Experimental Example 3 Sensitivity Detection of RT-qPCR Fully Premixed Reaction Reagent

[0119] For Example 1 Hybrid System 1, pseudovirus samples of four targets (respiratory syncytial virus, adenovirus, influenza A virus, internal standard, 500 copies / mL) quantified by ddPCR were detected respectively, repeated 10 times, and the detection rates were statistically analyzed. The results can be seen in Table 14 and Figure 14 It can be seen that the sensitivity to pseudovirus samples (500 copies / mL) of the four targets is relatively high, and the detection rate is 100%.

[0120] Table 14 Ct values and detection rates of each target

[0121] Number of Replicates Target 1 Target 2 Target 3 Target 4 1 36.81 34.23 35.06 36.97 2 36.73 35.61 35.57 37.09 3 35.81 35.54 35.82 37.55 4 37.07 35.33 34.94 37.16 5 37.98 36.12 35.40 35.87 6 35.98 34.88 35.61 35.95 7 35.92 34.47 35.68 36.31 8 36.87 35.42 36.31 36.46 9 37.76 35.63 36.44 36.89 10 36.45 35.25 36.41 36.94 Average 36.74 35.25 35.73 36.72 CV(%) 2.01 1.62 1.48 1.50 Detection Rate 10 / 10 10 / 10 10 / 10 10 / 10

[0122] Experimental Example 3 Digestion Ability Detection of RT-qPCR Fully Premixed Reaction Reagent

[0123] For Example 1 Hybrid System 4, 2×10 7 copies of U-containing contaminants were directly added, mixed well and then detected by the instrument. (The U-containing contaminants were obtained by using a dUTP system without UNG enzyme, paired with primer probe 4 to amplify pseudovirus, and then purified by PCR product and quantified by ddPCR), and the results are as Figure 15 shown. The RT-qPCR fully premixed reaction reagent of Example 1 has good digestion ability and can completely digest 2×10 7 copies of aerosol contamination. The control group was a control without U-containing contaminants.

[0124] Experimental Example 4 Detection of Anti-RNase Inhibition Ability

[0125] Refer to Table 15 to prepare Mixed System 1 of Example 1, Mixed System 1 of Comparative Example 1, and Mixed System 1 of Comparative Example 2 for the detection of anti-RNase inhibition ability.

[0126] Table 15 Preparation of Mixed Systems for the Detection of Anti-RNase Inhibition Ability

[0127]

[0128] Experimental group: 20 ng RNase A / rxn + Mixed System 1 of Example 1;

[0129] Control Group 1: 20 ng RNase A / rxn + Mixed System 1 of Comparative Example 1;

[0130] Control Group 2: 20 ng RNase A / rxn + Mixed System 1 of Comparative Example 2.

[0131] After mixing evenly, the experimental group or Control Group 1 - Control Group 2 were respectively tested on the machine. The results are shown in Table 16 and Figure 16 as follows. It can be seen that the RT-qPCR full premixed reaction reagent prepared in Example 1 of the present invention has better anti-RNase inhibition ability than Comparative Examples 1 - 2.

[0132] Table 16 qPCR Detection Results

[0133] Group Ct Value Experimental Group 27.47 Control Group 1 31.49 Control Group 2 34.74

[0134] The above detailed description is a specific description of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A RT-qPCR fully premixed reaction reagent, characterized in that: The RT-qPCR fully premixed reaction reagent includes a PCR reaction additive; the PCR reaction additive consists of 50-1000mM trehalose, 0.5-10.0% v / v DMSO, 50-500mM betaine, 50-500ng / μL SSB single-stranded binding protein and a stabilizer; the stabilizer consists of lauryl alcohol polyoxyethylene ether, glycerol and BSA.

2. The RT-qPCR premixed reaction reagent according to claim 1, characterized in that: The PCR reaction additive consists of 300-1000 mM trehalose, 2-10% v / v DMSO, 200-500 mM betaine, 100-500 ng / μL SSB single-stranded binding protein and a stabilizer.

3. The RT-qPCR premixed reaction reagent according to claim 1, characterized in that: The stabilizer consists of 0.05-0.3% v / v lauryl alcohol polyoxyethylene ether, 0.5-5.0% v / v glycerol and 20-400 ng / μL BSA.

4. The RT-qPCR premixed reaction reagent according to claim 1, characterized in that: The stabilizer consists of 0.15-0.3% v / v lauryl alcohol polyoxyethylene ether, 2.0-5.0% v / v glycerol and 100-400 ng / μL BSA.

5. The RT-qPCR premixed reaction reagent according to claim 1, characterized in that: The RT-qPCR fully premixed reaction reagents also include DNA polymerase, reverse transcriptase, thermosensitive uracil DNA glycosidase, RNase inhibitor, soluble cations and dNTPs.

6. The RT-qPCR premixed reaction reagent according to claim 5, characterized in that: The added amount of the DNA polymerase is 0.03-0.6U / μL, the added amount of the reverse transcriptase is 0.5-4U / μL, the added amount of the thermosensitive uracil DNA glycosidase is 0.005-0.05U / μL, and the added amount of the RNase inhibitor is 0.1-3U / μL.

7. The RT-qPCR premixed reaction reagent according to claim 5, characterized in that: The soluble cations include: 2-12mM Mg 2+ , 40-200mM K + and 10-100 mM NH 4+ .

8. The RT-qPCR premixed reaction reagent according to claim 5, characterized in that: The dNTPs include: 0.02-1.2 mM dATP, 0.02-1.2 mM dCTP, 0.02-1.2 mM dGTP, 0.02-1.2 mM dTTP and 0.04-2.4 mM dUTP.

9. Use of the RT-qPCR premixed reaction reagent according to any one of claims 1 to 8 in the preparation of an RT-qPCR kit.

10. A RT-qPCR kit, characterized in that: The kit comprises the RT-qPCR fully premixed reaction reagent according to any one of claims 1 to 8.

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