A direct qPCR amplification reagent, amplification kit and application for whole blood samples

Abstract

This invention discloses direct amplification qPCR reagents, kits, and applications for whole blood samples. The amplification reagents include: a composite hot-start DNA polymerase system, a buffer system, a surfactant, an anti-contamination system, and an anti-inhibition enhancer system. The composite hot-start DNA polymerase system is obtained by mixing antibody-modified hot-start Taq enzyme and antibody-modified Taq1C2 mutant Taq enzyme in a predetermined ratio. The buffer system includes Tris-HCl, MgCl2, (NH4)2SO4, and KCl. The surfactants include Tween-20 and NP-40. The anti-contamination system includes UDG enzyme and dNTPs, wherein the dNTPs are composed of dATP, dGTP, dCTP, dTTP, and dUTP. The anti-inhibition enhancer system includes D-trehalose, L-carnitine, DMSO, BSA, and SSB single-chain binding protein.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a direct amplification qPCR amplification reagent, amplification kit, and application for whole blood samples. Background Technology

[0002] Clinical molecular diagnostics often uses blood as the test sample. However, blood contains potent PCR inhibitors such as heme, immunoglobulins, lactoferrin, heparin, and EDTA, which can directly inhibit DNA polymerase activity, leading to decreased amplification efficiency, false negatives, and reduced sensitivity. Furthermore, some components in blood can severely interfere with fluorescence, causing distortion of the product fluorescence signal. Traditional detection procedures require nucleic acid extraction and purification from blood samples, which is cumbersome, time-consuming, and prone to cross-contamination and template loss, making it difficult to meet the needs of point-of-care testing (POCT) and high-throughput rapid screening. Whole blood direct amplification qPCR technology can eliminate the nucleic acid extraction step, directly amplifying from the original whole blood sample, significantly shortening detection time and reducing costs and contamination risks. However, currently available direct amplification reagents generally cannot achieve quantitative PCR, requiring nucleic acid electrophoresis to detect products. They also suffer from drawbacks such as low tolerance to whole blood (usually ≤20%), insufficient sensitivity, poor batch-to-batch stability, and weak anti-interference ability. Some direct amplification reagents cannot meet the high-reliability clinical testing requirements when faced with high-concentration whole blood samples.

[0003] Therefore, developing a direct amplification qPCR reagent that can tolerate high proportions of whole blood, requires no extraction, is highly stable, is contamination-resistant, and is suitable for routine clinical and POCT scenarios has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] In view of this, on the one hand, some embodiments disclose a direct amplification qPCR amplification reagent for whole blood samples, comprising: a complex hot-start DNA polymerase system, a buffer system, a surfactant, an anti-contamination system, and an anti-inhibition enhancer system; wherein,

[0005] The composite hot-start DNA polymerase system is obtained by mixing antibody-modified hot-start Taq enzyme and antibody-modified Taq1C2 enzyme in a set ratio.

[0006] The buffer system includes: Tris-HCl, MgCl2, (NH4)2SO4 and KCl;

[0007] Surfactants include: Tween-20 and NP-40;

[0008] The anti-pollution system includes: UDG enzymes and dNTPs, where dNTPs are composed of dATP, dGTP, dCTP, dTTP and dUTP;

[0009] The anti-inhibition enhancer system includes D-trehalose, L-carnitine, DMSO, bovine serum albumin (BSA), and single-chain binding protein (SSB).

[0010] Furthermore, some embodiments disclose direct amplification qPCR amplification reagents for whole blood samples. In these reagents, the concentration of the composite hot-start DNA polymerase system is 0.01–0.1 mg / mL; the buffer system is prepared by combining 20–100 mM Tris-HCl (pH 8.5–9.0), 1–8 mM MgCl2, 10–50 mM (NH4)2SO4, and 20–150 mM KCl; the surfactant is prepared by combining 0.05–0.2% Tween-20 and 0.6–0.9% NP-40; the anti-contamination system includes 0.05–0.15 U / μL UDG enzyme and 0.1–0.6 mM dNTPs, wherein the molar ratio of dATP, dGTP, dCTP, dTTP, and dUTP is 8:8:8:7:1; and the anti-inhibition enhancer system includes 0.4–0.8 M... D-trehalose, 0.2–0.7 M L-carnitine, 1–2% DMSO, and 0.01–0.1 mg / mL BSA and 0.05–0.15 U / μL SSB single-chain binding protein.

[0011] Some embodiments disclose a direct amplification qPCR amplification kit for whole blood samples, in which the composite hot-start DNA polymerase is composed of antibody-modified hot-start Taq1C2 mutant Taq enzyme and antibody-modified hot-start wild-type Taq enzyme, with final mass concentrations of 0.014 mg / mL and 0.036 mg / mL in the direct amplification qPCR amplification reagent, respectively, corresponding to a mass concentration ratio of 7:18.

[0012] Some embodiments disclose direct amplification qPCR reagents for whole blood samples. In these reagents, the composite hot-start DNA polymerase system comprises 0.014 mg / mL antibody-modified Taq1C2 enzyme and 0.036 mg / mL antibody-modified hot-start Taq enzyme, with a mass ratio of 7:18. The buffer system is prepared by mixing 100 mM Tris-HCl (pH 8.8), 8 mM MgCl2, 10 mM (NH4)2SO4, and 150 mM KCl. The surfactants include 0.2% Tween-20 and 0.8% NP-40. The anti-contamination system includes 0.1 U / μL UDG enzyme and 0.6 mM dNTPs. The anti-inhibition enhancement system includes 0.6 M D-trehalose, 0.48 M L-carnitine, 2% DMSO, 0.02 mg / mL bovine serum albumin (BSA), and 0.1 U / μL single-chain binding protein (SSB).

[0013] On the other hand, some embodiments disclose direct amplification qPCR amplification kits for whole blood samples, including the direct amplification qPCR amplification reagents for whole blood samples disclosed in the embodiments of the present invention.

[0014] Furthermore, some of the direct amplification qPCR kits for whole blood samples disclosed in the embodiments also include primers and probes.

[0015] On the other hand, some embodiments disclose the application of direct amplification qPCR amplification reagents for whole blood samples in infectious disease detection, genetic disease screening, fusion gene detection, rapid clinical molecular diagnosis, or point-of-care testing, including the use of direct amplification qPCR amplification reagents to directly amplify whole blood samples.

[0016] On the other hand, some embodiments disclose the application of direct amplification qPCR amplification kits for whole blood samples in infectious disease detection, genetic disease screening, fusion gene detection, rapid clinical molecular diagnostics, or point-of-care testing, including the direct amplification of whole blood samples using direct amplification qPCR amplification kits.

[0017] Furthermore, some of the applications disclosed in the embodiments include whole blood samples such as EDTA-anticoagulated whole blood, heparin-anticoagulated whole blood, finger-prick whole blood, and plasma.

[0018] On the other hand, some of the applications disclosed in the embodiments involve adding direct amplification qPCR amplification reagent to whole blood samples for amplification, with the final volume ratio of whole blood samples in the amplification system being 0.1-3%.

[0019] The direct amplification qPCR reagents, amplification kits, and applications for whole blood samples disclosed in the embodiments of this invention have at least the following beneficial technical effects:

[0020] 1. High resistance to inhibition: By combining hot-start Taq1C2 with anti-inhibition properties and hot-start Taq DNA polymerase with high amplification efficiency in a specific ratio, combined with the high osmotic pressure regulation of L-carnitine and D-trehalose, it can maintain high efficiency amplification in whole blood samples with up to 30-40% (v / v), which is significantly better than using wild-type Taq enzyme and commercially available direct amplification enzyme alone.

[0021] 2. High sensitivity: The lowest detection limit can reach 10 copies / μL.

[0022] 3. High stability: After being placed at 37℃ for 96 hours and subjected to 25 freeze-thaw cycles, the Ct value changed by less than 1.0, and the CV was less than 2%, indicating that the system has excellent stability.

[0023] 4. Contamination prevention: The dUTP / UDG system effectively prevents aerosol contamination of PCR products;

[0024] 5. Simple and efficient: Direct sample addition, no extraction required, the entire detection process takes ≤60 minutes, suitable for high-throughput and POCT.

[0025] Direct amplification qPCR reagents and kits for whole blood samples have promising applications in infectious disease detection, genetic disease screening, fusion gene detection, rapid clinical molecular diagnosis, or point-of-care testing. Attached Figure Description

[0026] Figure 1 Example 2: Schematic diagram of electrophoresis results;

[0027] Figure 2 Example 3: Result curve of the lowest detection limit for human genomic DNA;

[0028] Figure 3 Example 3: Limit of detection for whole blood samples;

[0029] Figure 4 Example 4: Accelerated stability test result curve;

[0030] Figure 5 Example 4: Freeze-thaw stability test result curve;

[0031] Figure 6 Example 5: Amplification efficiency result curve. Detailed Implementation

[0032] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in these embodiments of the invention, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in these embodiments is merely for describing particular implementations and is not intended to limit the scope of the disclosure of these embodiments.

[0033] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain; other experimental methods and technical means not specifically noted in the embodiments of this invention refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0034] The terms “basic” and “approximately” as used herein are used to describe small fluctuations. For example, they can mean less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%. Numerical data presented or expressed in range format herein are used for convenience and brevity only, and should therefore be interpreted flexibly to include not only the explicitly listed values ​​that define the range, but also all independent values ​​or subranges contained within that range. For example, a numerical range of “1–5%” should be interpreted to include not only the explicitly listed values ​​from 1% to 5%, but also the independent values ​​and subranges within the indicated range. Thus, this numerical range includes independent values ​​such as 2%, 3.5%, and 4%, and subranges such as 1%–3%, 2%–4%, and 3%–5%, etc. This principle also applies to ranges that list only one value. Furthermore, this interpretation applies regardless of the width of the range or the characteristics described.

[0035] In this document, including in the claims, conjunctions such as "comprising," "including," "with," "having," "containing," "involving," and "accommodating" are understood to be open-ended, meaning "including but not limited to." Only the conjunctions "consisting of" and "composed of" are closed conjunctions.

[0036] To better illustrate the content of this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the invention can be practiced even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail, in order to highlight the main points of the invention.

[0037] Without conflict, the technical features disclosed in the embodiments of the present invention can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of the present invention.

[0038] In some embodiments, the direct amplification qPCR amplification reagent for whole blood samples includes: a complex hot-start DNA polymerase system, a buffer system, a surfactant, an anti-contamination system, and an anti-inhibition enhancer system; wherein,

[0039] The composite hot-start DNA polymerase system is obtained by mixing antibody-modified hot-start Taq enzyme and antibody-modified Taq1C2 enzyme in a set ratio; typically, the mass concentration ratio of antibody-modified Taq1C2 enzyme to antibody-modified hot-start Taq enzyme is 7:18.

[0040] The buffer solution includes Tris-HCl, MgCl2, (NH4)2SO4, and KCl;

[0041] The surfactant system includes Tween-20 and NP-40;

[0042] The anti-pollution system consists of UDG enzymes and dNTPs, which are composed of dATP, dUTP, dTTP, dGTP, and dCTP.

[0043] The anti-inhibition enhancer system consists of D-trehalose, L-carnitine, DMSO, BSA, and single-chain binding protein SSB, which can alleviate the inhibition of fluorescence signals in the blood. L-carnitine, through amphiphilic binding to plasma lipoproteins / chylomicrons, significantly reduces light absorption in the 400–600 nm range (FAM / Alexa 488 excitation / emission region), minimizing the ineffective consumption of fluorescence energy by lipids and improving the signal-to-noise ratio of serum fluorescence detection. D-trehalose, as a chemical chaperone, locks the native conformation of plasma albumin / globulin, reducing tryptophan / tyrosine residue exposure, significantly reducing intrinsic fluorescence and light absorption in the 280–350 nm range, and suppressing background interference. DMSO competes with DNA bases for hydrogen bonds, weakening double-strand stability and making high-GC-content templates easier to denature and dissociate, thus lowering Tm by 2–3°C. BSA reduces the toxicity of PCR inhibitors such as heme, bile salts, and humic acid by binding to them, and competes with Taq enzymes for inhibitor binding, protecting enzyme activity. Single-stranded binding protein SSB improves primer binding efficiency by competing with PCR inhibitors for DNA stabilization of single-stranded templates.

[0044] The qPCR amplification reagents disclosed in this invention can directly use EDTA / heparin-anticoagulated whole blood, finger prick blood, plasma, swabs, etc., as templates to complete qPCR amplification without nucleic acid extraction. Typically, the qPCR amplification reagents should be stored at 4°C for at least 7 days, or at -20°C for at least 30 days.

[0045] Some embodiments disclose a hot-start DNA polymerase system in a direct amplification qPCR reagent for whole blood samples with a concentration of 0.01–0.1 mg / mL; the buffer system is prepared by mixing 20–100 mM Tris-HCl (pH 8.5–9.0), 1–8 mM MgCl2, 10–50 mM (NH4)2SO4, and 20–150 mM KCl; the surfactant is prepared by mixing 0.05–0.2% Tween-20 and 0.6–0.9% NP-40; the anti-contamination system includes 0.05–0.15 U / μL UDG enzyme and 0.1–0.6 mM dNTPs, with the molar ratio of dATP, dGTP, dCTP, dTTP, and dUTP in the dNTPs being 8:8:8:7:1; and the anti-inhibition enhancer system includes 0.4–0.8 M... D-trehalose, 0.2–0.7M L-carnitine, 1–2% DMSO, 0.1–0.5 mg / mL BSA, and 0.05–0.15 U / μL SSB single-chain binding protein; wherein the concentration of each component is its final concentration in the direct amplification qPCR detection reagent.

[0046] In a preferred embodiment, the composite hot-start DNA polymerase system in the direct amplification qPCR amplification reagent comprises 0.014 mg / mL antibody-modified Taq1C2 enzyme and 0.036 mg / mL antibody-modified hot-start Taq enzyme; the buffer system consists of 100 mM Tris-HCl (pH 8.8), 8 mM MgCl2, 10 mM (NH4)2SO4, and 150 mM KCl; the surfactant consists of 0.2% Tween-20 and 0.8% NP-40; the anti-contamination system comprises 0.1 U / μL UDG enzyme and 0.6 mM dNTPs; the anti-inhibition enhancer system comprises 0.6 M D-trehalose, 0.48 M L-carnitine, 2% DMSO, and 0.02 mg / mL BSA and 0.1 U / μL single-stranded binding protein SSB; wherein the concentration of each component is its final concentration in the direct amplification qPCR amplification reagent.

[0047] Some embodiments disclose direct amplification qPCR amplification kits for whole blood samples, including the direct amplification qPCR amplification reagents for whole blood samples disclosed in the embodiments of the present invention.

[0048] Some embodiments of the disclosed direct amplification qPCR kits for whole blood samples also include primers and probes. Typically, the direct amplification qPCR kits for whole blood samples are in 2× premix form and are compatible with user-defined primers and probes.

[0049] Some embodiments disclose direct amplification qPCR reagents or kits for whole blood samples, which are applied to infectious disease detection, genetic disease screening, fusion gene detection, rapid clinical molecular diagnosis, or point-of-care testing.

[0050] Some embodiments disclose direct amplification qPCR amplification reagents or kits for whole blood samples, which are used for the detection of whole blood samples, including EDTA-anticoagulated whole blood, heparin-anticoagulated whole blood, finger-prick whole blood, and plasma.

[0051] When testing whole blood samples, the direct amplification qPCR amplification reagent or kit disclosed in some embodiments is added to the whole blood sample for amplification, and the final volume ratio of the whole blood sample in the amplification system is 0.1-3%.

[0052] The technical details are further illustrated below with reference to the embodiments.

[0053] Example 1

[0054] Preparation of 2× Whole Blood Direct Amplification qPCR Amplification Reagent

[0055] (a) Prepare 2× whole blood direct amplification qPCR amplification reagents according to the following final concentration:

[0056] 100mM Tris-HCl (pH 8.8), 150mM KCl, 8mM MgCl2, 10mM (NH4)2SO4, 0.2% Tween-20, 0.8% NP-40, 0.02mg / mL BSA, 2% DMSO, 0.48M L-carnitine, 0.6M D-trehalose, 0.1U / μL LSB, 0.6mM dNTPs / (dATP, dGTP, dCTP, dTTP, dUTP molar ratio of 8:8:8:7:1), 0.1U / μL UDG enzyme, 0.014mg / mL hot-start Taq1C2 enzyme, 0.036 mg / mL hot-start Taq enzyme.

[0057] (ii) 20 μL of reaction system

[0058] When using, take 10 μL of 2× whole blood direct amplification qPCR amplification reagent, add 2-5 μL of diluted whole blood sample, and add primers, probes and water to 20 μL to obtain the reaction system, and then perform qPCR reaction.

[0059] (III) Amplification Procedure

[0060] 37℃ for 2 min; 95℃ for 5 min; 95℃ for 15 s; 60℃ for 45 s; 40 cycles.

[0061] Example 2

[0062] Comparative experiment on inhibitor resistance

[0063] (I) Experimental Methods

[0064] Group A: wild-type Taq enzyme; Group B: commercially resistant whole blood Taq enzyme; Group C: Taq1C2 enzyme alone. Groups A, B, and C were added to 2× reaction buffer with final concentrations of 100 mM Tris-HCl (pH = 8.8), 150 mM KCl, 8 mM MgCl2, 10 mM (NH4)2SO4, 0.2% Tween 20, 0.02 mg / mL BSA, and 0.6 mM dNTPs, respectively. Whole blood containing EDTA at different concentrations (0%, 0.1%, 1%, 5%, 10%, 20%, 30%, and 40%) was used to detect the amplified human internal reference gene (120 bp). The reaction program was: 95℃; pre-denaturation for 3 min, 95℃ denaturation for 15 s, 55℃ annealing for 15 s, and 72℃ extension for 40 s, for a total of 35 cycles. After the reaction was completed, the products were analyzed by 1.5% (w / v) agarose gel electrophoresis.

[0065] (II) Results

[0066] Figure 1 The electrophoresis results showed that: Group A: amplification was possible only with pure template and could not tolerate blood inhibition; Group B: amplification was possible with ≤20% whole blood, but amplification failed at a higher rate; Group C: amplification was possible with 40% whole blood, with clear amplification bands and no inhibition.

[0067] Figure 1 In the diagram, markers 1 through 8 represent 0 (gDNA), 0.1%, 1%, 5%, 10%, 20%, 30%, and 40% whole blood, respectively.

[0068] Example 3

[0069] Detection limit test

[0070] (I) Experimental Methods

[0071] The lowest detection limit of the reagent of the present invention was investigated using human genomic DNA and whole blood samples, respectively.

[0072] (1) Detection limit of genomic DNA: Genomic DNA was diluted in the following order: 50 ng / μL, 30 ng / μL, 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, and 1 pg / μL. The reagents of this invention were used for quantitative real-time PCR amplification. The reaction program was set as follows: UDG enzyme digestion at 37℃ for 2 min; pre-denaturation at 95℃ for 3 min, followed by denaturation at 95℃ for 15 s.

[0073] (2) Extend at 60℃ for 40 seconds, for a total of 40 cycles.

[0074] (2) Limit of detection of whole blood: Fresh whole blood was serially diluted and added to the reaction system at final volume fractions of 4%, 3%, 2%, 1%, 0.1%, 0.01%, and 0.001%. The reagents of this invention were used for amplification, and the reaction procedure was the same as above.

[0075] (II) Results

[0076] like Figure 2 , Figure 3 As shown, in genomic DNA samples, the 0.1 ng / μL group consistently exhibited typical amplification curves with a Ct value < 35; amplification became unstable when the concentration was reduced to 0.01 ng / μL. In whole blood samples, the 0.1% group consistently showed specific amplification signals with a Ct value < 35, and the 4% group was still detectable; these results meet clinical testing requirements.

[0077] Example 4

[0078] Accelerated stability and freeze-thaw stability

[0079] (I) Experimental Methods

[0080] The reagents of this invention were divided into two groups. One group was placed in a 37°C metal bath and amplified at 0 h, 12 h, 24 h, 48 h, and 96 h, respectively. The other group underwent repeated freeze-thaw cycles (-20°C to room temperature) and amplified after 0, 5, 10, 15, 20, and 25 freeze-thaw cycles, respectively. The reaction program was set as follows: UDG enzyme digestion at 37°C for 2 min; pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s; extension at 60°C for 45 s, for a total of 45 cycles.

[0081] (II) Results

[0082] Figure 4 , Figure 5 The results showed that after storage at 37℃ for 96 h, the Ct change was <0.5 and the CV was <5%, indicating no significant decrease in performance. After 25 freeze-thaw cycles, the Ct change was <1.0, demonstrating excellent system stability. This indicates that the anti-inhibition enhancer system provided excellent protection for enzyme activity.

[0083] Example 5

[0084] Amplification efficiency test

[0085] (I) Experimental Methods

[0086] The standard was serially diluted 10-fold (10 9 ~10 1 qPCR amplification was performed using copies / μL, following the same reaction procedure as above.

[0087] Calculate the slope of the standard curve.

[0088] (II) Results

[0089] Figure 6 As shown, the amplification efficiency was 97.3% and R² = 0.9979, indicating that the reagent maintains extremely high amplification kinetics performance under direct amplification conditions.

[0090] The present invention discloses a reagent and kit for direct amplification qPCR of whole blood samples. The core enzyme system is a combination of antibody-modified hot-start Taq enzyme and antibody-modified Taq1C2 enzyme, along with a dedicated buffer system, nonionic surfactants, dNTPs (including dUTP), UDG enzyme, and enhancers (D-trehalose, L-carnitine, DMSO, BSA, and single-stranded binding protein SSB). It allows direct quantitative PCR amplification using whole blood samples as templates without nucleic acid purification. Through the synergistic effect of the complex enzymes and multiple anti-inhibition protections, it can tolerate 30-40% (v / v) whole blood samples, overcoming interference from inhibitors such as heme, EDTA, and heparin. The detection limit is as low as 0.1 ng genomic DNA and 0.1% whole blood, with an amplification efficiency of 97.3%. It also exhibits excellent thermal stability and anti-contamination capabilities, showing no significant performance degradation after 96 hours at 37°C and 25 freeze-thaw cycles. This invention is simple, rapid, sensitive, and specific to operate, and is suitable for rapid detection scenarios such as clinical infectious diseases, genetic diseases, and target-related fusion genes, and has important clinical application and market value.

[0091] The technical solutions and technical details disclosed in the embodiments of this invention are merely illustrative of the inventive concept of this invention and do not constitute a limitation on the technical solutions of the embodiments of this invention. Any conventional changes, substitutions, or combinations made to the technical details disclosed in the embodiments of this invention have the same inventive concept as this invention and are within the protection scope of the claims of this invention.

Claims

1. A direct amplification qPCR amplification reagent for whole blood samples, characterized in that, include: The system comprises a composite hot-start DNA polymerase system, a buffer system, a surfactant system, an anti-fouling system, and an anti-inhibition enhancer system; among which, The composite hot-start DNA polymerase system is obtained by mixing antibody-modified hot-start Taq enzyme and antibody-modified Taq1C2 enzyme in a set ratio. The buffer system comprises: Tris-HCl, MgCl2, (NH4)2SO4, and KCl; The surfactants include: Tween-20 and NP-40; The anti-pollution system includes: UDG enzyme and dNTPs, wherein the dNTPs are composed of dATP, dGTP, dCTP, dTTP and dUTP; The anti-inhibition enhancers include: D-trehalose, L-carnitine, DMSO, bovine serum albumin (BSA), and single-chain binding protein (SSB).

2. The direct amplification qPCR amplification reagent for whole blood samples according to claim 1, characterized in that, In the direct amplification qPCR amplification reagent: The concentration of the composite hot-start DNA polymerase system is 0.01–0.1 mg / mL; The buffer system comprises 20–100 mM Tris-HCl with a pH of 8.5–9.0, 4–8 mM MgCl2, 10–50 mM (NH4)2SO4, and 20–150 mM KCl; The surfactant comprises 0.05–0.2% Tween-20 and 0.6–0.9% NP-40; The anti-pollution system includes 0.05–0.15 U / μL UDG enzyme and 0.1–0.6 mM dNTPs, wherein the molar ratio of dATP, dGTP, dCTP, dTTP and dUTP in the dNTPs is 8:8:8:7:1; The anti-inhibition enhancement system comprises 0.4–0.8 M D-trehalose, 0.2–0.7 M L-carnitine, 1–2% DMSO, 0.01–0.1 mg / mL BSA, and 0.05–0.15 U / μL SSB single-chain binding protein.

3. The direct amplification qPCR amplification reagent for whole blood samples according to claim 1, characterized in that, The composite hot-start DNA polymerase is composed of antibody-modified hot-start Taq1C2 mutant Taq enzyme and antibody-modified hot-start wild-type Taq enzyme, with final mass concentrations of 0.014 mg / mL and 0.036 mg / mL in the direct amplification qPCR reagent, respectively, corresponding to a mass concentration ratio of 7:

18.

4. The direct amplification qPCR amplification reagent for whole blood samples according to claim 1, characterized in that, In the direct amplification qPCR amplification reagent: The final concentrations of the antibody-modified hot-start Taq1C2 enzyme and the hot-start Taq enzyme in the direct amplification qPCR amplification reagent of the composite hot-start DNA polymerase system are 0.014 mg / mL and 0.036 mg / mL, respectively, with a corresponding mass concentration ratio of 7:

18. The buffer system comprises 100 mM Tris-HCl at pH 8.8, 8 mM MgCl2, 10 mM (NH4)2SO4 and 150 mM KCl; The surfactant comprises 0.2% Tween-20 and 0.8% NP-40; The anti-pollution system includes 0.1 U / μL UDG enzyme and 0.6 mM dNTPs; The anti-inhibition enhancer comprises 0.6M D-trehalose, 0.48M L-carnitine, 2% DMSO, 0.02 mg / mL bovine serum albumin (BSA), and 0.1 U / μL single-chain binding protein (SSB).

5. A direct amplification qPCR amplification kit for whole blood samples, characterized in that, Includes the direct amplification qPCR amplification reagent for whole blood samples as described in any one of claims 1 to 4.

6. The direct amplification qPCR amplification kit for whole blood samples according to claim 5, characterized in that, It also includes primers and probes.

7. The application of the direct amplification qPCR amplification reagent for whole blood samples according to any one of claims 1 to 4 in infectious disease detection, genetic disease screening, fusion gene detection, rapid clinical molecular diagnosis, or point-of-care testing, characterized in that, include: Whole blood samples were directly amplified using direct amplification qPCR reagents.

8. The application of the direct amplification qPCR amplification kit for whole blood samples as described in claim 5 or 6 in infectious disease detection, genetic disease screening, fusion gene detection, rapid clinical molecular diagnosis, or point-of-care testing, characterized in that, include: Whole blood samples were directly amplified using a direct amplification qPCR amplification kit.

9. The application according to claim 7 or 8, characterized in that, The whole blood samples include EDTA-anticoagulated whole blood, heparin-anticoagulated whole blood, finger-prick whole blood, and plasma.

10. The application according to claim 9, characterized in that, The direct amplification qPCR amplification reagent was added to whole blood samples for amplification, and the final volume ratio of whole blood samples in the amplification system was 0.1-3%.

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