QPCR reaction system, product thereof and application of qPCR reaction system in virus detection

Abstract

The invention provides a qPCR reaction system, a product thereof and application of the qPCR reaction system in virus detection, and relates to the technical field of qPCR. The invention provides a qPCR (quantitative polymerase chain reaction) buffer solution. The qPCR buffer solution comprises the following components: SYBR Green I, a hot start Taq enzyme, Tris, KCl, MgCl2, (NH4) 2SO4, dNTPs (deoxyribonucleoside triphosphates), choline chloride and hexadecyl trimethyl ammonium bromide. When the qPCR buffer solution is used for detection, the repeatability is good, the detection limit is low, the anti-interference capability is high, and the application potential in virus detection is huge.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of qPCR, and relates to a qPCR reaction system, in particular to a qPCR reaction system, a product thereof and application thereof in virus detection. BACKGROUND

[0002] Real-time fluorescence quantitative polymerase chain reaction (qPCR) technology, as a core means for nucleic acid quantitative detection in the field of molecular biology, has been widely used in clinical pathogen screening, gene expression analysis, gene mutation detection, environmental microbial monitoring and other fields due to its advantages of rapidness, sensitivity and real-time monitoring. The technology introduces a fluorescence signal reporter molecule (such as SYBR Green I dye) into the reaction system, so that the fluorescence signal intensity is real-time correlated with the amount of amplification product, and the precise quantification of target nucleic acid is realized by combining with the cycle threshold (Ct value), which greatly improves the accuracy and efficiency of detection compared with the traditional end-point PCR technology.

[0003] The detection performance of qPCR technology is highly dependent on the overall adaptability of the reaction system, in which the buffer components, enzyme activity, fluorescence dye compatibility and concentration ratio of each component jointly determine the amplification efficiency, detection specificity, quantitative accuracy and anti-interference ability. At present, SYBR Green I dye has become one of the most widely used fluorescence reporter molecules in scientific research and clinical detection due to its low cost, simple operation and no need for specific probe design. However, the SYBR Green I-based qPCR system still faces many technical bottlenecks in practical application, which limits its application effect in low-abundance nucleic acid detection, complex matrix sample analysis and other scenarios.

[0004] Firstly, the quantitative accuracy, repeatability and detection limit are difficult to balance. SYBR Green I is a non-specific intercalating dye, which can not only bind to the target amplification product, but also bind to primer dimers and non-specific amplification products to produce background fluorescence, resulting in Ct value deviation and affecting the accuracy of quantitative results. At the same time, in the detection of low-copy target nucleic acid (such as tumor circulating tumor DNA, trace pathogen detection), the existing system often has the problems of insufficient amplification efficiency and low signal-to-noise ratio, and the detection limit is usually only 100 copies / μL, which is difficult to capture extremely low concentration of target molecules, and cannot meet the needs of early diagnosis and high-sensitivity detection in clinic.

[0005] Secondly, the anti-interference ability is weak, and it is difficult to adapt to complex samples. Clinical samples (such as blood, tissue homogenate), environmental samples (such as soil, feces) often contain hemoglobin, humic acid, polysaccharide, lipid and other impurities, which can inhibit the activity of Taq enzyme, destroy the ion balance of the reaction system, and cause amplification failure or result distortion. Although the existing qPCR system can improve the anti-interference ability by adding bovine serum albumin (BSA), surfactant and other components, the effect is limited, and the failure rate of direct amplification of complex samples is still high, often requiring tedious sample purification pretreatment steps, increasing the detection cost and time-consuming.

[0006] Researchers improve the comprehensive performance of the qPCR system by optimizing the performance of the enzyme, adjusting the composition and ratio of the buffer, etc. For example, the application of hot-start Taq enzyme can reduce non-specific amplification at low temperature, betaine can reduce the DNA double-strand melting temperature and improve the amplification effect of high GC template, and Tween-20 and other surfactants can reduce the non-specific adsorption of enzymes and tube wall. However, there is no qPCR reaction system that can simultaneously achieve high quantitative repeatability, low detection limit, strong anti-interference ability, and adapt to various template types. Therefore, it is an urgent technical problem to develop a qPCR reaction system with reasonable component ratio, good synergy of each component, and significantly improved detection effect.

[0007] The insect baculovirus expression vector system uses baculovirus as an exogenous gene vector and insect cells as a host for genome self-amplification and expression of the target protein. The insect baculovirus expression vector needs a plasmid vector and a wild-type baculovirus to integrate into a recombinant baculovirus containing an exogenous gene to realize the expression of the exogenous gene. The key to constructing a recombinant baculovirus is to construct a shuttle transfer vector: inserting the target gene into the multiple cloning site downstream of the ph or p10 promoter. Since the transfer vector contains homologous sequences for recombination with the wild-type baculovirus, the transfer vector and the parent virus DNA will obtain a carrier with an exogenous gene when they are recombined. Researchers often use recombinant baculovirus plasmids as viral gene quality control products to verify the detection effect of the qPCR detection reagent for viruses, and the buffer of the qPCR reaction has a significant impact on the detection effect.

[0008] Chinese patent CN120310887A discloses a qPCR method for detecting the recombination rate of baculovirus plasmid. The extracted baculovirus DNA is used as a template for detection by fluorescence dye qPCR method, and the Ct value of total baculovirus (Cttotal) and the Ct value of recombinant baculovirus (Ctrecombinant) are obtained. The difference ΔCt=Cttotal-Ctrecombinant, and the recombinant proportion%=2 ΔCtX100%. Meanwhile, a new method for detecting the proportion of recombinant bacmids is provided. The total bacmid primer pair Bacmid-F and Bacmid-R can specifically amplify non-recombinant bacmids and recombinant bacmids, and the recombinant bacmid primer pair Tn7L-F and M13-R can only amplify samples containing recombinant bacmids, so that the proportion of recombinant bacmids can be detected by qPCR, and the proportion of recombinant bacmids can be used to guide subsequent experiments; however, the qPCR reaction system in the technology uses commercial reagents, and the repeatability and detection limit of the detection are poor.

[0009] Chinese patent CN114807085A discloses a qPCR reaction system, and components thereof are 1X SYBR Green I, 2.5U hot start Taq enzyme, 40mM Tris, 10mM (NH4)2SO4, 50mM K2SO4, 1% Tween-20, 2.5mM MgSO4, 50mM betaine and 0.35mM dNTPs, pH=9.0; however, the detection accuracy and anti-interference ability of the reaction system are poor. SUMMARY

[0010] The present application provides a qPCR reaction system, products thereof and applications in virus detection. The present application provides a qPCR buffer, and components thereof include SYBR Green I, hot start Taq enzyme, Tris, KCl, MgCl2, (NH4)2SO4, dNTPs, choline chloride and cetyltrimethylammonium bromide. The qPCR buffer has good repeatability, low detection limit and strong anti-interference ability when used for detection, and has great application potential in virus detection.

[0011] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows: In one aspect, the present application provides a qPCR buffer, and components thereof include SYBR Green I, hot start Taq enzyme, Tris, KCl, MgCl2, (NH4)2SO4, dNTPs, choline chloride and cetyltrimethylammonium bromide.

[0012] Preferably, components of the qPCR buffer include 1.6X-2.4X SYBR Green I, 3.0-5.0U hot start Taq enzyme, 56-100mM Tris, 44-96mM KCl, 2.5-3.6mM MgCl2, 4.2-10.5mM (NH4)2SO4, 0.5-1.0mMdNTPs, 68-100mM choline chloride and 0.01%-0.03%cetyltrimethylammonium bromide, and pH is 8.0-9.0.

[0013] Preferably, the qPCR buffer components include 1.8X-2.2X SYBR Green I, 3.5-4.5 U hot start Taq enzyme, 60-90 mM Tris, 60-90 mM KCl, 2.8-3.4 mM MgCl2, 6.0-8.0 mM (NH4)2SO4, 0.6-0.8 mM dNTPs, 80-90 mM choline chloride, and 0.015%-0.025% cetyltrimethylammonium bromide, at a pH of 8.0-9.0.

[0014] In some embodiments, the qPCR buffer components include 2.0X SYBR Green I, 4.0 U hot start Taq enzyme, 80 mM Tris, 70 mM KCl, 3.0 mM MgCl2, 7.0 mM (NH4)2SO4, 0.75 mM dNTPs, 85 mM choline chloride, and 0.02% cetyltrimethylammonium bromide, at a pH of 8.5.

[0015] In some embodiments, the qPCR buffer components include 1.6X SYBR Green I, 5.0 U hot start Taq enzyme, 56 mM Tris, 96 mM KCl, 2.5 mM MgCl2, 10.5 mM (NH4)2SO4, 0.5 mM dNTPs, 100 mM choline chloride, and 0.01% cetyltrimethylammonium bromide, at a pH of 9.0.

[0016] In some embodiments, the qPCR buffer components include 2.4X SYBR Green I, 3.0 U hot start Taq enzyme, 100 mM Tris, 44 mM KCl, 3.6 mM MgCl2, 4.2 mM (NH4)2SO4, 1.0 mM dNTPs, 68 mM choline chloride, and 0.03% cetyltrimethylammonium bromide, at a pH of 8.0.

[0017] In another aspect, the present application provides a qPCR reaction system, which comprises the above-mentioned qPCR buffer, and further comprises a forward primer, a reverse primer, or a template DNA.

[0018] Preferably, 10 μL of the qPCR buffer is contained in each 20 μL qPCR reaction system.

[0019] Preferably, the final concentration of the forward primer or the reverse primer is 0.1-0.5 μM.

[0020] In some embodiments, the final concentration of the forward primer or the reverse primer is 0.25 μM.

[0021] In another aspect, the present application provides a qPCR kit comprising the above-mentioned qPCR buffer or the above-mentioned qPCR reaction system.

[0022] In another aspect, the present application provides the above-mentioned qPCR buffer, the above-mentioned qPCR reaction system or the above-mentioned qPCR kit for use in in vitro detection, which is a non-disease diagnosis or treatment use.

[0023] Preferably, the use comprises detection of viruses.

[0024] Compared with the prior art, the present application has at least the following beneficial effects: The present application provides a qPCR buffer, the components of which include SYBR Green I, hot-start Taq enzyme, Tris, KCl, MgCl2, (NH4)2SO4, dNTPs, choline chloride and cetyltrimethylammonium bromide. The choline chloride and cetyltrimethylammonium bromide in the qPCR buffer have a synergistic effect, making the detection reproducible, low in detection limit and strong in anti-interference ability, and having great application potential in virus detection. DETAILED DESCRIPTION

[0025] Unless otherwise specified, the various raw materials and reagents in the present application are purchased from commercial suppliers, and experiments are performed according to the operating instructions. Unless otherwise specified, the instruments, equipment, devices, etc. used in the present application are conventional instruments, equipment, devices, etc. and experiments are performed according to the operating instructions and the supporting reagents.

[0026] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is further described in detail below in combination with examples. Unless otherwise specified in the examples, the experiments are performed according to the conventional conditions or the conditions recommended by the manufacturers. Unless otherwise specified, all reagents or instruments are conventional products that can be purchased on the market. In order to better illustrate the present application, numerous specific details are given in the specific embodiments below. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application.

[0027] SYBR Green I is a commonly used non-specific double-stranded DNA (dsDNA) intercalating fluorescent dye, which is an asymmetric cyanine compound in nature. The fluorescence signal of the dye in free state is weak, and the fluorescence intensity can be enhanced by 800-1000 times when it is embedded in the small groove region of the dsDNA double helix. The fluorescence signal intensity is positively correlated with the content of dsDNA. In the qPCR system, SYBR Green I does not need to be combined with specific primers or probes, and can be adapted to the amplification and detection of any double-stranded DNA. It has the characteristics of low cost and simple operation, but has the limitation of easy combination with primer dimers and non-specific amplification products, which can cause background fluorescence to rise.

[0028] The cycle threshold (Ct value) is a core parameter for quantifying target nucleic acids in qPCR technology, which refers to the number of cycles experienced when the fluorescence signal intensity reaches the preset threshold during the amplification reaction. Ct value is negatively correlated with the initial copy number of target nucleic acids, i.e. the higher the initial copy number, the smaller the Ct value. By constructing a Ct value-copy number standard curve of known concentration standard, the precise quantification of target nucleic acids in unknown samples can be achieved, which is a key indicator for evaluating the sensitivity, accuracy and repeatability of qPCR detection.

[0029] Hot-start Taq enzyme is a class of Taq DNA polymerase variants modified by chemical modification, antibody modification or ligand modification. Its core feature is that it has no activity at low temperature and can restore enzyme activity at high temperature. Unmodified ordinary Taq enzyme can start DNA amplification when the reaction system is configured at room temperature, which can easily lead to the formation of primer dimers and non-specific amplification products. Hot-start Taq enzyme needs to be activated by high temperature (usually 94-95°C for several minutes), which can effectively inhibit non-specific amplification at low temperature and improve the specificity and amplification efficiency of qPCR reaction. It is one of the core components of high-performance qPCR system.

[0030] Data analysis and statistical analysis were performed using professional data processing software, and single-factor ANOVA test was used for significance analysis. P<0.05 represents significant difference.

[0031] Preparation example: materials and experimental methods The primer design and detection method of this preparation example refers to Chinese patent CN120310887A, which is shown as follows: (1) Total bacmid primer design: The bacmid is derived from AcMNPV virus. According to the partial gene sequence encoding AcMNPV virus obtained from GenBank (KF022001.1), the kanamycin resistance gene encoded by AcMNPV virus was selected to design the total bacmid primer pair, which was used to detect the total bacmid; (2) qPCR detection method: SYBR Green qPCR method was used, and the extracted bacmid DNA was used as a template. Total bacmid primer pairs were used for detection, and the Ct value of the total bacmid was obtained.

[0032] The specific detection method is as follows: 1) Prepare primers according to Table 1. The total bacmid primer pair includes Bacmid-F (SEQ ID No. 1) and Bacmid-R (SEQ ID No. 2). Table 1 Primer explanation

[0033] 2) Prepare the qPCR reaction system according to Table 2. The extracted bacmid DNA is diluted with a diluent. The diluent is RNase-free water. The diluent (RNase-free water) is used as a no-template control (NTC). Each sample is set in duplicate wells.

[0034] Table 2 qPCR reaction system

[0035] 3) Perform qPCR reaction according to the reaction program shown in Table 3. Calculate the Ct value of the sample.

[0036] Table 3 qPCR reaction program

[0037] Example 1: Preparation of qPCR buffer SYBR Green I: purchased from Shanghai Creative Science and Technology Co., Ltd., product number PR01021; Hot-start Taq enzyme: purchased from Beijing Bai'oleibo Science and Technology Co., Ltd., product number QN0871.

[0038] Prepare qPCR buffer: components are 2.0X SYBR Green I, 4.0U hot-start Taq enzyme, 80mM Tris, 70mM KCl, 3.0mM MgCl2, 7.0mM (NH4)2SO4, 0.75mM dNTPs, 85mM choline chloride and 0.02% cetyltrimethylammonium bromide, pH 8.5.

[0039] Example 2: Preparation of qPCR buffer Preparation of qPCR buffer: the components are 1.6X SYBR Green I, 5.0U hot start Taq enzyme, 56mM Tris, 96mM KCl, 2.5mM MgCl2, 10.5mM (NH4)2SO4, 0.5mM dNTPs, 100 mM choline chloride and 0.01% cetyltrimethylammonium bromide, pH 9.0.

[0040] Example 3: Preparation of qPCR buffer Preparation of qPCR buffer: the components are 2.4X SYBR Green I, 3.0U hot start Taq enzyme, 100mM Tris, 44mM KCl, 3.6mM MgCl2, 4.2mM (NH4)2SO4, 1.0mM dNTPs, 68mM choline chloride and 0.03% cetyltrimethylammonium bromide, pH 8.0.

[0041] Comparative Example 1: Preparation of qPCR buffer The difference from Example 1 is only that no choline chloride is added to the qPCR buffer, and the others are the same as Example 1.

[0042] Comparative Example 2: Preparation of qPCR buffer The difference from Example 1 is only that no cetyltrimethylammonium bromide is added to the qPCR buffer, and the others are the same as Example 1.

[0043] Comparative Example 3: Preparation of qPCR buffer The difference from Example 1 is only that neither choline chloride nor cetyltrimethylammonium bromide is added to the qPCR buffer, and the others are the same as Example 1.

[0044] Comparative Example 4: Preparation of qPCR buffer The difference from Example 1 is only that cetyltrimethylammonium bromide is replaced by an equimolar amount of cetyltrimethylammonium chloride, and the others are the same as Example 1.

[0045] Comparative Example 5: Preparation of qPCR buffer The difference from Example 1 is only that the concentration of choline chloride is 120mM, and the others are the same as Example 1.

[0046] Test Example: qPCR detection effect After the plasmid is prepared according to the preparation example, it is gradient diluted with nuclease-free water to obtain standard samples of different concentrations (1 copy / μL-1×10 7 Then, the qPCR buffers prepared in Examples 1-3 and Comparative Examples 1-5 are used to test different samples according to the method of the preparation example.

[0047] (1) repeatability The qPCR detection was performed on different concentrations of standard samples, and 3 replicates were set for each concentration. The Ct detection results are shown in Table 4: Table 4 Detection results of different qPCR buffers

[0048] As can be seen from Table 4, the detection CV values of Examples 1-3 are significantly reduced compared to the comparative examples, and choline chloride and cetyltrimethylammonium bromide have a synergistic effect.

[0049] (2) Detection limit The standard sample was further diluted to 1 copy / μL, 5 copies / μL, 10 copies / μL, and 20 copies / μL and detected, and 10 replicates were detected for each concentration. After detection, the results were summarized and the detection rate of each concentration was calculated. The lowest concentration at which all 10 replicates were detected was the detection limit of the established method, and the number of detection results in 10 replicates is shown in Table 5.

[0050] Table 5 Detection results of different buffers

[0051] As can be seen from Table 5, the detection limit of Examples 1-3 is significantly reduced compared to the comparative examples, and the detection sensitivity is higher.

[0052] (3) Anti-interference verification 100 μL of 1 x 10 4 After mixing 100 μL of 1 x 10

[0053] The simulated samples were divided into 4 groups, and the first and second groups were exogenous interferents: The first group contained cefoxitin sodium for injection (300 μL), florfenicol (30 μL), tilmicosin premix (30 μL), and physiological saline (240 μL); The second group contained dexamethasone acetate (2.5 μL), gentamicin sulfate (30 μL), and physiological saline (567.5 μL); The third and fourth groups were endogenous interferents: The third group was a CHO-S cell genomic DNA sample; The fourth group was a mucin solution (300 μL) and physiological saline (300 μL); In addition, a positive control group and a negative control group were set up. The negative control group was nuclease-free water, and the positive control group was a mixture of 100 μL of 1 x 10 4 copies / μL standard sample and 100 μL of nuclease-free water.

[0054] Table 6 Results of Anti-interference Test

[0055] Note: "Relative deviation" = (average Ct value of the interfering group - average Ct value of the positive control group) / average Ct value of the positive control group × 100%; "-" indicates not detected.

[0056] As can be seen from Table 6, compared with the comparative examples, the relative deviation values ​​of Examples 1-3 were significantly reduced. Except for the third group of interfering substances, in the other three interfering substance experiments: compared with the comparative example 3, the relative deviations of the comparative examples 1 and 2 (corresponding to the addition of choline chloride or hexadecyltrimethylammonium bromide alone) were reduced. Examples 1-3 (with the addition of choline chloride and hexadecyltrimethylammonium bromide at the same time) further reduced the relative deviation on the basis of the comparative examples 1-2. Moreover, the reduction in relative deviation after the combination of choline chloride and hexadecyltrimethylammonium bromide was greater than the sum of the effects of choline chloride or hexadecyltrimethylammonium bromide alone, indicating that choline chloride and hexadecyltrimethylammonium bromide have a synergistic effect in anti-interference (reducing relative deviation).

[0057] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A qPCR buffer, characterized in that, The components include SYBR Green I, hot-start Taq enzyme, Tris, KCl, MgCl2, (NH4)2SO4, dNTPs, choline chloride, and hexadecyltrimethylammonium bromide.

2. The qPCR buffer according to claim 1, characterized in that, The components include 1.6X-2.4X SYBR GreenI, 3.0-5.0U hot-start Taq enzyme, 56-100mM Tris, 44-96mM KCl, 2.5-3.6mM MgCl2, 4.2-10.5mM (NH4)2SO4, 0.5-1.0mM dNTPs, 68-100mM choline chloride, and 0.01%-0.03% cetyltrimethylammonium bromide, with a pH of 8.0-9.

0.

3. The qPCR buffer according to claim 2, characterized in that, The components include 1.8X-2.2X SYBR GreenI, 3.5-4.5U hot-start Taq enzyme, 60-90mM Tris, 60-90mM KCl, 2.8-3.4mM MgCl2, 6.0-8.0mM (NH4)2SO4, 0.6-0.8mM dNTPs, 80-90mM choline chloride, and 0.015%-0.025% cetyltrimethylammonium bromide, with a pH of 8.0-9.

0.

4. The qPCR buffer according to claim 3, characterized in that, The components include 2.0X SYBR Green I, 4.0U hot-start Taq enzyme, 80mM Tris, 70mM KCl, 3.0mM MgCl2, 7.0mM (NH4)2SO4, 0.75mM dNTPs, 85mM choline chloride and 0.02% cetyltrimethylammonium bromide, with a pH of 8.

5.

5. The qPCR buffer according to claim 1 or 2, characterized in that, The components include 1.6X SYBR Green I, 5.0U hot-start Taq enzyme, 56mM Tris, 96mM KCl, 2.5mM MgCl2, 10.5mM (NH4)2SO4, 0.5mM dNTPs, 100 mM choline chloride and 0.01% cetyltrimethylammonium bromide, with a pH of 9.

0.

6. The qPCR buffer according to claim 1 or 2, characterized in that, The components include 2.4X SYBR Green I, 3.0U hot-start Taq enzyme, 100mM Tris, 44mM KCl, 3.6mM MgCl2, 4.2mM (NH4)2SO4, 1.0mM dNTPs, 68mM choline chloride and 0.03% cetyltrimethylammonium bromide, with a pH of 8.

0.

7. A qPCR reaction system, characterized in that, The PCR reaction system includes the qPCR buffer according to any one of claims 1-6, and further includes forward primers, reverse primers, or template DNA.

8. The qPCR reaction system according to claim 7, characterized in that, Each 20 μL qPCR reaction system contains 10 μL qPCR buffer.

9. A qPCR kit, characterized in that, The qPCR kit comprises the qPCR buffer according to any one of claims 1-6 or the qPCR reaction system according to any one of claims 7-8.

10. The use of the qPCR buffer according to any one of claims 1-6, the qPCR reaction system according to any one of claims 7-8, or the qPCR kit according to claim 9 in in vitro detection, characterized in that, The application is not a disease diagnosis or treatment application; the application includes virus detection.

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