RT-qPCR full premix reaction reagent, application and product thereof

Abstract

The application provides a RT-qPCR full premix reaction reagent and application and product thereof, and belongs to the technical field of biochemical reagents.The RT-qPCR full premix reaction reagent provided by the application comprises PCR reaction additives, DNA polymerase, reverse transcriptase, heat-sensitive uracil DNA glycosylase, RNAase inhibitor, soluble cation and dNTPs; the PCR reaction additives are 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; and the stabilizer is composed of lauryl alcohol polyoxyethylene ether, glycerol and BSA.The RT-qPCR full premix reaction reagent provided by the application is not only good in universality, high in specificity and sensitivity, but also has good stability and can be stably stored in a-20 DEG C refrigerator for one year.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biochemical reagents, and particularly relates to an RT-qPCR full premix reaction reagent, application and product thereof. BACKGROUND

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

[0003] The commonly used RT-qPCR reaction system is composed of nucleic acid amplification buffer, enzyme mixture, primer probe reaction liquid and the like and needs to be stored separately. When used, each component needs to be dissolved and mixed uniformly at room temperature before use. When used, each component needs to be mixed uniformly according to a fixed proportion and then divided into tubes. The whole process needs to be calculated and carefully operated, and the wrong combination often occurs. Moreover, the reaction system needs to be prepared and used immediately. If the nucleic acid amplification buffer, enzyme mixture and primer probe reaction liquid are premixed, frozen or refrigerated for storage, stability problems will occur, 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, a nucleic acid aptamer, a stabilizer and an enhancer. The full premix RT-qPCR reaction system can be used for 7 days at 37℃ high temperature and pressure without affecting the detection ability, and can be stably stored for 12-15 months under-20℃. However, the applicability and sensitivity of the premix system cannot be determined. SUMMARY

[0005] To solve the above problems, the application provides an RT-qPCR full premix reaction reagent, application and product thereof. The RT-qPCR full premix reaction reagent provided by the application comprises PCR reaction additives, DNA polymerase, reverse transcriptase, heat-sensitive uracil DNA glycosylase, RNase inhibitor, soluble cation and dNTPs; the PCR reaction additives are 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 stabilizers; the stabilizers are composed of lauryl alcohol polyoxyethylene ether, glycerol and BSA. The RT-qPCR full premix reaction reagent provided by the application has good universality, high specificity and sensitivity, and good stability, and can be stably stored in a refrigerator at-20℃ for one year.

[0006] The technical scheme of the application comprises:

[0007] In the first aspect, the application provides an RT-qPCR full premix reaction reagent, which comprises PCR reaction additives; the PCR reaction additives are 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 stabilizers; the stabilizers are composed of lauryl alcohol polyoxyethylene ether, glycerol and BSA.

[0008] Preferably, the PCR reaction additives are 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 stabilizers.

[0009] Further preferably, the PCR reaction additives are composed of 500 mM trehalose, 3.5 %v / v DMSO, 200 mM betaine, 200 ng / μL SSB single-strand binding protein and stabilizers.

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

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

[0012] Further preferably, the stabilizers are composed of 0.15 %v / v lauryl alcohol polyoxyethylene ether, 2.4 %v / v glycerol and 200 ng / μL BSA.

[0013] Specifically, the RT-qPCR full premix reaction reagent further comprises a DNA polymerase, a reverse transcriptase, a heat-sensitive uracil DNA glycosylase, an RNase inhibitor, a soluble cation and dNTPs.

[0014] Further specifically, the DNA polymerase is added in an amount of 0.03-0.6 U / μL, the reverse transcriptase is added in an amount of 0.5-4 U / μL, the heat-sensitive uracil DNA glycosylase is added in an amount of 0.005-0.05 U / μL, and the RNase inhibitor is added in an amount of 0.1-3 U / μL.

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

[0016] Further specifically, the soluble cation consists of Mg 2+ , K + and NH 4+ .

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

[0018] Further preferably, the soluble cation comprises 4-12 mM Mg 2+ , 50-200 mM K + and 10-100 mM NH 4+ .

[0019] Further specifically, the dNTPs comprise 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 comprise 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 application provides use of the RT-qPCR full premix reaction reagent in the preparation of an RT-qPCR kit.

[0022] In a third aspect, the present application provides an RT-qPCR kit, which comprises the aforementioned RT-qPCR full premix reaction reagent.

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

[0024] (1) The RT-qPCR full premix reaction reagent of the present application can be stored at -20℃ for 1 year and at 37℃ for 7 days after mixing with primers;

[0025] (2) The RT-qPCR full premix reaction reagent of the present application can be adapted to various primers and probes, and has strong universality;

[0026] (3) The RT-qPCR full premix reaction reagent of the present application can stably detect nucleic acid samples with an abundance of 500 copies / mL, and has high sensitivity;

[0027] (4) The RT-qPCR full premix reaction reagent of the present application has high specificity, strong RNase inhibition ability, and can digest 2x10 7 copies of aerosol pollution. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

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

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

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

[0038] Figure 11 Universal test results of the mixed system 1-4 of Example 1 and the mixed system 1-4 of Comparative Example 1; A in the figure is the mixed system 1 of Example 1 and the mixed system 1 of Comparative Example 1; B is the mixed system 2 of Example 1 and the mixed system 2 of Comparative Example 1; C is the mixed system 3 of Example 1 and the mixed system 3 of Comparative Example 1; D is the mixed system 4 of Example 1 and the mixed system 4 of Comparative Example 1.

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

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

[0041] Figure 14 Sensitivity test results of the mixed system 1 of Example 1; A in the figure 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 Example 1 mixed system 4.

[0043] Figure 16 RNase inhibition ability test results of Example 1; A in the figure is the experimental group; B is the control 1 group; C is the control 2 group. DETAILED DESCRIPTION

[0044] The application will be further described in conjunction with specific examples, and the following examples are not used to limit the application, but only to illustrate the application. The experimental methods used in the following examples are not specifically described, and the experimental methods not specifically described in the examples are generally performed under conventional conditions. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0045] Preparation of primer probe 1-4 in basic example 1

[0046] The primer probe 1-4 prepared in the application is used to verify the effect of RT-qPCR full premix reaction reagent in the application. The composition of primer probe 1-4 is shown in Table 1-Table 4.

[0047] Table 1 primer probe 1

[0048]

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

[0050] Table 2 primer probe 2

[0051]

[0052]

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

[0054] Table 3 primer probe 3

[0055]

[0056]

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

[0058] Table 4 primer probe 4

[0059]

[0060] Preparation of RT-qPCR whole premix reaction reagent and its mixed system of Example 1

[0061] 1. Preparation of RT-qPCR whole premix reaction reagent (2XZCHS RT-qPCR Mix-1)

[0062] The RT-qPCR whole premix reaction reagent of Example 1 was prepared according to Table 5, and was denoted as 2XZCHS RT-qPCR Mix-1. The final concentration in the table is the final concentration of each component in the RT-qPCR whole premix reaction reagent.

[0063] Table 5. Components and concentrations of RT-qPCR whole premix reaction reagent

[0064]

[0065]

[0066] 2. Preparation of mixed system of Example 1

[0067] The RT-qPCR whole premix reaction reagent 2XZCHS RT-qPCR Mix-1 of Example 1 was combined with primer probe 1, and was denoted as the mixed system of Example 1. The mixed system of Example 1 was prepared according to Table 6.

[0068] Table 6. Mixed system of Example 1

[0069] Component Addition 2x ZCHS RT-qPCR Mix-1 12.5 μL 10x Primer Probe 1 2.5 μL Sterile Nuclease-free Water 5 μL

[0070] 3. Effect verification

[0071] The mixed system of Example 1 was subjected to two treatments: a, storage at -20℃ for 1 year; b, storage at 37℃ for 7 days. Freshly prepared mixed system of Example 1 was used as a control, and the amplification ability of 2XZCHS RT-qPCR Mix-1 after treatment was detected.

[0072] The amplification ability of the RT-qPCR whole premix reaction reagent (2XZCHS RT-qPCR Mix-1) was detected by qPCR, and the results are shown in Table 7. Figure 1 It can be seen that the RT-qPCR whole premix reaction reagent of Example 1 has strong stability, and the amplification ability is not affected after storage at -20℃ for 1 year.

[0073] The amplification ability, specificity, and sensitivity of the mixed system of Example 1 freshly prepared, stored at -20℃ for 1 year, or stored at 37℃ for 7 days were detected, and the experimental results are shown in Table 8. Figure 2 - Figure 4 It can be seen that the RT-qPCR whole premix reaction reagent (2XZCHS RT-qPCR Mix-1) of Example 1 can be stored for a long time, and the amplification ability, specificity, and sensitivity are not affected by time.

[0074] Preparation of RT-qPCR whole premix reaction reagent and its mixed system of Example 2

[0075] 1. Preparation of RT-qPCR whole premix reaction reagent (2xZCHS RT-qPCR Mix-2)

[0076] The RT-qPCR whole premix reaction reagent of Example 2 was prepared according to Table 7, and was denoted as 2xZCHS RT-qPCR Mix-2. The final concentration in the table is the final concentration of each component in the RT-qPCR whole premix reaction reagent.

[0077] Table 7 Components and concentrations of RT-qPCR whole premix reaction reagent

[0078]

[0079] 2. Preparation of mixed system of Example 2

[0080] The RT-qPCR whole premix reaction reagent 2xZCHS RT-qPCR Mix-2 of Example 2 was matched with primer probe 2, and was denoted as mixed system of Example 2. The mixed system of Example 2 was prepared according to Table 8.

[0081] Table 8 Mixed system of Example 1

[0082] Component Addition 2x ZCHS RT-qPCR Mix-2 12.5 μL 10x Primer Probe 2 2.5 μL Sterile Nuclease-free Water 5 μL

[0083] 3. Effect verification

[0084] The amplification capacity, specificity and sensitivity of the mixed system of Example 2 freshly prepared, stored at -20℃ for 1 year or stored at 37℃ for 7 days were detected, and the experimental results are shown in Table 10. Figure 5 - Figure 7 It can be seen that the RT-qPCR whole premix reaction reagent (2xZCHS RT-qPCR Mix-2) of Example 2 can be stored for a long time, and the amplification capacity, specificity and sensitivity are not affected by time.

[0085] Preparation of RT-qPCR whole premix reaction reagent and its mixed system of Example 3

[0086] 1. Preparation of RT-qPCR whole premix reaction reagent (2xZCHS RT-qPCR Mix-3)

[0087] The RT-qPCR whole premix reaction reagent of Example 3 was prepared according to Table 9, and was denoted as 2xZCHS RT-qPCR Mix-3. The final concentration in the table is the final concentration of each component in the RT-qPCR whole premix reaction reagent.

[0088] Table 9 Components and concentrations of RT-qPCR whole premix reaction reagent

[0089]

[0090] 2. Preparation of Example 3 mixed system

[0091] The RT-qPCR master mix 2xZCHS RT-qPCR Mix-3 with primers and probes of Example 3 is called Example 3 mixed system. The Example 3 mixed system was prepared according to Table 10.

[0092] Table 10 Example 1 mixed system

[0093]

[0094]

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

[0096] The amplification ability, specificity and sensitivity of the Example 3 mixed system freshly prepared, stored at -20℃ for 1 year or stored at 37℃ for 7 days were detected, and the experimental results are shown in Table 12. Figure 8 - Figure 10 It can be seen that the RT-qPCR master mix (2xZCHS RT-qPCR Mix-3) of Example 3 can be stored for a long time, and the amplification ability, specificity and sensitivity are not affected by time.

[0097] Preparation of RT-qPCR master mix of Comparative Example 1

[0098] The difference between the RT-qPCR master mix of Comparative Example 1 and the RT-qPCR master mix (2xZCHS RT-qPCR Mix-1) of Example 1 is only that the PCR reaction additives are different.

[0099] The composition and concentration of the PCR reaction additives of Comparative Example 1 are shown in Table 11. The final concentration in the table is the final concentration of each component in the RT-qPCR master mix. The RT-qPCR master mix of Comparative Example 1 is called 2xRT-qPCR Mix-Comparative Example 1.

[0100] Table 11 Composition and concentration of PCR reaction additives of 2xRT-qPCR Mix-Comparative Example 1

[0101]

[0102] Preparation of RT-qPCR master mix of Comparative Example 2

[0103] The RT-qPCR master mix reagent of Comparative Example 2 and the RT-qPCR master mix reagent (2xZCHS RT-qPCR Mix-1) of Example 1 differ only in PCR reaction additives.

[0104] The composition and concentration of the PCR reaction additives of Comparative Example 2 are shown in Table 12. The final concentration in the table is the final concentration of each component in the RT-qPCR master mix reagent. The RT-qPCR master mix reagent of Comparative Example 2 is referred to as 2xRT-qPCR Mix-Comparative Example 2.

[0105] Table 12 Components and concentrations of PCR reaction additives of 2xRT-qPCR Mix-Comparative Example 2

[0106]

[0107]

[0108] Experiment Example 1 Detection of universality of RT-qPCR master mix reagent

[0109] Examples 1-3 above show that the RT-qPCR master mix reagents 2xZCHS RT-qPCR Mix-1, 2xZCHS RT-qPCR Mix-2, and 2xZCHS RT-qPCR Mix-3 provided by the present application have good long-term stability, and the amplification ability, specificity, and sensitivity are not affected by time. In this experiment example, the RT-qPCR master mix reagent 2xZCHS RT-qPCR Mix-1 of Example 1 was selected to prepare the corresponding mixed systems to verify the universality of the RT-qPCR master mix reagent of the present application.

[0110] The RT-qPCR master mix reagent 2xZCHS RT-qPCR Mix-1 of Example 1 is referred to as Example 1 mixed system 1 with primer probe 1. The RT-qPCR master mix reagent 2xZCHS RT-qPCR Mix-1 in Example 1 was used to prepare Example 1 mixed systems 1-4 according to Table 13.

[0111] Table 13 Composition of Example 1 mixed systems 1-4

[0112]

[0113] Replace 2xZCHS RT-qPCR Mix-1 in Table 13 with equal volume of 2xRT-qPCR Mix-Comparative Example 1 to obtain Comparative Example 1 mixed systems 1-Comparative Example 1 mixed system 4 as controls.

[0114] The results of the universality detection of the mixed system 1 of Example 1, the mixed system 4 of Example 1, the mixed system 1 of Comparative Example 1 and the mixed system 4 of Comparative Example 1 are shown in Table 1 and Table 2, respectively. Figure 11 As shown in Table 1 and Table 2, the results show that the RT-qPCR full premix reaction reagent of Example 1 can be adapted to various primer probes, and has strong universality.

[0115] Experimental Example 2: Specificity detection of RT-qPCR full premix reaction reagent

[0116] The results of the specificity detection of the mixed system 1 of Example 1, the mixed system 4 of Example 1, the mixed system 1 of Comparative Example 1 and the mixed system 4 of Comparative Example 1 are shown in Table 3 and Table 4, respectively. Figure 12 Figure 13 As shown in Table 3 and Table 4, the results show that the RT-qPCR full premix reaction reagent (2×ZCHS RT-qPCR Mix-1) provided by Example 1 of the present application has good specificity when matched with primer probes 1-4, respectively. Compared with Comparative Example 1, it has a significant advantage in specificity.

[0117] As shown in Table 3 and Table 4, the results show that the RT-qPCR full premix reaction reagent (2×ZCHS RT-qPCR Mix-1) provided by Example 1 of the present application has good specificity when matched with primer probes 1-4, respectively. Compared with Comparative Example 1, it has a significant advantage in specificity.

[0118] Experimental Example 3: Sensitivity detection of RT-qPCR full premix reaction reagent

[0119] The mixed system 1 of Example 1 was used to detect the four target pseudovirus samples (respiratory syncytial virus, adenovirus, influenza A virus, internal standard, 500 copies / mL) quantified by ddPCR, 10 times of repetition, and the detection rate was calculated. The results can be seen in Table 14 and Table 15. Figure 14 It can be seen that the sensitivity of the four target pseudovirus samples (500 copies / mL) is relatively high, and the detection rate is 100%.

[0120] Table 14: Ct value and detection rate 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 capacity detection of RT-qPCR full premix reaction reagent

[0123] The mixed system 4 of Example 1 was used to directly add 2×10 7 copies of U-containing contaminants, mix well and then detect. (The U-containing contaminants are dUTP-containing systems without UNG enzyme, matched with primer probe 4 to amplify pseudovirus, and the PCR product is purified and quantified by ddPCR), and the results are shown in Table 16. Figure 15 As shown in Table 16, the RT-qPCR full premix reaction reagent of Example 1 has good digestion capacity and can completely digest 2×10 7 copies of aerosol contamination. The control group is the control without U-containing contaminants.

[0124] ​Experimental Example 4 Anti-RNase inhibition ability detection

[0125] Referring to Table 15, the mixture system 1 of Example 1, the mixture system 1 of Comparative Example 1 and the mixture system 1 of Comparative Example 2 were used for anti-RNase inhibition ability detection.

[0126] Table 15 Preparation of mixture system for anti-RNase inhibition ability detection

[0127]

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

[0129] Control 1 group: 20 ng RNase A / rxn + mixture system 1 of Comparative Example 1;

[0130] Control 2 group: 20 ng RNase A / rxn + mixture system 1 of Comparative Example 2.

[0131] After mixing, the experimental group or control 1 group to control 2 group were detected respectively. The results are shown in Table 16 and Figure 16 It can be seen that the anti-RNase inhibition ability of the RT-qPCR full premix reaction reagent prepared by Example 1 is better than that of Comparative Examples 1-2.

[0132] Table 16 qPCR detection results

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

[0134] The above detailed description is a specific description of one of the feasible embodiments of the present application, which is not used to limit the patent scope of the present application. It should be noted that any equivalent implementation or change without departing from the present application shall be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. An RT-qPCR master mix reaction reagent, characterized in that, The RT-qPCR full premix reaction reagent comprises a PCR reaction additive; 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-stranded binding protein and a stabilizer; the stabilizer is composed of 0.15-0.3 % v / v lauryl alcohol polyoxyethylene ether, 2.0-5.0 % v / v glycerol and 100-400 ng / μL BSA.

2. The RT-qPCR master mix according to claim 1, characterized in that, The RT-qPCR full premix reaction reagent further comprises a DNA polymerase, a reverse transcriptase, a heat-sensitive uracil DNA glycosylase, an RNase inhibitor, a soluble cation and dNTPs.

3. The RT-qPCR master mix reagent according to claim 2, wherein, The DNA polymerase is added in an amount of 0.03-0.6 U / μL, the reverse transcriptase is added in an amount of 0.5-4 U / μL, the heat-sensitive uracil DNA glycosylase is added in an amount of 0.005-0.05 U / μL, and the RNase inhibitor is added in an amount of 0.1-3 U / μL.

4. The RT-qPCR master mix according to claim 2, wherein, The soluble cations include: 2-12 mM Mg 2+ , 40-200 mM K + , and 10-100 mM NH 4+ .

5. The RT-qPCR master mix according to claim 2, wherein, The dNTPs comprise 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.

6. Use of the RT-qPCR full premix reaction reagent according to any one of claims 1-5 in the preparation of an RT-qPCR kit.

7. An RT-qPCR kit characterized in that, The kit comprises the RT-qPCR full premix reaction reagent according to any one of claims 1-5.

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