Reaction system, kit and detection method applied to RNA amplification of complex whole blood sample

Abstract

The invention belongs to the technical field of molecular biological detection, and discloses a reaction system, a kit and a detection method applied to RNA amplification of a complex whole blood sample. The reaction system comprises a buffer component and an enzyme protection component; the buffer component consists of Tris and Bicine and is used for cooperatively maintaining the stability of the pH value of a reaction system in the RNA amplification process of a complex whole blood sample; and the enzyme protection component is composed of AEBSF and dextran sulfate. Through collaborative innovation of a double-buffer system and an active enzyme protective agent, the integrated challenges that the pH is easy to drift, the template compatibility is poor and the enzyme activity is easy to damage when a complex whole blood sample is faced in the prior art are effectively overcome, and a key technical support is provided for rapid, stable and reliable bedside molecular diagnosis.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and in particular to a reaction system, kit, and detection method for RNA amplification in complex whole blood samples. Background Technology

[0002] Reverse transcription polymerase chain reaction (RT-PCR) and other nucleic acid amplification technologies are routine methods for gene detection. However, when these technologies are directly applied to complex whole blood samples, their reliability faces multiple severe challenges from the sample matrix, reaction kinetics, and the target itself. These samples are extremely complex, containing not only common PCR inhibitors such as hemoglobin, immunoglobulins, and lactoferrin, but also unique interfering substances introduced by systemic inflammatory responses or treatments, such as elastase released by neutrophils and antibiotics (e.g., gentamicin), collectively forming an extreme environment of "multiple inhibitors." These substances not only disrupt the chemical microenvironment of the amplification reaction but also directly damage enzyme activity and the integrity of the target nucleic acid, often leading to insufficient detection sensitivity, poor repeatability, and even false negative results.

[0003] Existing technologies face multi-dimensional limitations in addressing this complex scenario, and various optimization strategies are difficult to coordinate effectively. Firstly, regarding pH dynamic stability, traditional amplification reactions widely rely on buffer systems such as Tris, but these systems have high temperature coefficients, causing significant pH shifts as the reaction temperature increases. Furthermore, endogenous substances in complex whole blood samples (such as lactic acid and urea) can further perturb pH through chemical reactions. + The concentration, combined with the temperature effect, causes the system pH to deviate significantly from the optimal activity range of reverse transcriptase and DNA polymerase. Traditional ionic additives (such as ammonium sulfate and potassium chloride) have limited buffering capacity against this synergistic perturbation.

[0004] Secondly, regarding the protection of key enzyme activities, conventional stabilizers (such as trehalose and DTT) mainly maintain the structural stability of enzyme proteins, but cannot provide active protection against the threat of degradation by proteases specifically present in the sample (such as elastase). A more prominent challenge lies in the severe lack of compatibility with complex RNA templates. The quality of RNA extracted from complex whole blood is questionable: on the one hand, residual inhibitors (such as heparin and high salt ions) severely interfere with the kinetics of primer annealing and extension; on the other hand, abundant RNases and inflammation-related proteases in the sample may cause degradation or chemical modification of the target RNA, impairing its integrity. Traditional primer design is usually optimized for pure, intact RNA templates. When faced with such "challenging templates" that may be broken, truncated, or contain non-specifically binding inhibitors, their annealing specificity and extension efficiency decrease significantly, easily leading to primer dimerization, non-specific amplification, or amplification failure. This has become one of the core bottlenecks restricting detection sensitivity.

[0005] The three major problems mentioned above (dynamic pH drift, passive loss of enzyme activity, and primer incompatibility with complex templates) do not exist in isolation, but rather intertwine and exacerbate each other in complex whole blood samples. For example, suboptimal pH further reduces the polymerase's ability to extend damaged templates; while mismatched primer extension failures increase reaction byproducts and alter the local chemical environment. Existing technical solutions often address single problems with piecemeal optimizations. Simply stacking different additives may lead to unpredictable antagonistic effects among components in complex matrices (such as certain buffer components chelating key Mg). 2+ However, changes in ionic strength affect primer annealing, leading to a decrease in overall performance instead of an increase. Summary of the Invention

[0006] In view of the above-mentioned problems, this invention provides a reaction system, kit, and detection method for RNA amplification in complex whole blood samples. Through the synergistic innovation of a double-buffered system and an active enzyme protectant, this invention effectively overcomes the integrated challenges of existing technologies when dealing with complex whole blood samples, such as easy pH drift, poor template compatibility, and easily impaired enzyme activity. This provides key technical support for achieving rapid, stable, and reliable point-of-care molecular diagnostics.

[0007] To address the aforementioned problems, this invention provides a reaction system for RNA amplification in complex whole blood samples, the reaction system comprising a buffer component and an enzyme protection component; The buffer component consists of Tris (tris(hydroxymethyl)aminomethane) and Bicine (N,N-bis(2-hydroxyethyl)glycine), which are used to synergistically maintain the pH stability of the reaction system during RNA amplification of complex whole blood samples. The enzyme protection component consists of AEBSF and dextran sulfate.

[0008] In this invention, the bicine in the dual buffer system composed of Tris and bicine has a lower pKa temperature coefficient, which can effectively counteract the negative pH drift caused by the inherent properties of Tris during the transition from low-temperature storage to high-temperature reaction. Simultaneously, this dual buffer system effectively counteracts the release or conversion of H+ by endogenous inhibitors such as hemoglobin, urea, and lactic acid in complex whole blood samples. + With a stronger buffer capacity, it can synergistically overcome the dual effects of "temperature drift" and "sample chemical interference", and dynamically stabilize the pH of the reaction system within the optimal activity range of reverse transcriptase and DNA polymerase, thus fundamentally ensuring the stability of amplification efficiency.

[0009] In this invention, AEBSF (serine protease inhibitor) is used instead of conventional stabilizers such as trehalose and DTT. This actively inhibits the activity of proteases (such as neutrophil elastase) specific to complex whole blood samples at the source, preventing their degradation of reverse transcriptase and DNA polymerase, and providing a "clean" enzyme working environment for the reaction. Simultaneously, the synergistically added dextran sulfate not only further stabilizes the enzyme protein but also improves the local concentration and efficiency of the amplification system.

[0010] An exemplary reaction system for RNA amplification in complex whole blood samples includes a buffer component, an enzyme protection component, and a primer and probe set targeting the STOM gene, ABHD14B gene, and FLT3LG gene. The buffer component consists of Tris (tris(hydroxymethyl)aminomethane) and Bicine (N,N-bis(2-hydroxyethyl)glycine), which are used to synergistically maintain the pH stability of the reaction system during RNA amplification of complex whole blood samples. The enzyme protection component consists of AEBSF and dextran sulfate.

[0011] In the above example, the bicine in the Tris-Bicine dual buffer system has a lower pKa temperature coefficient, effectively offsetting the negative pH drift caused by the inherent properties of Tris during the transition from low-temperature storage to high-temperature reaction. Simultaneously, this dual buffer system effectively counteracts the release or conversion of H+ by endogenous inhibitors such as hemoglobin, urea, and lactic acid in complex whole blood samples. + It has a stronger buffer capacity, which can overcome the dual effects of "temperature drift" and "sample chemical interference" and keep the pH of the reaction system dynamically stable within the optimal activity range of reverse transcriptase and DNA polymerase, thus fundamentally ensuring the stability of amplification efficiency and being compatible with complex RNA templates.

[0012] An exemplary reaction system for RNA amplification in complex whole blood samples includes a buffer component, an enzyme protection component, and a primer and probe set targeting the STOM gene, ABHD14B gene, and FLT3LG gene. The buffer component consists of Tris (tris(hydroxymethyl)aminomethane) and Bicine (N,N-bis(2-hydroxyethyl)glycine), which are used to synergistically maintain the pH stability of the reaction system during RNA amplification of complex whole blood samples. The enzyme protection component consists of AEBSF and dextran sulfate.

[0013] The primer and probe set consists of specific primers and probes for the STOM, ABHD14B, and FLT3LG genes in whole blood samples from patients with bloodstream infections.

[0014] In the above examples, the upstream primer, downstream primer, and probe for the STOM gene are shown in SEQ ID NO: 1-3, respectively; the upstream primer, downstream primer, and probe for the ABHD14B gene are shown in SEQ ID NO: 4-6, respectively; and the upstream primer, downstream primer, and probe for the FLT3LG gene are shown in SEQ ID NO: 7-9, respectively.

[0015] The above examples demonstrate multiple primer and probe sets targeting human genes (such as STOM, ABHD14B, and FLT3LG genes) in whole blood samples from patients with bloodstream infections. These primers and probes are specially designed and screened, exhibiting higher specificity and annealing stability. Even in cases of partial degradation of sample RNA or interference from residual inhibitors, they can still effectively bind to the target sequence, significantly reducing primer dimer formation and non-specific amplification. This improves compatibility and detection success rate for complex, low-quality RNA templates in whole blood samples, solving the problem of drastically decreased sensitivity of traditional primers in challenging samples.

[0016] Preferably, in the buffer component, the final concentration of Tris is 10-30 mM and the final concentration of Bicine is 30-50 mM.

[0017] Preferably, in the buffer components, the final concentration of Tris is 15-20 mM and the final concentration of Bicine is 30-45 mM.

[0018] Preferably, in the buffer component, the molar ratio of Tris to Bicine is 1:(1.5-3).

[0019] Preferably, the reaction system further includes 4-8 mM of magnesium salt.

[0020] It should be noted that Bicine reacts with Mg, a key component of PCR, during the PCR reaction. 2+ Chelation, reducing Mg 2+ Concentration; This invention compensates for the chelated Mg by using a high concentration of magnesium salts. 2+ .

[0021] Preferably, in the enzyme-protected component, the final concentration of AEBSF is 0.1-1.0 mM, and the final mass-volume concentration of dextran sulfate is 0.01%-0.1%.

[0022] Preferably, the reaction system further includes a mixture of dNTPs, reverse transcriptase, thermostable DNA polymerase, and nucleic acid template.

[0023] Preferably, the dNTPs mixture comprises equal concentrations of dATP, dTTP, dCTP, and dGTP, each at a concentration of 0.2-0.5 mM.

[0024] For example, the concentration of the dNTPs mixture is 0.3 mM each of dATP, dTTP, dCTP and dGTP, the concentration of reverse transcriptase is 1 mM, the concentration of thermostable DNA polymerase is 1 mM, and the concentration of nucleic acid template is 20 mM.

[0025] Preferably, the pH value of the reaction system at 25°C is 8.4-8.6.

[0026] Based on the same inventive concept, the present invention also provides a kit for simultaneously detecting the STOM gene, ABHD14B gene and FLT3LG gene, including the reaction system for RNA amplification applied to complex whole blood samples as described above.

[0027] Preferably, the kit further includes negative and positive controls.

[0028] Preferably, the kit further includes a nucleic acid release reagent, a nucleic acid extraction reagent, and an impurity washing and purification reagent.

[0029] Preferably, the kit also includes specific primers and probes for the STOM gene, ABHD14B gene, and FLT3LG gene.

[0030] In the above examples, the upstream primer, downstream primer, and probe for the STOM gene are shown in SEQ ID NO: 1-3, respectively; the upstream primer, downstream primer, and probe for the ABHD14B gene are shown in SEQ ID NO: 4-6, respectively; and the upstream primer, downstream primer, and probe for the FLT3LG gene are shown in SEQ ID NO: 7-9, respectively.

[0031] Based on the same inventive concept, the present invention also provides a method for detecting multiple genes for non-diagnostic purposes, using any of the above-described reaction systems for RNA amplification of complex whole blood samples or the above-described kits to perform reverse transcription and real-time fluorescent PCR amplification of RNA from the whole blood sample to be tested; wherein, the multiple genes are STOM gene, ABHD14B and FLT3LG gene.

[0032] In this article, the term "non-diagnostic purpose" refers to something not intended to obtain information about whether an individual has a disease such as a bloodstream infection. For example, the presence of the aforementioned target gene expression can be detected in a test culture (e.g., blood).

[0033] Preferably, the RNA extraction method for complex whole blood samples is extraction using magnetic beads (a commonly used RNA extraction method in the art).

[0034] An exemplary method for RNA extraction from complex whole blood samples includes the following steps: (1) Sample pretreatment and lysis release: Take 100-200 μL of complex whole blood sample and mix it with lysis buffer containing protein denaturant (such as guanidine isothiocyanate) and surfactant at a volume ratio of 1:3 to 1:5, and vortex thoroughly. Let it stand at room temperature (15-25℃) for 5-10 minutes to completely destroy the cell membrane and nuclear membrane and denature the proteins, thereby releasing the total nucleic acid (including DNA and RNA) in the cells into the lysis buffer; (2) Specific binding of nucleic acids to magnetic beads: Add 20-50 μL of a suspension of magnetic nanospheres (magnetic beads) modified with silanol or carboxyl groups, and a high-concentration alcohol solution (such as isopropanol, final concentration of about 20-40%) to the lysis mixture. Invert and mix at room temperature for 10-15 minutes. During this process, the high salt and alcohol environment of the solution causes the nucleic acids to expose their phosphate backbone due to dehydration, and they are specifically adsorbed onto the surface of the magnetic beads through hydrogen bonds and cation bridges, while denatured proteins and other impurities remain in the solution; (3) Multi-step washing and purification of impurities: The magnetic bead-nucleic acid complex is immobilized using an external magnetic field, and the supernatant is discarded. Two to three washes are performed sequentially: First wash: a washing buffer containing a high concentration of dissociative salt (such as guanidine hydrochloride) and alcohol is used to remove residual protein and lipid impurities; Second wash: a 70-80% ethanol solution is used to thoroughly remove salt ions and residual organic solvents. Each time, the magnetic beads should be fully resuspended in the washing buffer and allowed to stand for about 30 seconds before being separated by a magnetic field to ensure purification effect.

[0035] (4) Elution and preservation of nucleic acids: After washing, allow the magnetic bead complex to stand at room temperature for 3-5 minutes with the cap open to allow residual ethanol to evaporate completely. Add 50-100 μL of nuclease-free water or low-concentration Tris-EDTA buffer (pH 8.0-8.5) and incubate at 55-65℃ for 5-10 minutes with intermittent vortexing. Under these conditions, the low ionic strength of the elution buffer disrupts the hydrogen bonds between the nucleic acids and the magnetic beads, allowing high-purity nucleic acids to dissolve in the elution buffer. Finally, collect the supernatant containing nucleic acids under the action of a magnetic field and immediately place it in an ultra-low temperature environment of -70℃ or below for long-term storage to prevent nucleic acid (especially RNA) degradation.

[0036] Preferably, the reverse transcription reaction temperature is 55℃ and the time is 15 min; the real-time fluorescence PCR amplification reaction includes 95℃ pre-denaturation for 5 min, 95℃ denaturation for 1 min, 65℃ annealing and extension for 40 s.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention employs a dual buffer system composed of Tris and Bicine, creatively replacing the traditional "single Tris buffer + ammonium / potassium salt" approach. Bicine has a lower pKa temperature coefficient, effectively offsetting the negative pH shift caused by the inherent properties of Tris during the transition from low-temperature storage to high-temperature reaction. Simultaneously, this dual buffer system effectively counteracts the release or conversion of H+ by endogenous inhibitors such as hemoglobin, urea, and lactic acid in complex whole blood samples. + With a stronger buffer capacity, it can overcome the dual effects of "temperature drift" and "sample chemical interference" and keep the pH of the reaction system dynamically stable within the optimal activity range of reverse transcriptase and DNA polymerase, thus fundamentally ensuring the stability of amplification efficiency. (2) This invention has specially designed multiple sets of primers and probes targeting STOM-based, ABHD14B, and FLT3LG genes. These primers and probes have been specially designed and screened, and have higher specificity and annealing stability. Even when the sample RNA is partially degraded or interfered with by residual inhibitors, they can still effectively bind to the target sequence, significantly reduce primer dimer formation and non-specific amplification, thereby improving the compatibility and detection success rate of complex, low-quality RNA templates from complex whole blood sources, and solving the problem of the sharp decrease in sensitivity of traditional primers in challenging samples; (3) This invention uses AEBSF (serine protease inhibitor) instead of conventional stabilizers such as trehalose and DTT, which can actively inhibit the activity of proteases (such as neutrophil elastase) specific to complex whole blood samples from the source, preventing their degradation of reverse transcriptase and DNA polymerase, and providing a "clean" enzyme working environment for the reaction. Simultaneously, the synergistically added dextran sulfate not only further stabilizes the enzyme protein but also improves the local concentration and efficiency of the amplification system. Regarding the Bicine chelation of Mg in this invention... 2+ Due to its characteristics, this invention provides optimized Mg... 2+ The concentration range is compensated to ensure the overall balance and efficiency of each component under synergistic effect. Attached Figure Description

[0038] Figure 1 The amplification curves of the STOM gene obtained by RT-PCR from complex whole blood sample 1 in Example 1 and Comparative Example 1 of this invention are shown. Figure 2 The amplification curves of the ABHD14B gene obtained by RT-PCR from complex whole blood sample 1 in Example 1 and Comparative Example 1 of this invention are shown. Figure 3 The amplification curves of the FLT3LG gene obtained by RT-PCR from complex whole blood sample 1 in Example 1 and Comparative Example 1 of this invention are shown. Figure 4The amplification curves of the STOM gene obtained by RT-PCR from complex whole blood sample 2 in Example 1 and Comparative Example 2 of this invention are shown. Figure 5 The amplification curves of the ABHD14B gene obtained by RT-PCR from complex whole blood sample 2 in Example 1 and Comparative Example 2 of this invention are shown. Figure 6 This is an amplification curve of the FLT3LG gene obtained by RT-PCR from complex whole blood sample 2 in Example 1 and Comparative Example 2 of the present invention. Figure 7 The amplification curves of the STOM gene obtained by RT-PCR of complex whole blood samples 3 (3 concentrations) in Example 1 and Comparative Example 3 of this invention are shown. Figure 8 The amplification curves of the ABHD14B gene obtained by RT-PCR of complex whole blood samples 3 (3 concentrations) in Example 1 and Comparative Example 3 of this invention are shown. Figure 9 The amplification curves of the FLT3LG gene obtained by RT-PCR from complex whole blood samples 3 (3 concentrations) in Example 1 and Comparative Example 3 of this invention are shown. Detailed Implementation

[0039] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art; unless otherwise specified, the raw materials and reagents involved herein can be purchased commercially or obtained by known methods.

[0040] To address the technical problems mentioned in the background section, this invention provides a reaction system, reagent kit, and detection method for RNA amplification in complex whole blood samples. Through the synergistic innovation of a double-buffered system and an active enzyme protectant, this invention effectively overcomes the integrated challenges of existing technologies, such as pH drift, poor template compatibility, and easily impaired enzyme activity, when dealing with complex whole blood samples. This provides key technical support for achieving rapid, stable, and reliable point-of-care molecular diagnostics.

[0041] The following examples and comparative models further illustrate this point.

[0042] Example 1 A method for detecting multiple genes (STOM gene, ABHD14B gene, and FLT3LG gene) for non-diagnostic purposes, comprising the following steps: (1) Nucleic acid extraction: The complex whole blood sample (whole blood sample from patients with bloodstream infection) was extracted using the magnetic bead method. The main steps included sample nucleic acid release, nucleic acid binding to magnetic beads, washing and purification of impurities such as proteins and polysaccharides, and nucleic acid elution (see the steps of the RNA extraction method for complex whole blood samples in the previous example of this application). The eluted nucleic acid was stored at -70°C or below.

[0043] (2) The reaction system for RNA amplification in complex whole blood samples (whole blood samples from patients with bloodstream infections) includes Tris-HCl (20mM), Bicine (30mM), AEBSF (0.1mM), dextran sulfate (0.01% (w / v)), MgCl2 (4mM), a mixture of dNTPs (equal concentrations of dATP, dTTP, dCTP and dGTP, each at 0.3mM), reverse transcriptase (mutant M-MLV reverse transcriptase (Hifair III Reverse Transcriptase), 1mM), thermostable DNA polymerase (HotStart Taq DNA Polymerase, 1mM), and nucleic acid template. STOM gene The primer and probe sets for the FLT3LG gene and the FLT3LG gene (each primer / probe concentration is 50 μmol / L) are shown in Table 1 below; among them, The molar ratio was 1:1.5; the pH of the reaction system (at 25°C) was 8.3, and the pH value stabilized at the optimal range of enzyme activity between 8.3 and 8.8 at the final reaction temperature.

[0044] (3) The nucleic acid extracted in step (1) is subjected to RNA amplification in the reaction system of step (2); wherein, the amplification procedure is: reverse transcription Pre-denaturation (95℃, 5 min); Denaturation Annealing and extension .

[0045] Table 1: ; Example 2 The difference between this embodiment and Example 1 is the concentration of each component in the reaction system. Specifically, the components are: Tris-HCl (20mM), Bicine (40mM), AEBSF (0.8mM), dextran sulfate (0.06% (w / v)), MgCl2 (6mM), a mixture of dNTPs (equal concentrations of dATP, dTTP, dCTP, and dGTP, each at 0.3mM), reverse transcriptase (mutant M-MLV reverse transcriptase (Hifair III Reverse Transcriptase), 1mM), thermostable DNA polymerase (HotStart Taq DNA Polymerase, 1mM), nucleic acid template (mRNA, 20mM), and primer and probe sets for the STOM, ABHD14B, and FLT3LG genes, as shown in Table 1. The molar ratio of Tris to Bicine is 1:2. The pH of the reaction system (at 25°C) is 8.5, and the final reaction temperature stabilizes the pH in the optimal enzyme activity range of 8.3-8.8. The other steps and parameters are the same as in Example 1.

[0046] Example 3 The difference between this embodiment and Example 1 is the concentration of each component in the reaction system. Specifically, the components are: Tris-HCl (15mM), Bicine (45mM), AEBSF (1mM), dextran sulfate (0.1% (w / v)), MgCl2 (8mM), a mixture of dNTPs (equal concentrations of dATP, dTTP, dCTP, and dGTP, each at 0.3mM), reverse transcriptase (mutant M-MLV reverse transcriptase (Hifair III Reverse Transcriptase), 1mM), and thermostable DNA polymerase (HotStart Taq). The primer and probe sets for DNA Polymerase (1 mM), nucleic acid template (mRNA, 20 mM), STOM gene, ABHD14B gene, and FLT3LG gene (each primer / probe concentration was 50 μmol / L) are shown in Table 1. The molar ratio of Tris to Bicine was 1:3. The pH of the reaction system (at 25°C) was 8.7, and the final reaction temperature stabilized at the optimal enzyme activity range of 8.3-8.8. Other steps and parameters were the same as in Example 1.

[0047] Comparative Example 1 The difference between this comparative example and Example 1 is that AEBSF was replaced with trehalose. Other steps and parameters were the same as in Example 1. The pH of the reaction system (at 25°C) was 8.8, and the pH at the final reaction temperature was 7.5, indicating that as the reaction temperature increased, the inhibitors in the reaction solution released more H+. +This causes a change in the pH value of the reaction solution, indicating that trehalose cannot effectively maintain the stability of the reaction system.

[0048] Comparative Example 2 The difference between this comparative example and Example 1 is that Bicine was replaced with ammonium sulfate. Other steps and parameters were the same as in Example 1. The pH of the reaction system at 25°C was 8.8, and the pH at the final reaction temperature was 7.7, indicating that ammonium sulfate could not effectively maintain the pH of the reaction system as the reaction temperature increased.

[0049] Comparative Example 3 The difference between this comparative example and Example 1 is that the concentration of MgCl2 is 3 mM. Other steps and parameters are the same as in Example 1. This is because the Bicine chelate in the reaction system contains Mg... 2+ , so that the Mg in the reaction system 2+ The reduced content means that DNA polymerase and reverse transcriptase activities cannot be fully activated.

[0050] Performance testing and results analysis: The amplification curves obtained by performing real-time fluorescence quantitative PCR amplification on complex whole blood sample 1 (whole blood sample from patient 1 with bloodstream infection) in Example 1 and Comparative Example 1 are shown below. Figure 1-3 As shown, by Figure 1-3 (The black solid line represents Example 1, and the red dashed line represents Comparative Example 1.) It can be seen that, compared with the amplification curve of Comparative Example 1, the amplification Ct values ​​of STOM gene, ABHD14B gene, and FLT3LG gene in the amplification curve of Example 1 are significantly earlier, indicating that Example 1 of the present invention can effectively improve the anti-interference ability of the reaction system. The AEBSF of Example 1 can specifically eliminate the inhibitors in complex whole blood sample RNA during the reaction process, so that the activities of polymerase and reverse transcriptase are not affected, thereby improving the anti-interference ability of the buffer system.

[0051] The amplification curves obtained by performing real-time fluorescence quantitative PCR amplification on complex whole blood sample 2 (whole blood sample from patient 2 with bloodstream infection) in Example 1 and Comparative Example 2 are shown below. Figure 4-6 As shown, by Figure 4-6 (The black solid line represents Example 1, and the red dashed line represents Comparative Example 2.) It can be seen that, compared to the amplification curve of Comparative Example 2, the amplification Ct values ​​of the STOM gene, ABHD14B gene, and FLT3LG gene in the amplification curve of Example 1 were significantly earlier, indicating that Bincine in Example 1 can effectively maintain the stability of the PCR reaction solution; while in Comparative Example 2, ammonium sulfate and other interfering substances such as lactic acid and urea hydrolyze to produce more H+ during the reaction process as the reaction temperature increases. +This causes the pH of the reaction system to drop, affecting the activity of polymerase and reverse transcriptase; however, Bincine in Example 1, due to its extremely low temperature coefficient, can effectively cope with pH changes caused by reaction temperature and interfering substances, thereby keeping polymerase and reverse transcriptase in a state of maximum activity and maintaining the stability of the entire reaction.

[0052] Complex whole blood sample 3 (whole blood sample from patient 3 with bloodstream infection) was serially diluted 5-fold to obtain RNA stock solution 3, RNA stock solution 3-5TX (5-fold dilution concentration), and RNA stock solution 3-25TX (25-fold dilution concentration). The amplification curves obtained by performing real-time quantitative PCR amplification on the samples of the above three concentrations as described in Example 1 and Comparative Example 3 are shown below. Figure 7-9 As shown, by Figure 7-9 (The black solid line represents Example 1, and the red dashed line represents Comparative Example 3.) It can be seen that, compared to the amplification curve of Comparative Example 3, the amplification Ct values ​​of the STOM, ABHD14B, and FLT3LG genes in the amplification curve of Example 1 were significantly earlier, indicating a significant improvement in amplification efficiency; this suggests that more Mg needs to be added to this reaction system. 2+ To supplement the Mg chelated by Bicine 2+ .

[0053] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A reaction system for RNA amplification in complex whole blood samples, characterized in that, The reaction system includes a buffer component and an enzyme protection component; The buffer component consists of Tris and Bicine, which are used to synergistically maintain the pH stability of the reaction system during RNA amplification of complex whole blood samples. The enzyme protection component consists of AEBSF and dextran sulfate.

2. The reaction system for RNA amplification of complex whole blood samples according to claim 1, characterized in that, In the buffer components, the final concentration of Tris is 10-30 mM, and the final concentration of Bicine is 30-50 mM.

3. The reaction system for RNA amplification in complex whole blood samples according to claim 2, characterized in that, In the buffer components, the final concentration of Tris is 15-20 mM, and the final concentration of Bicine is 30-45 mM.

4. The reaction system for RNA amplification of complex whole blood samples according to claim 1, characterized in that, In the buffer component, the molar ratio of Tris to Bicine is 1:(1.5-3).

5. The reaction system for RNA amplification of complex whole blood samples according to claim 1, characterized in that, The reaction system also includes 4-8 mM magnesium salt.

6. The reaction system for RNA amplification of complex whole blood samples according to claim 1, characterized in that, In the enzyme protection component, the final concentration of AEBSF is 0.1-1.0 mM, and the final mass-volume concentration of dextran sulfate is 0.01%-0.1%.

7. The reaction system for RNA amplification of complex whole blood samples according to claim 3, characterized in that, The reaction system also includes a mixture of dNTPs, reverse transcriptase, thermostable DNA polymerase, and nucleic acid template.

8. The reaction system for RNA amplification applied to complex whole blood samples according to claim 1, characterized in that, The pH value of the reaction system at 25°C is 8.4-8.

6.

9. A kit for simultaneously detecting the STOM gene, ABHD14B, and FLT3LG gene, characterized in that, The reaction system for RNA amplification of complex whole blood samples as described in any one of claims 1-8.

10. A method for detecting multiple genes for non-diagnostic purposes, characterized in that, Using the reaction system for RNA amplification of complex whole blood samples as described in any one of claims 1-8 or the kit as described in claim 9, reverse transcription and real-time fluorescence PCR amplification are performed on RNA derived from the whole blood sample to be tested; wherein the multiple genes are STOM gene, ABHD14B and FLT3LG gene.

Images