qPCR kits and rapid qPCR protocol systems and methods

Abstract

The application provides a qPCR kit, a rapid qPCR reaction system and a method. Through improvement of an enzyme system and a buffer system composition in the qPCR kit and optimization of an amplification reaction program, the entire RT-qPCR program operation can be completed within 30 min, the result can be consistent with that of a conventional amplification program, multiple models and programs can be compatible, and Ct value backoff cannot exceed 1.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, specifically to a qPCR kit and a rapid qPCR procedure and method. Background Technology

[0002] In the past five years, research on isothermal amplification methods has progressed rapidly. Because isothermal amplification does not rely on complex heating and cooling systems and autoluminescence technology, it can eliminate the need for expensive reagents and complex instruments for operation and analysis, and has therefore attracted much attention.

[0003] However, currently available mainstream qPCR reagents can only handle amplification programs lasting ≥1 hour, which is time-consuming and labor-intensive, making them unsuitable for large-scale screening and testing, as well as applications such as customs. In particular, when these qPCR reagents are used in rapid qPCR programs (amplification program length ≤0.5 hours), they exhibit decreased sensitivity or poor compatibility. Summary of the Invention

[0004] Therefore, it is necessary to provide qPCR kits and rapid qPCR program systems and methods that can be applied not only to rapid (amplification program time ≤ 0.5h) qPCR programs, but also to ensure sensitivity and compatibility.

[0005] The present invention adopts the following technical solution:

[0006] This invention provides a qPCR kit, comprising an enzyme system and a buffer system. The enzyme system consists of: RT enzyme, hot-start Taq enzyme, UDG enzyme, RNase inhibitor, and diluent; wherein the diluent is an ion combination solution containing only potassium ions, EDTA, and glycerol.

[0007] Preferably, the enzyme system comprises: 5U-190U RT enzyme, 0.5U-80U hot-start Taq enzyme, 0.1U-5U UDG enzyme, 1U-5U RNase inhibitor, and diluent; wherein the diluent is an ion combination solution containing 20mM-100mM potassium ions, 0.05mM-1mM EDTA, and 5%-50% (v / v) glycerol at a final concentration.

[0008] The buffer system is composed of:

[0009]

[0010] This invention provides a rapid qPCR reaction program for the above-mentioned qPCR kit:

[0011] Reverse transcription: 50℃ for 2 min; pre-denaturation: 95℃ for 2 s; cycling reaction: annealing at 95℃ for 1 s; extension at 60℃ for 3–20 s, cycle number 40–55.

[0012] The Ct values ​​obtained by the above rapid qPCR reaction procedure and the conventional procedure will not differ by more than 0.5.

[0013] The present invention also provides a preferred qPCR reaction system composition for the above-mentioned qPCR kit as follows: 0.5-2 μL of enzyme system, 4-8 μL of buffer system, 0.4-1 μL of forward prime (10 μM), 0.4-1 μL of reverse prime (10 μM), 0.4-1 μL of probe (10 μM), 1-2 μL of RNA template, and Nuclease-free water added to a total system volume of 25-50 μL.

[0014] The rapid qPCR reaction program and amplification reaction system described herein are compatible with the ABI QuantStudio instrument. TM 5. Real-time quantitative PCR system Q5, ABI Step One Plus TM The CT values ​​of the real-time quantitative PCR system, CFX-96Touch Real-Time PCR Detection System, Quant Gene 9600 real-time quantitative PCR instrument, and AGS8830 real-time quantitative PCR instrument differ by no more than 1.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] This invention achieves the following core technical advantages by improving and optimizing the enzyme and buffer systems, reaction systems, and procedures in the qPCR kit: 1) Rapid: The entire RT-qPCR program can be completed within 30 minutes; 2) Good consistency with conventional procedures: Compared with its own conventional procedures, the Ct value decline cannot exceed 0.5; 3) High universality: It has amplification effects on human, rat, mouse, and COVID-19 samples, and the amplified Ct values ​​and curves are at least consistent with the RK20412 kit or the conventional kits of competitors; 4) Extremely high detection rate: The detection rate in the LOT part is significantly higher than that of conventional commercially available rapid kits, with a statistically significant difference; 5) Compatible with multiple instruments and programs: It is compatible with the rapid and conventional programs of instruments such as ABI Q5, Step One, CFX-96, Gene-9600, and AGS8830, with the Ct value difference between models not exceeding 1; 6) Ensures consistent amplification effect after the reagent volume is scaled up to a specific ratio. Attached Figure Description

[0017] Figure 1 This is a test diagram of the reaction system using 4 μL of enzyme combination three and buffer combination three in Example 2.

[0018] Figure 2 This is a test diagram of the reaction system using enzyme combination 3L and buffer combination 35μL in Example 2.

[0019] Figure 3 This is a comparison chart of the standard procedure and the rapid qPCR procedure for human RNA in Example 3.

[0020] Figure 4 This is a comparison chart of the standard procedure and the rapid qPCR procedure for COVID-19 in Example 3.

[0021] Figure 5 This is a comparison chart of qPCR tests for human RNA performed using the Quant Gene 9600 real-time quantitative PCR analyzer in Example 4.

[0022] Figure 6 This is a comparison chart of qPCR tests for COVID-19 using the Quant Gene 9600 real-time quantitative PCR analyzer in Example 4.

[0023] Figure 7 This is a comparison image of the qPCR test for human RNA using the Stepone instrument in Example 4.

[0024] Figure 8This is a comparison chart of qPCR tests for COVID-19 using the Stepone instrument in Example 4.

[0025] Figure 9 This is a comparison diagram of the qPCR test of the amplification system in Example 5.

[0026] Figure 10 This is a comparison chart of qPCR tests of ABclonal and TOROIVD in Example 6.

[0027] Figure 11 This is a comparison chart of qPCR tests of ABclonal and Vazyme in Example 6.

[0028] It is worth noting that in the above figures, the title at the top of the figure is "Amplification Plot" or "Amplification," the horizontal axis unit is "Cycle," and the vertical axis unit is "△R." n "Or fluorescence intensity." Detailed Implementation

[0029] The technical concept of this invention is to address the problem of decreased sensitivity or poor compatibility when conventional qPCR reagents are used in rapid (amplification program duration ≤ 0.5h) qPCR programs, and to optimize and develop new qPCR reagent compositions, reaction systems, and programs.

[0030] Specifically, the improved and optimized qPCR reagent (kit) of the present invention includes an enzyme system and a buffer system.

[0031] The optimized composition of the enzyme system is as follows: RT enzyme 5-190U, hot-start Taq enzyme 0.5U-80U, UDG enzyme 0.1U-5U, and diluent; wherein the diluent is an ion combination solution containing potassium ions at a final concentration of 20mM-100mM, 0.05mM-1mM EDTA, and 5%-50% (V / V) glycerol.

[0032] The components of the buffer system are shown in the table below:

[0033]

[0034]

[0035] Further details regarding the rapid qPCR reaction procedure used in the aforementioned qPCR kit:

[0036] Reverse transcription: 50℃ for 2 min; pre-denaturation: 95℃ for 2 s; cycling reaction: annealing at 95℃ for 1 s; extension at 60℃ for 3–20 s, cycle number 40–55.

[0037] The Ct values ​​obtained by the above rapid qPCR reaction procedure and the conventional procedure will not differ by more than 0.5.

[0038] The preferred qPCR reaction system composition for the above-mentioned qPCR kit is further provided as follows: 0.5-2 μL enzyme system, 4-8 μL buffer system, 0.4-1 μL forward prime (10 μM), 0.4-1 μL reverse prime (10 μM), 0.4-1 μL probe (10 μM), 1-2 μL RNA template (10 pg-1 ug), and Nuclease-free water added to a total system volume of 25-50 μL.

[0039] The above rapid qPCR reaction procedure and amplification reaction system are applicable to instruments such as ABI Q5, Step One, CFX-96, Gene-9600, and AGS8830. The CT value between different instruments should not differ by more than 1.

[0040] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0041] Example 1

[0042] This example explores the effect of different enzyme system combinations on qPCR amplification results, specifically including the following steps:

[0043] S1, prepare the enzyme system shown in the table below:

[0044]

[0045] Prepare the following buffer combination:

[0046]

[0047] S2 uses ABI Step One Plus TM The real-time quantitative PCR system should be used according to the following reaction system and procedure:

[0048] qPCR reaction mixture (25 μL)

[0049] Buffer Combination 1 5μL Enzyme combination 1.5μL Introducing FAM VIC ROX (purchase commercial kit) 1μL Template (10x dilution) (COVID-19 pseudovirus standard) 12.5μL Nuclease-free Water To 25μL

[0050] Rapid qPCR reaction program

[0051]

[0052] The amplification results of using Buffer Combination 1 with different enzyme systems are statistically analyzed in the table below:

[0053]

[0054] Note: "--" indicates no amplification.

[0055] The test results in the table above show that the optimized enzyme combination 3 is the most effective for qPCR amplification and detection.

[0056] S3. Further, using the selected enzyme combination three as the enzyme mix, we explored the effect of different buffer system combinations on qPCR amplification results.

[0057] The different buffer systems are composed as shown in the table below:

[0058]

[0059] The testing instruments, reaction system quantities, and reaction procedures are the same as those described above. The results of the tests using enzyme combination three with different buffer systems are shown in the table below:

[0060]

[0061] The test results in the table above show that the combination of the optimized enzyme combination 3 and the buffer combination 3 results in the best qPCR amplification and detection effect. Therefore, the combination of enzyme combination 3 and buffer combination 3 is the preferred choice for the rapid qPCR kit.

[0062] Example 2

[0063] Using the CFX-96Touch Real-Time PCR Detection System and a commercially available kit from 3DMed as a control, this example further investigates the effect of different ratios of enzyme combination three and buffer combination three in the qPCR reagent reaction system on the amplification results.

[0064] In a 25 μL system, following the reaction system and procedure of Example 1, the ratio of enzyme system to buffer system input was examined to be (0.1-3 μL):(2.5-8 μL). The results showed that the ratio of enzyme system to buffer system input was (0.5-2 μL):(4-5 μL) with excellent effect.

[0065] Some test results are as follows Figure 1 and 2 And as shown in the table below:

[0066]

[0067] More preferably, the optimal combination of enzyme system and buffer system input ratio is 1 μL:4 μL, which yields the best sensitivity and fluorescence intensity.

[0068] Example 3

[0069] Using a CFX-96Touch Real-Time PCR Detection System, this example further tests the compatibility of the qPCR reagent system containing enzyme combination three and buffer combination three with the reaction program. The specific method steps are as follows:

[0070] Prepare the qPCR reaction system as shown in the table below:

[0071] qPCR reaction mixture (25 μL)

[0072]

[0073]

[0074] The reactions were carried out according to the following standard procedure and rapid procedure respectively:

[0075] Standard qPCR reaction procedure

[0076]

[0077] Rapid qPCR reaction program

[0078]

[0079] Among them, the qPCR test results for human RNA (commercially available) are as follows: Figure 3 As shown, the results of the commercially available qPCR test for COVID-19 are as follows: Figure 4 As shown.

[0080] The Ct value of the rapid qPCR program differs from that of the conventional program by no more than 0.5, and the sensitivity is consistent with that of the conventional program. This demonstrates that the optimization of the improved enzyme system and buffer system can adapt to the amplification of the rapid program and is compatible with the amplification of the conventional program, and the sensitivity is not affected by the reaction program.

[0081] Example 4

[0082] Using a CFX-96Touch Real-Time PCR Detection System, and referring to Example 3, this example further tests the compatibility of the qPCR reagent system containing enzyme combination three and buffer combination three with different instruments. The test results are shown in […]. Figures 5 to 8 .

[0083] The test results show that the qPCR reagents using enzyme combination 3 and buffer combination 3 are compatible with different instruments, including ABI Step One Plus. TM The CT values ​​of the real-time quantitative PCR system, CFX-96Touch Real-Time PCR Detection System, and Quant Gene 9600 real-time quantitative PCR instrument differed by no more than 1.

[0084] Example 5

[0085] Using the CFX-96Touch Real-Time PCR Detection System, and referring to Example 3, this example further tests the qPCR test effect after amplification (30μL and 50μL) of the qPCR reagent system containing enzyme combination three and buffer combination three.

[0086] qPCR reaction system

[0087] Buffer Combination Three 4μL 4.8μL 8μL Enzyme Combination Three 1μL 1.2μL 2μL Forward Primer (10μM) (Commercially Available) 0.4μL 0.48μL 0.8μL Reverse Primer (10μM) (Commercially Available) 0.4μL 0.48μL 0.8μL Probe (10μM) (commercially available) 0.4μL 0.48μL 0.8μL RNA Template (commercially available) 2μL 2μL 2μL Nuclease-free Water To 25μL To 30μL To 50μL

[0088] The test results for the above three systems are shown in [link to test results]. Figure 9 .

[0089] The test data for some channels is summarized in the table below:

[0090] 25μL system 28.2 33.3 30μL system 28.34 33.7 50μL system 28.32 33.52

[0091] The results above show that, compared to the 25 μL system, when the qPCR reagent systems containing enzyme combination 3 and buffer combination 3 are scaled up to 30 μL and 50 μL, the difference in ct values ​​between the two channels is within 0.5, demonstrating a very good scale-up effect.

[0092] Example 6

[0093] Using a CFX-96Touch Real-Time PCR Detection System, this example further compares the qPCR reagent containing enzyme combination 3 and buffer combination 3 (ABclonal) with commercially available rapid qPCR reagents.

[0094] Commercially available rapid qPCR reagents are as follows:

[0095] ROROIVD: Probe 1-step RT-qPCR 5G Kit 2.0, catalog number QPR-303UD, lot number: G22030210.

[0096] The 25 μL reaction system is as follows:

[0097] 2×5G qPCR Buffer BB 12.5μL RT-qPCR Enzyme Mix UD 1.3μL Forward Prime (10μM) 0.4μL Forward Prime (10μM) 0.4μL Probe (10μM) 0.4μL RNA Template 2μL Nuclease-free Water To 25μL

[0098] The reaction procedure is as follows:

[0099]

[0100] Vazyme: AccurSTART U + One Step RT-qPCR Probe Kit (FOR FAST), catalog number Q231-C1, batch number: C17E2292HA.

[0101] The 20 μL reaction system is as follows:

[0102]

[0103]

[0104] The reaction procedure is as follows:

[0105]

[0106] Test results are available Figure 10 and Figure 11 .

[0107] The results above show that, using commercially available human and COVID-19 primers, probes, and templates, at high, medium, and low template concentrations, and under the conditions of ROROIVD and Vazyme reaction system dosage and reaction procedure, the qPCR reagent containing enzyme combination three and buffer combination three exhibits superior overall sensitivity and fluorescence signal values.

[0108] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A qPCR kit, characterized in that, It includes an enzyme system and a buffer system. The enzyme system consists of: RT enzyme, hot-start Taq enzyme, UDG enzyme, and diluent. The diluent is an ionic combination solution containing potassium ions, EDTA, and glycerol. The ratio of the enzyme system to the buffer system is 1:4; The enzyme system consists of: 60 U of RT enzyme, 20 U of hot-start Taq enzyme, 1 U of UDG enzyme, 2 U of RNase inhibitor, and diluent; wherein the diluent is an ion combination solution containing only 60 mM potassium ions, 0.5 mM EDTA, and 30% V / V glycerol. The buffer system is composed of: Tris-HCl 30mM at a pH of 7.5–8.9 DNTP containing U 20mM, KCl 3M, MgCl2 3mM Tween-20 0.02% V / V Tetrahydrothiophene 1-oxide 0.02% V / V, Tetramethyl ammonium chloride 20mM, Betaine 20mM, DMSO 0.05% V / V and Triton X-100 0.01% V / V.

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