Application of melezitose and reversible binding factor oligo in improving the storage stability of fluorescent quantitative PCR full premix system

Abstract

The application discloses application of melezitose and a reversible binding factor Oligo in improving the storage stability of a fluorescent quantitative PCR full premix system. The application discloses application of melezitose and the reversible binding factor Oligo in improving the storage stability of the fluorescent quantitative PCR full premix system. Further, the application discloses a fluorescent quantitative PCR full premix system with improved storage stability. The application firstly adds the melezitose and the reversible binding factor Oligo into the fluorescent quantitative PCR full premix system, effectively improves the storage stability of the fluorescent quantitative PCR full premix system, improves the amplification performance of the fluorescent quantitative PCR full premix system, and has obvious synergistic effects on the Ct value and the end-point fluorescence value of the fluorescent quantitative PCR.

Description

Technical Field

[0001] This invention relates to the field of molecular biology. More specifically, it relates to the application of menotriose and the reversible binding factor Oligo in improving the storage stability of fully premixed systems for quantitative real-time PCR. Background Technology

[0002] Polymerase chain reaction (PCR) is a technique for amplifying specific DNA fragments and is widely used in many fields related to medicine and biology, such as gene cloning, pathogen detection, gene diagnosis, forensics, and the food industry. Formally, it evolved from early techniques that relied on electrophoresis to determine the final concentration of the product to the currently widely used quantitative real-time PCR (qPCR) technique, which allows for the assessment of amplification progress during and at the reaction endpoint by detecting fluorescence.

[0003] Before performing a PCR reaction, a PCR reaction solution needs to be prepared. Typically, this involves mixing primers or primers and probes with buffer, enzymes, dNTPs, and the template, and then immediately performing PCR detection. A fully premixed system is a system in which all components except the template are mixed. Premixed systems can be stored and used simply by aliquoting them into PCR wells and adding the template, significantly saving time and labor costs. However, fully premixed systems often suffer from poor stability, leading to a decrease in amplification performance during storage, resulting in higher Ct values ​​or lower fluorescence values ​​at the amplification curve endpoint.

[0004] The reversible binding factor Oligo possesses a core sequence on a single-stranded deoxyribonucleic acid molecule that binds both Taq enzyme and reverse transcriptase (Takahisa Noma, Kazunori Ikebukuro. Aptamer selection based on inhibitory activity using an evolution-mimicking algorithm. Biochemical and Biophysical Research Communications 347(2006)226–231). Pintotriose is a trisaccharide compound with a polyhydroxy structure. Polyhydroxy compounds have the ability to stabilize protein structures and protect enzyme activity. However, there are no reports on simultaneously adding the reversible binding factor Oligo and pintotriose to a fully mixed system for quantitative real-time PCR to improve its preservation stability. Summary of the Invention

[0005] One objective of this invention is to provide the application of menotriose and the reversible binding factor Oligo in the preparation of a fully premixed system for quantitative real-time PCR. Adding menotriose and Oligo to the fully premixed system for quantitative real-time PCR effectively improves the storage stability of the fully premixed system and significantly enhances the Ct value and endpoint fluorescence value of quantitative real-time PCR.

[0006] Another objective of this invention is to provide a fully premixed system for quantitative real-time PCR with improved preservation stability.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention first provides the application of mesotriose and / or the reversible binding factor Oligo in any of the following:

[0009] 1) Application in the preparation of fully premixed systems for quantitative real-time PCR;

[0010] 2) Application in improving the storage stability of fully premixed systems for quantitative real-time PCR;

[0011] 3) Application in improving the amplification performance of fully premixed systems for quantitative real-time PCR.

[0012] Furthermore, the sequence of the reversible binding factor Oligo is the single-stranded deoxyribonucleic acid shown in SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6; wherein the 3' end of the sequence is blocked with SpacerC3.

[0013] In a preferred embodiment of the present invention, the sequence of the reversible binding factor Oligo is the single-stranded deoxyribonucleic acid shown in SEQ ID NO.4; wherein the 3' end of the sequence is blocked with SpacerC3.

[0014] Furthermore, the final concentration of menotriose in the fully premixed system for quantitative real-time PCR is 20-80 mM; preferably, the final concentration of menotriose in the fully premixed system for quantitative real-time PCR is 40 mM.

[0015] Furthermore, the final concentration of the reversible binding factor Oligo in the fully premixed system for quantitative real-time PCR is 10-40 nM; preferably, the final concentration of the reversible binding factor Oligo in the fully premixed system for quantitative real-time PCR is 20 nM.

[0016] Furthermore, the storage temperature of the fully premixed fluorescent quantitative PCR system is -20℃ to 37℃ (for example, it can be -20℃, 4℃, 25℃, 37℃, or any range between two values).

[0017] Furthermore, the quantitative PCR is quantitative real-time PCR using DNA as a template (qPCR) and / or one-step reverse transcription quantitative real-time PCR using RNA as a template (qRT-PCR).

[0018] This invention first adds menotriose and the reversible binding factor Oligo to a fully mixed system for quantitative real-time PCR (qPCR), obtaining a fully mixed qPCR system with added menotriose and Oligo. Then, the fully mixed qPCR system with added menotriose and Oligo is stored, improving its storage stability. Finally, the fully mixed qPCR system with added menotriose and Oligo is aliquoted and template is added for qPCR, improving the amplification performance of the fully mixed qPCR system. The reversible binding factor Oligo of this invention can bind to and inhibit the spatial structure of DNA polymerase or reverse transcriptase activity in the fully mixed qPCR system at low temperatures. At the enzyme's operating temperature, Oligo cannot form the spatial structure that binds to and inhibits enzyme activity, thus releasing the inhibition effect. Furthermore, through its synergistic effect with menotriose, this invention achieves the goal of improving the storage stability and amplification performance of the fully mixed qPCR system.

[0019] The present invention further provides a fully premixed system for quantitative real-time PCR with improved preservation stability, wherein the fully premixed system for quantitative real-time PCR includes menotriose and / or the reversible binding factor Oligo.

[0020] Furthermore, the sequence of the reversible binding factor Oligo is the single-stranded deoxyribonucleic acid shown in SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6; wherein the 3' end of the sequence is blocked with SpacerC3.

[0021] In a preferred embodiment of the present invention, the sequence of the reversible binding factor Oligo is the single-stranded deoxyribonucleic acid shown in SEQ ID NO.4; wherein the 3' end of the sequence is blocked with SpacerC3.

[0022] Furthermore, the final concentration of menotriose in the fully premixed system for quantitative real-time PCR is 20-80 mM; preferably, the final concentration of menotriose in the fully premixed system for quantitative real-time PCR is 40 mM.

[0023] Furthermore, the final concentration of the reversible binding factor Oligo in the fully premixed system for quantitative real-time PCR is 10-40 nM; preferably, the final concentration of the reversible binding factor Oligo in the fully premixed system for quantitative real-time PCR is 20 nM.

[0024] Furthermore, the fluorescence quantitative PCR is fluorescence quantitative PCR using DNA as a template (qPCR) or one-step reverse transcription fluorescence quantitative PCR using RNA as a template (qRT-PCR).

[0025] Furthermore, the fully premixed system for quantitative real-time PCR also includes buffer components, DNA polymerase components, and / or primer components for quantitative real-time PCR using DNA as a template, or includes buffer components, DNA polymerase components, reverse transcriptase components, and / or primer components for one-step reverse transcription quantitative real-time PCR using RNA as a template.

[0026] Furthermore, the buffer components include a buffer system, magnesium ions, and / or dNTPs, etc.

[0027] Furthermore, the DNA polymerase component includes DNA polymerase; preferably, the DNA polymerase is Taq DNA polymerase.

[0028] Furthermore, the reverse transcriptase component includes reverse transcriptase; preferably, the reverse transcriptase is... IV Reverse Transcriptase[M-MLV,RNase H-].

[0029] Furthermore, the primer components include an upstream primer, a downstream primer, and a fluorescent probe.

[0030] Furthermore, the storage temperature of the fully premixed fluorescent quantitative PCR system is -20℃ to 37℃ (for example, it can be -20℃, 4℃, 25℃, 37℃, or any range between two values).

[0031] The beneficial effects of this invention are as follows:

[0032] This invention is the first to add menotriose and the reversible binding factor Oligo to the fully premixed system of quantitative real-time PCR, which effectively improves the storage stability of the fully premixed system of quantitative real-time PCR, enhances the amplification performance of the fully premixed system of quantitative real-time PCR, and has a significant synergistic effect on the Ct value and endpoint fluorescence value of quantitative real-time PCR. Attached Figure Description

[0033] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0034] Figure 1 The amplification effect of fully premixed real-time PCR systems (i.e., qPCR fully premixed systems) with different concentrations of melitriose or different concentrations of different reversibly binding Oligo was measured.

[0035] Figure 2The amplification effect of qPCR premixed systems with different concentrations of melitriose after being incubated at 37°C for 7 days was measured.

[0036] Figure 3 The amplification effect of qPCR fully premixed systems with different reversible Oligo binding additives after being incubated at 37°C for 7 days was measured.

[0037] Figure 4 The amplification effect of a fully premixed qPCR system containing 40 mM melitriose and different reversibly binding Oligo was measured after being incubated at 37°C for 1 week.

[0038] Figure 5 The amplification effect of a fully premixed qPCR system containing 40 mM melitriose and different reversibly binding Oligo was measured after being incubated at 4°C for 1 week.

[0039] Figure 6 The amplification effect of a fully premixed qPCR system containing 40 mM melitriose and reversibly binding Oligo1 after being stored at -20°C for 6 months was determined.

[0040] Figure 7 The amplification effect of a fully premixed reverse transcription real-time PCR system (qRT-PCR fully premixed system) with 40 mM melitriose and different reversibly binding Oligo was measured after being incubated at 37°C for 1 week.

[0041] Figure 8 The amplification effect of a fully premixed qRT-PCR system containing 40 mM melitriose and different reversibly binding Oligo was measured after being incubated at 4°C for 1 week.

[0042] Figure 9 The amplification effect of a fully premixed qRT-PCR system containing 40 mM melitriose and reversibly binding Oligo1 after being stored at -20°C for 6 months was determined. Detailed Implementation

[0043] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1: A fully premixed system for quantitative real-time PCR (qPCR) was prepared by adding one or both of melatonin and the reversible binding factor Oligo for DNA-based quantitative PCR amplification.

[0045] The reversible binding factor Oligo binds to the core sequence of Taq enzyme at 37°C and below, forming a specific spatial structure that binds to Taq enzyme and inhibits its activity. When extended to the Taq enzyme's operating temperature of 60°C or 72°C, the spatial structure of the Oligo core, which binds to the Taq enzyme core sequence, is disrupted and dissociates from the Taq enzyme, thus relieving the inhibitory effect on enzyme activity.

[0046] The fully premixed system for real-time PCR used in this embodiment includes the following basic components: 5U Taq DNA polymerase, 40mM Tris-Cl (pH=8.5), 62.5mM KCl, 6mM (NH4)2SO4, 5mM MgCl2, 0.125mM dNTPs, 250nM upstream primer for detecting African swine fever (5'-TTAAAACCCCCGATGATCC-3', as shown in SEQ ID NO.1), 250nM downstream primer (5'-GGTTGGTATTCCTCCCG-3', as shown in SEQ ID NO.2), and 250nM CY5-labeled detection probe (5'CY5-TGCGATGATGATTACCTTT-3'BHQ3, sequence shown in SEQ ID NO.3).

[0047] I. Effects of adding melatonin or the reversible binding factor Oligo to a fully premixed system for real-time PCR on PCR

[0048] 1. Based on the above-mentioned basic components in this embodiment, 20mM, 40mM, or 80mM melitriose, or 10nM, 20nM, or 40nM of reversible binding factor Oligo 1 (5'-GAATCCTCGCGCACAATTCAGCATGCGCCTAGGCCTTTTCA-3'-SpacerC3, sequence as shown in SEQ ID NO.4), Oligo 2 (5'-CAATGGTGCCCGAGAATTGACGATCGCGGTACCGGTTTTGA-3'-SpacerC3, sequence as shown in SEQ ID NO.5), or Oligo 3 (5'-GAACAAGAATGGTGCCCGAGAATTGACGATCGCGGTACCGGTTTTGATTGTTC-3'-SpacerC3, sequence as shown in SEQ ID NO.4) are added respectively. Prepare a fully premixed PCR system (i.e., qPCR fully premixed system) by adding either menotriose or no menotriose and the reversible binding factor Oligo (i.e., no additives), and immediately perform the qPCR reaction after preparation. Compare the Ct values ​​to detect whether different concentrations of menotriose and different concentrations of the three reversible binding factors Oligo inhibit PCR.

[0049] The results are as follows Figure 1 As shown, 20 mM or 40 mM menotriose had no significant effect on the Ct value, while the Ct value increased slightly (delayed) after the addition of 80 mM menotriose. The three reversible binding factors, Oligo, had no significant effect on the Ct value at concentrations of 10 nM, 20 nM, or 40 nM.

[0050] 2. Based on the basic components described in this embodiment, 20 mM melitoxylate, 40 mM melitoxylate, 80 mM melitoxylate, or no melitoxylate (i.e., no additive) were added to prepare a fully premixed qPCR system. The fully premixed qPCR system was stored at 37°C for 7 days before the qPCR reaction. A fully premixed qPCR system without melitoxylate was prepared as a control before the PCR reaction.

[0051] Add 20 μl of the premixed real-time PCR mixture to each PCR well, then add 5 μL of African swine fever positive template to complete the PCR reaction preparation. Perform the PCR reaction using a two-step method: pre-denaturation at 95℃ for 5 minutes; followed by 45 cycles of 95℃ for 15 seconds and 60℃ for 45 seconds.

[0052] The results are as follows Figure 2 As shown, the amplification performance of the fully premixed real-time PCR systems with different concentrations of melatonin added after being placed at 37℃ for 7 days was slightly increased compared to the fully premixed real-time PCR system without melatonin added. The 40mM system showed the best performance, but it did not reach the expected level.

[0053] 3. Based on the basic components described in this embodiment, add 20 nM of the reversible binding factor Oligo1, Oligo2, or Oligo3, or do not add the reversible binding factor Oligo (i.e., no additive), to prepare a fully premixed system for quantitative real-time PCR. The fully premixed system for quantitative real-time PCR is stored at 37°C for 7 days before the quantitative real-time PCR reaction. A fully premixed system for quantitative real-time PCR without the added reversible binding factor Oligo is prepared as a control before the PCR reaction.

[0054] Add 20 μl of the premixed real-time PCR mixture to each PCR well, then add 5 μL of African swine fever positive template to complete the PCR reaction preparation. Perform the PCR reaction using a two-step method: pre-denaturation at 95℃ for 5 minutes; followed by 45 cycles of 95℃ for 15 seconds and 60℃ for 45 seconds.

[0055] The results are as follows Figure 3As shown, the amplification performance of the fully premixed real-time PCR system with added Oligo1, Oligo2, or Oligo3 after being stored at 37°C for 7 days was slightly increased compared to the fully premixed real-time PCR system without added reversible binding factor Oligo, but still did not meet expectations.

[0056] II. Effects of simultaneous addition of melatonin and reversible binding factor Oligo to a fully premixed system for real-time quantitative PCR on PCR

[0057] In this embodiment, based on the above-mentioned basic components, 40 mM menotriose and 20 nM reversible binding factor Oligo 1, Oligo 2, or Oligo 3 are added, or neither menotriose nor reversible binding factor Oligo is added (i.e., no additives) to prepare a fully premixed system for quantitative real-time PCR. The fully premixed system for quantitative real-time PCR is stored at 37°C and 4°C for 7 days before the quantitative real-time PCR reaction. A freshly prepared fully premixed system without additives is used as a control before the PCR reaction. 20 μl of the fully premixed system for quantitative real-time PCR is added to the PCR well, followed by 5 μL of African swine fever positive template to complete the PCR reaction solution. A two-step PCR reaction is performed: pre-denaturation at 95°C for 5 minutes; followed by 95°C for 15 seconds and 60°C for 45 seconds, for 45 cycles.

[0058] The results are as follows Figure 4 and 5 As shown, adding 40 mM melitriose and 20 nM reversible binding factor Oligo 1 and storing at 37℃ and 4°C for 7 days significantly improved the storage stability of the fully premixed system for quantitative PCR. However, the combination of adding melitriose with Oligo 2 or Oligo 3 did not significantly improve the storage stability of the fully premixed system.

[0059] In this embodiment, based on the above-mentioned basic components, 40 mM menotriose and 20 nM reversible binding factor Oligo1 were added, or neither menotriose nor Oligo1 were added (i.e., no additives) to prepare a fully premixed system for quantitative real-time PCR. The fully premixed system was stored at -20℃ for 6 months before quantitative real-time PCR. A freshly prepared, additive-free fully premixed system was used as a control before the PCR reaction. 20 μl of the fully premixed system was added to each PCR well, followed by 5 μL of African swine fever positive template to complete the PCR reaction. A two-step PCR reaction was performed: pre-denaturation at 95℃ for 5 minutes; followed by 45 cycles of 95℃ for 15 seconds and 60℃ for 45 seconds.

[0060] The results are as follows Figure 6As shown, the performance of the fully premixed real-time PCR system with added mesotriose and Oligo 1 after storage at -20℃ for 6 months is significantly better than that of the fully premixed real-time PCR system without additives.

[0061] Example 2: A fully premixed system for quantitative real-time PCR was prepared by adding one or both of melatonin and the reversible binding factor Oligo to RNA-based reverse transcription quantitative real-time PCR (qRT-PCR) amplification.

[0062] This example describes the addition of menotriose and reversible binding factors Oligo 1, Oligo 2, and Oligo 3 to a fully premixed system containing reverse transcriptase for RNA-based quantitative PCR amplification. It is speculated that the reversible binding factor Oligo, in addition to binding to Taq DNA polymerase, may also bind to reverse transcriptase at 37°C. However, at an operating temperature of 45-55°C, the fragile spatial structure of the core sequence binding to reverse transcriptase is disrupted, causing Oligo to dissociate from the reverse transcriptase and thus relieving its inhibitory effect on enzyme activity.

[0063] The fully premixed system for real-time PCR used in this embodiment includes the following basic components: 5U Taq DNA polymerase, 2U reverse transcriptase (…). IV Reverse Transcriptase [M-MLV, RNase H-] (TransGen, AW101) , 12U RNase inhibitor, 25mM Tris-Cl (pH=8.5), 62.5mM KCl, 5mM (NH4)2SO4, 4.375mM MgCl2, 0.125mM dNTPs, 250nM upstream primer for porcine reproductive and respiratory syndrome virus (5'-CTCGTCCTGGTACGTCAG-3', as shown in SEQ ID NO.7), 250nM downstream primer (5'-CACCTCTGAGTGAGCC-3', as shown in SEQ ID NO.8), and 250nM Texas Red-labeled detection probe (5'Texas Red-CACTCAAAGGTGTCATCAG-3'BHQ2, sequence shown in SEQ ID NO.9).

[0064] 1. Based on the basic components described in this embodiment, add 40 mM menotriose and 20 nM reverse binding factor Oligo 1, Oligo 2, or Oligo 3, or do not add menotriose and reversible binding factor Oligo (i.e., no additives) to prepare a fully premixed reverse transcription quantitative PCR system (i.e., qRT-PCR fully premixed system). Store the fully premixed reverse transcription quantitative PCR system at 37°C or 4°C for 7 days. Prepare a fresh, additive-free fully premixed qRT-PCR system as a control before the reaction. Add 20 μl of the fully premixed qRT-PCR system to the PCR wells, then add 5 μL of template containing porcine reproductive and respiratory syndrome virus (PRRSV) RNA to complete the PCR reaction. The qRT-PCR reaction program is: 50°C for 5 min; 95°C for 30 s; 95°C for 5 s; 60°C for 30 s, 45 cycles.

[0065] The results are as follows Figure 7 and 8 As shown, the addition of mesotriose and the reversible binding factor Oligo 1 significantly improved the amplification performance of the fully premixed system after storage at 37°C and 4°C for 7 days.

[0066] 2. Based on the basic components described in this embodiment, add 40 mM menotriose and 20 nM reverse binding factor Oligo1, or do not add menotriose and reversible binding factor Oligo1 (i.e., no additives) to prepare a fully premixed reverse transcription quantitative PCR system (qRT-PCR fully premixed system). The fully premixed reverse transcription quantitative PCR system is stored at -20℃ for 6 months. Prepare a fresh, additive-free fully premixed PCR system as a control before the reaction. Add 20 μl of the fully premixed reverse transcription quantitative PCR system to the PCR wells, then add 5 μL of template containing porcine reproductive and respiratory syndrome virus (PRRSV) RNA to complete the PCR reaction. The qRT-PCR reaction program is: 50℃ for 5 min; 95℃ for 30 s; 95℃ for 5 s; 60℃ for 30 s, 45 cycles.

[0067] The results are as follows Figure 9 As shown, the amplification performance of the fully premixed reverse transcription quantitative RT-PCR system with added mesotriose and Oligo 1 after storage at -20℃ for 6 months was significantly better than that of the fully premixed reverse transcription quantitative RT-PCR system without additives.

[0068] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. Use of melezitose and a reversible binding factor Oligo in any one of the following: 1) in the preparation of a fluorescent quantitative PCR full premix system; 2) in improving the storage stability and amplification performance of a fluorescent quantitative PCR full premix system. The sequence of the reversible binding factor Oligo is a single-stranded deoxyribonucleic acid as shown in SEQ ID NO. 4; wherein, the 3' end of the sequence is closed with SpacerC3; the final concentration of melezitose in the fluorescent quantitative PCR full premix system is 20-80 mM; the final concentration of the reversible binding factor Oligo in the fluorescent quantitative PCR full premix system is 10-40 nM.

2. Use according to claim 1, characterized in that, the final concentration of melezitose in the fluorescent quantitative PCR full premix system is 40 mM.

3. Use according to claim 1, characterized in that, the final concentration of the reversible binding factor Oligo in the fluorescent quantitative PCR full premix system is 20 nM.

4. Use according to claim 1, characterized in that, The storage temperature of the fluorescent quantitative PCR full premix system is -20℃-37℃.

5. The use according to claim 1, characterized in that, The fluorescent quantitative PCR is a fluorescent quantitative PCR with DNA as a template and / or a one-step reverse transcription fluorescent quantitative PCR with RNA as a template.

6. A fluorescent quantitative PCR total premix system with improved storage stability and amplification performance, characterized in that, The fluorescent quantitative PCR full premix system comprises melezitose and a reversible binding factor Oligo; the sequence of the reversible binding factor Oligo is a single-stranded deoxyribonucleic acid as shown in SEQ ID NO. 4; wherein the 3' end of the sequence is closed with SpacerC3; the final concentration of melezitose in the fluorescent quantitative PCR full premix system is 20-80 mM; the final concentration of the reversible binding factor Oligo in the fluorescent quantitative PCR full premix system is 10-40 nM.

7. The real-time PCR master mix according to claim 6, characterized in that the final concentration of melezitose in the fluorescent quantitative PCR full premix system is 40 mM; the final concentration of the reversible binding factor Oligo in the fluorescent quantitative PCR full premix system is 20 nM.

8. The real-time PCR master mix according to claim 6, characterized in that The fluorescent quantitative PCR full premix system further comprises a Buffer component, a DNA polymerase component, and / or a primer component for a fluorescent quantitative PCR with DNA as a template, or comprises a Buffer component, a DNA polymerase component, a reverse transcriptase component, and / or a primer component for a one-step reverse transcription fluorescent quantitative PCR with RNA as a template.

9. The real-time PCR master mix according to claim 8, characterized in that The Buffer component comprises a buffer system, magnesium ions, and / or dNTPs; the primer component comprises an upstream primer, a downstream primer, and a fluorescent probe.

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