Whole blood sample processing reagents and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular to a whole blood sample processing reagent and application thereof. The present application provides a whole blood sample processing reagent, which has simple components, and when used for processing a whole blood sample, only needs to be premixed with the sample to be detected, so that the pretreatment of the whole blood sample can be realized, and the influence of the inhibitors of the whole blood sample on nucleic acid extraction and nucleic acid amplification can be removed. Compared with the prior art, the present application does not need additional centrifugation, waste liquid removal, thorough mixing, heating and dissolving operations and the corresponding complex equipment, greatly simplifies the pretreatment process of the whole blood sample, fully covers the needs of basic laboratories and automatic operation, and is suitable for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to reagents for processing whole blood samples and their applications. Background Technology

[0002] With the rapid development of molecular detection technology, the types of samples being tested are becoming increasingly diversified. Among them, whole blood samples, as an important source of genomic DNA purification, have seen a continuous increase in demand for efficient processing. Although there are various dedicated nucleic acid extraction kits for whole blood samples on the market, existing technologies have significant limitations: on the one hand, these kits are usually only compatible with a single type of whole blood sample and are difficult to be compatible with other samples (such as swabs, saliva, sputum, etc.), resulting in frequent kit changes when testing multiple types of samples, which is cumbersome and hinders the integration of automated processes; on the other hand, while developing universal reagents can simplify operations, the significant differences in the physicochemical properties of different samples require compromises on core performance aspects such as lysis efficiency and inhibitor removal, and complex samples often require additional pretreatment steps. For example, existing pretreatment methods for whole blood samples usually rely on centrifugation to separate red blood cells or heating to lyse white blood cells, which are complex and highly dependent on equipment, further limiting the expansion of practical application scenarios.

[0003] Current common whole blood pretreatment techniques require adding red blood cell lysis buffer and centrifuging to remove red blood cells, followed by adding suspension and heating to dissolve white blood cell precipitates. This process involves multiple steps, including centrifugation, waste liquid removal, mixing, and heating. It requires not only conventional equipment like pipettes and mixers but also non-portable instruments such as centrifuges, metal baths, or water baths. This multi-step, equipment-intensive process significantly increases operational complexity and time costs, and demands high skill levels from laboratory personnel, making it difficult to adapt to the efficient integration requirements of automated platforms. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a whole blood sample processing reagent and its application. The whole blood sample processing reagent provided by the present invention has simple components, simplifies the whole blood sample processing process, and is more suitable for the nucleic acid extraction needs of clinical whole blood samples in molecular diagnostic processes.

[0005] A first aspect of the invention relates to a treatment reagent for preparing and processing whole blood samples, and for using whole blood samples for nucleic acid lysis and purification, comprising a detergent, a cationic surfactant and a reducing agent, wherein the pH of the treatment reagent is 7 to 9.

[0006] In some embodiments, the treatment reagent may further include an antibacterial agent and a buffer solution, wherein the buffer solution maintains the pH of the treatment reagent at 7 to 9.

[0007] In some embodiments, it comprises 0.4–0.8 M buffer solution, 1%–6% (v / v) detergent, 0.5%–1.5% (w / v) cationic surfactant, 0.2%–7.25% (w / v) reducing agent, 0.5%–1.5% (w / v) antioxidant and 0.05%–0.1% (v / v) antibacterial agent.

[0008] The percentages indicated in this article, v / v, refer to volume ratio. For example, 1% (v / v) means that the substance contains 0.01 ml of the substance per 1 ml of treatment solution. The percentages indicated, w / v, refer to mass ratio (g / ml). For example, 1% (w / v) means that the substance contains 0.01 g of the substance per 1 ml of treatment solution.

[0009] In some embodiments, the detergent includes at least one of Tween 20, Triton X-100, and SDS;

[0010] The cationic surfactant includes at least one of CTAB, cetylpyridine bromide and polyoxyethylene fatty amine;

[0011] The reducing agent includes at least one of DTT, thioglycerol, cysteine, and hydroxylamine hydrochloride;

[0012] The antioxidants include at least one of vitamin C, sodium vitamin C, isoascorbic acid, sodium isoascorbate, erythritol derivative, ethylenediaminetetraacetic acid, and phytic acid;

[0013] The antibacterial agent includes at least one of Proclin-950, Proclin 300, IPBC-II, and sodium azide;

[0014] The buffer solution includes at least one of Tris-HCl buffer, PBS buffer, MES buffer, and Hepes buffer.

[0015] In some specific embodiments, the detergent includes Tween 20 and / or Triton X-100;

[0016] The cationic surfactant is CTAB;

[0017] The reducing agent includes thioglycerol and / or hydroxylamine hydrochloride;

[0018] The antioxidant is sodium isoascorbate;

[0019] The antibacterial agent is Proclin-950;

[0020] The buffer solution is Tris-HCl buffer.

[0021] In some embodiments, the detergent comprises 1% to 6% (v / v) Tween 20 and / or 1% to 6% (v / v) Triton X-100;

[0022] The reducing agent comprises 1.25% to 7.25% (w / v) thioglycerol and / or 0.2% to 1% (w / v) hydroxylamine hydrochloride.

[0023] Specifically, the detergent is 1% to 6% (v / v) Tween 20; or

[0024] The detergent is 1% to 6% (v / v) Tween 20 and 0.5% to 3% (v / v) Triton X-100.

[0025] In some specific embodiments, the treatment reagents include: 0.6M Tris-HCl buffer at pH 8, 3% (v / v) Tween 20, 0.5% (v / v) Triton X-100, 1% (w / v) CTAB, 6.25% (w / v) thioglycerol, 1% (w / v) hydroxylamine hydrochloride, 1% (w / v) sodium isoascorbate, and 0.05% (v / v) Proclin-950.

[0026] The present invention provides the use of the treatment reagent in at least one of the following (1) to (4):

[0027] (1) Preparation of a kit for whole blood sample processing;

[0028] (2) Preparation of nucleic acid extraction kit;

[0029] (3) Preparation of nucleic acid detection kits;

[0030] (4) Prepare a reagent kit for nucleic acid amplification.

[0031] The present invention provides a kit for at least one of whole blood sample processing, nucleic acid extraction, nucleic acid detection and nucleic acid amplification, comprising the processing reagents and acceptable adjuvants or carriers.

[0032] This invention provides a method for processing whole blood samples, including:

[0033] A whole blood sample is mixed with the aforementioned processing reagent to obtain a processed whole blood sample, which can be directly used for nucleic acid lysis and purification; or

[0034] Whole blood samples were processed using the kit described above.

[0035] In some embodiments, the volume ratio of the whole blood sample to the processing reagent is 1:(1-5).

[0036] In some specific embodiments, the volume ratio of the whole blood sample to the processing reagent is 1:3.

[0037] This invention provides a simple reagent for processing whole blood samples. When processing whole blood samples, it only requires premixing with the sample to be tested to achieve pretreatment, removing the influence of whole blood sample inhibitors on nucleic acid extraction and amplification. Compared with existing technologies, it eliminates the need for additional centrifugation, waste liquid removal, thorough mixing, heating to dissolve, and other complex equipment, greatly simplifying the whole blood sample pretreatment process. It comprehensively covers the convenient operation needs of basic laboratories and automated platforms, making it suitable for widespread application. Detailed Implementation

[0038] This invention provides a whole blood sample processing reagent and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0039] The purpose of this invention is to provide a simple whole blood processing solution that, in conjunction with the lysis buffer in subsequent nucleic acid extraction reagents, achieves cell lysis, protein denaturation, and nucleic acid release. The main functional components of this processing solution include: detergent, cationic surfactant, and reducing agent.

[0040] In some embodiments of the present invention, the treatment solution contains 1% to 6% (v / v) detergent, 0.5% to 1.5% (w / v) cationic surfactant, and 0.2% to 7.25% (w / v) reducing agent.

[0041] In some embodiments of the invention, the detergent includes at least one of Tween 20, Triton X-100, and SDS.

[0042] In some embodiments of the present invention, the cationic surfactant includes at least one of CTAB (hexadecyltrimethylammonium bromide), hexadecylpyridine bromide, and polyoxyethylene fatty amine.

[0043] In some embodiments of the present invention, the reducing agent includes at least one of DTT, thioglycerol, cysteine, and hydroxylamine hydrochloride.

[0044] In some embodiments of the present invention, the solvent of the treatment solution is water. Ultrapure water is preferred.

[0045] In some embodiments of the present invention, a buffer solution is included. The buffer solution maintains the pH of the treatment reagent at 7–9.

[0046] In some embodiments of the present invention, the buffer includes at least one of Tris-HCl buffer, PBS buffer, MES buffer, and Hepes buffer.

[0047] In some embodiments of the present invention, the treatment solution comprises a 0.4–0.8 M buffer solution.

[0048] In some embodiments of the present invention, an antioxidant is included. The antioxidant functions as both an antioxidant and a preservative, preventing the oxidation of reducing agents and other substances, thus improving the long-term preservation effect of the treated solution.

[0049] In some embodiments of the present invention, the antioxidant includes at least one selected from vitamin C, sodium vitamin C, isoascorbic acid, erythritol derivative, ethylenediaminetetraacetic acid, and phytic acid.

[0050] In some embodiments of the present invention, the antioxidant is preferably a erythritol derivative.

[0051] In some embodiments of the present invention, the antioxidant is preferably sodium isoascorbate.

[0052] In some embodiments of the present invention, the treatment solution includes 0.5 to 1.5% (w / v) of antioxidant.

[0053] In some embodiments of the present invention, the treatment fluid includes an antibacterial agent, which can be used to improve the effect of preventing mold growth in pipes.

[0054] In some embodiments of the present invention, the antibacterial agent includes at least one of Proclin-950, Proclin 300, IPBC-II, and sodium azide.

[0055] In some embodiments of the present invention, the treatment solution contains 0.05% to 0.1% (v / v) of antibacterial agent.

[0056] This invention provides a ratio of whole blood sample to whole blood sample processing reagent, where whole blood sample : whole blood sample processing reagent = 1 : (1 to 5). Within this range, the whole blood sample processing reagent of this application can achieve pretreatment of whole blood samples.

[0057] In some embodiments, the whole blood sample and whole blood sample processing reagent are mixed in a ratio of 1:3 and thoroughly mixed (inverted 5 times or shaken for about 5-10 seconds). The whole blood sample mixed with the whole blood sample processing reagent can be directly subjected to nucleic acid lysis and purification.

[0058] In some embodiments, the subsequent nucleic acid lysis and purification process employs magnetic bead nucleic acid extraction. In some embodiments, when combined with the Antu fully automated platform: AutoMolec 1600 or 3000 fully automated nucleic acid purification and real-time fluorescence PCR analyzer, nucleic acid extraction and amplification detection can be completed.

[0059] In some embodiments, the pH of the treatment reagent is 7 to 9.

[0060] In some embodiments, the pH of the treatment reagent is 8.

[0061] In some embodiments, the basic formulation of the treatment solution includes: 0.4–0.8 M buffer solution, 0.1%–6% (v / v) detergent, 0.5%–1.5% (w / v) cationic surfactant and 0.2%–7.25% (w / v) reducing agent.

[0062] In some embodiments, the basic formulation of the treatment solution includes: 0.4–0.8 M buffer solution, 1%–6% (v / v) detergent, 0.5%–1.5% (w / v) cationic surfactant, 0.2%–7.25% (w / v) reducing agent and 0.5%–1.5% (w / v) antioxidant.

[0063] In some embodiments, the basic formulation of the treatment solution includes: 0.4–0.8 M buffer solution, 0.1%–6% (v / v) detergent, 0.5%–1.5% (w / v) cationic surfactant, 0.2%–7.25% (w / v) reducing agent and 0.05%–0.1% (v / v) antibacterial agent.

[0064] In some embodiments, the basic formulation of the treatment solution includes: 0.4–0.8 M buffer solution, 0.1%–6% (v / v) detergent, 0.5%–1.5% (w / v) cationic surfactant, 0.2%–7.25% (w / v) reducing agent, 0.5%–1.5% (w / v) antioxidant, and 0.05%–0.1% (v / v) antibacterial agent.

[0065] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0066] Example 1

[0067] In this embodiment, the following steps 1-6 are modified by changing the components and dosage settings in the treatment solution formulation. The treatment solution formulation includes: pH adjusted to 8, and the specific contents of each component are: 0.6M Tris-HCl buffer, 3% (v / v) Tween 20, 1% (w / v) CTAB, and 6.25% (w / v) thioglycerol.

[0068] The following 1 to 7 tests are performed on the same whole blood sample (human ALDH2 gene GG genotype) to verify and optimize different components and contents of the treatment solution.

[0069] The extraction and amplification kits used herein are the nucleic acid extraction or purification reagent (Yu Zheng Medical Device Preparation 20180037) and the human ALDH2 gene detection kit (PCR-fluorescent probe method) (National Medical Device Approval 20243400144) from Autobio Diagnostics Co., Ltd.

[0070] 1. Optimization of the pH of the treatment solution, with other components unchanged: The pretreatment of the present invention is applicable to extraction reagents for magnetic bead-based extraction, and the adsorption capacity of magnetic beads is closely related to the pH value of the environment. In this study, the pH of the treatment solution was optimized, with pH values set to 4.5, 6, 7, 8, and 9 respectively. The experimental results are shown in Table 1 below. When the pH is 7 to 9, the difference between Ct values is small; when the pH is 6, the Ct value is delayed by more than 3.25; when the pH is 4.5, the verification is delayed and even missed. There is a linear relationship between the Ct value and the logarithm of the initial template, which can accurately characterize the effective extraction amount of the target nucleic acid. A 10-fold difference in the target nucleic acid corresponds to a Ct value difference of about 3.3 cycles; corresponding to the present invention, the preferred pH of the present invention is 7 to 9, and the effective extraction amount of the target nucleic acid is increased by more than 10 times.

[0071] Table 1

[0072]

[0073] 2. Optimization of the buffer solution, with other components unchanged: Tris-HCl can stably provide a buffer environment of pH 6.5-9.0. Optimizing the amount of the buffer solution is beneficial to pH stability. Therefore, in this study, Tris-HCl at concentrations of 1M, 0.8M, 0.6M, 0.4M, and 0.2M were set for testing, and the experimental results are shown in Table 2 below. The results show that excessively high buffer content leads to delayed Ct, while excessively low buffer content leads to missed Ct detection, both affecting the treatment effect, and the preferred concentration is 0.4 to 0.8M.

[0074] Table 2

[0075]

[0076] 3. Optimization of nonionic detergents while keeping other components unchanged: Nonionic detergents can dissolve lipids, increase cell membrane permeability, dissolve erythrocytes, and prevent nonspecific protein adsorption. Three test scenarios were set up: different concentration gradients of Tween 20, different concentration gradients of other types of nonionic detergents Triton X-100, and a combination of Tween 20 and Triton X-100. Specific dosages and experimental results are shown in Table 3 below. It was found that omitting or overdosing Tween 20 resulted in poor repeatability between replicates, with Ct values ​​dropping by more than two values. Therefore, 1–6% Tween 20 and / or Triton X-100 is preferred.

[0077] Table 3

[0078]

[0079] 4. CTAB Optimization, Other Components Remain Unchanged: Cationic surfactants can dissolve cell membrane and nuclear membrane proteins, causing nuclear proteins to depolymerize and release nucleic acids. However, because CTAB is prone to precipitation at low temperatures, optimization was performed for low concentrations of CTAB. Tests were conducted with 0%, 0.5%, 1%, and 1.5% CTAB concentrations, and the results are shown in Table 4. Without CTAB, the Ct value decreased or was missed; adding a trace amount (0.5–1.5%) (w / v) had a significant effect, increasing the Ct value by more than 2.04 Ct values ​​compared to the solution without CTAB.

[0080] Table 4

[0081]

[0082] 5. Reducing agent optimization, other components unchanged: The reducing agent can be used to reduce disulfide bonds in proteins and nucleic acids. Different gradients of thioglycerol and other types of reducing agents, such as hydroxylamine hydrochloride, were tested. The experimental results are shown in Table 5 below. It was found that omitting or overdosing the addition of thioglycerol resulted in delayed or missed detection of Ct. Therefore, 1.25%–7.25% thioglycerol (w / v) or 0.2%–1% (w / v) hydroxylamine hydrochloride are preferred.

[0083] Table 5

[0084]

[0085]

[0086] 6. Reducing Agent Validation: This study screened other potential reducing agents, testing DTT, thioglycerol, cysteine, and hydroxylamine hydrochloride. The experimental results are shown in Table 6 below. Except for cysteine, all other reducing agents showed some effect. Considering the poor stability of DTT, which is not conducive to long-term reagent storage, it is suitable for immediate preparation or short-term use, or for long-term storage by combining it with antioxidants. Thioglycerol and hydroxylamine hydrochloride, on the other hand, have relatively better stability and can be used alone or in combination, making them suitable for long-term storage.

[0087] Table 6

[0088]

[0089] 7. In this embodiment, adding an antioxidant to the treatment solution formulation can prevent oxidation reactions and increase the stability of the solution system. Sodium D-isoascorbate (isoascorbate sodium) and sodium isoascorbate were tested at gradients of no addition, 0.5%, 1%, 1.5%, and 3%. Only the 3% value showed a slight decrease; the others were not significantly different from the no-addition value. The purpose of this antioxidant is to prevent the reducing agent in the reagent components from being oxidized, and it also has a preservative function. 0.5% to 1.5% (w / v) of antioxidant can be added.

[0090] In this embodiment, an antibacterial agent is added to the treatment solution formulation to increase the antibacterial properties of the solution system. When the amount of Proclin-950 added is below 0.10% v / v, it has no negative effect on the treatment of whole blood samples.

[0091] The results of the validation experiments for antioxidants and antibacterial agents are shown in Table 7 below.

[0092] Table 7

[0093]

[0094] Example 2

[0095] Three experimental groups were set up to test whole blood samples 1, 2, and 3 of the human ALDH2 gene GG genotype. Among them:

[0096] Method 1 is a whole blood sample processing reagent. In this embodiment, the processing reagent includes: pH adjusted to 8, and the specific contents of each component are: 0.6M Tris-HCl buffer, 3% (v / v) Tween 20, 0.5% (v / v) Triton X-100, 1% (w / v) CTAB, 6.25% (w / v) thioglycerol, 1% (w / v) hydroxylamine hydrochloride, 1% (w / v) sodium isoascorbate, and 0.05% (v / v) Proclin-950.

[0097] The specific usage method of the whole blood sample processing solution includes: mixing the whole blood sample with the whole blood sample processing solution (the basic formulation of Example 1) at a volume ratio of 1:3 evenly. The processed sample processing solution is subjected to nucleic acid extraction using the nucleic acid extraction or purification reagent (Yu Zheng Xie Bei 20180037 No.) from Autobio Diagnostics Co., Ltd., and then subjected to amplification and detection using the Human ALDH2 Gene Detection Kit (PCR-Fluorescent Probe Method) (Guo Xie Zhu Zhun 20243400144).

[0098] Method 2 is the pretreatment method of the CN 110747255 A patent, and the specific pretreatment steps are as follows:

[0099] 1. Take 0.2-0.4 mL of whole blood sample, add 1.2 mL of the above red blood cell lysate, mix by vortexing, lyse red blood cells for at least 1 min to obtain a clear and transparent solution;

[0100] 2. Centrifuge at 12000 rpm for 2 min, discard the supernatant, and collect the precipitate;

[0101] 3. Add 0.8 mL of whole blood precipitate suspension, fully resuspend the precipitate by vortexing, and perform a brief centrifugation;

[0102] 4. Incubate at 90°C for 5 min, vortex for 5 s, and set aside for later use;

[0103] The processed sample processing solution is subjected to nucleic acid extraction using the nucleic acid extraction or purification reagent (Yu Zheng Xie Bei 20180037 No.) all purchased from Autobio Diagnostics Co., Ltd., and then subjected to amplification and detection using the Human ALDH2 Gene Detection Kit (PCR-Fluorescent Probe Method) (Guo Xie Zhu Zhun 20243400144).

[0104] Method 3 uses the Tiangen Biotech Blood Genomic DNA Extraction Kit (Spin Column Type, Cat. No. YDP348, Jing Chang Xie Bei 20200001 No.), nucleic acid extraction is performed according to the kit's instruction manual, and amplification and detection are carried out with the Human ALDH2 Gene Detection Kit (PCR-Fluorescent Probe Method) (Guo Xie Zhu Zhun 20243400144) purchased from Autobio Diagnostics Co., Ltd.

[0105] The specific concentration detection data are shown in Table 8, and the CT values detected by matching the nucleic acids extracted by different methods with the amplification kit are shown in Table 9.

[0106] Table 8 Nucleic acid concentration test of different methods

[0107]

[0108]

[0109] Table 9. Detection results (Ct values ​​at G sites) of nucleic acid extracted by different methods combined with amplification kits

[0110] Sample 1 21.62 21.46 24.12 Sample 2 21.26 21.36 23.20 Sample 3 21.28 21.78 23.53

[0111] The results show that all three methods in the example meet the requirements of the amplification detection kit. Furthermore, the detection values ​​Ct of methods 1 and 2 are higher than those of method 3, indicating that the whole blood sample processing reagent provided in this application can effectively remove the influence of whole blood sample inhibitors on nucleic acid extraction efficiency and PCR amplification. At the same time, compared with method 2, this application only requires one operation to achieve the corresponding effect, without the need for complex equipment such as centrifugation or heating, which greatly simplifies the pretreatment process of whole blood samples.

[0112] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for processing whole blood samples, characterized in that, include: The whole blood sample is mixed with the whole blood sample processing reagent to obtain the processed whole blood sample; The whole blood sample processing reagent consists of 0.4–0.8 M buffer, 0.1%–6% (v / v) detergent, 0.5%–1.5% (w / v) cationic surfactant, and 0.2%–7.25% (w / v) reducing agent; or The whole blood sample processing reagent consists of 0.4–0.8 M buffer solution, 1%–6% (v / v) detergent, 0.5%–1.5% (w / v) cationic surfactant, 0.2%–7.25% (w / v) reducing agent, and 0.5%–1.5% (w / v) antioxidant; or The whole blood sample processing reagent consists of 0.4–0.8 M buffer, 1%–6% (v / v) detergent, 0.5%–1.5% (w / v) cationic surfactant, 0.2%–7.25% (w / v) reducing agent, and 0.05%–0.1% (v / v) antibacterial agent; or The whole blood sample processing reagent consists of 0.4-0.8M buffer, 1%-6% (v / v) detergent, 0.5%-1.5% (w / v) cationic surfactant, 0.2%-7.25% (w / v) reducing agent, 0.5-1.5% (w / v) antioxidant and 0.05%-0.1% (v / v) antibacterial agent; The pH of the treatment reagent is 7-9; The detergents include Tween 20 and / or Triton X-100; The cationic surfactant is CTAB; The reducing agent includes at least one of DTT, thioglycerol, and hydroxylamine hydrochloride; The antioxidant is sodium isoascorbate; The antibacterial agent is Proclin-950; The buffer solution is Tris-HCl buffer.

2. The processing method according to claim 1, characterized in that, The detergent comprises 1% to 6% (v / v) Tween 20 and / or 1% to 6% (v / v) Triton X-100; The reducing agent comprises 1.25% to 7.25% (w / v) thioglycerol and / or 0.2% to 1% (w / v) hydroxylamine hydrochloride.

3. The processing method according to claim 1, characterized in that, The volume ratio of the whole blood sample to the processing reagent is 1:(1~5).

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