A pre-treatment reagent, method and kit for whole blood sample RNA purification

Abstract

The present application relates to the technical field of RNA extraction, and particularly relates to a pretreatment reagent, a method and a kit for RNA purification of whole blood samples. By introducing hydrophobic DESs as the core pretreatment component, the DESs are mixed with the whole blood sample in advance, the DESs form an independent phase layer after centrifugation by using the unique physicochemical properties, and the liposoluble drug components, inflammatory proteins and pigments are selectively extracted to the hydrophobic DESs phase and the RNA is preliminarily purified; the upper water phase of the pretreatment and purification is taken and added into a lysis solution containing a chaotropic salt to fully release the nucleic acid, and DNase I is introduced for digestion. The present application combines the pretreatment of hydrophobic DESs and the digestion of DNase I, and is used in combination with PVP (targeting adsorption of residual pigments and phenolic compounds), reduces the impurities of the lysis system through the front-end pretreatment, creates a low-interference and high-efficiency purification environment for the specific binding of RNA by the magnetic beads, and forms a synergistic removal network for the interference of complex samples.

Description

Technical Field

[0001] This invention relates to the field of RNA extraction technology, and in particular to a pretreatment reagent, method, and kit for purifying RNA from whole blood samples. Background Technology

[0002] RNA extraction from complex biological samples (such as whole blood in an infected or inflammatory state) is a key prerequisite for pathogen detection and host response analysis. Although existing technologies focus on the discovery and application of diagnostic biomarkers for specific diseases, these technologies often do not delve into how to effectively process such extremely complex starting samples to obtain high-quality RNA.

[0003] Due to the extreme complexity of such sample matrices, RNA extraction faces three main universal challenges: (1) Coexistence of diverse and potent inhibitors: Samples not only contain high levels of conventional inhibitors (such as hemoglobin), but may also contain exogenous drug intervention products (such as various antibiotics and their metabolites) and endogenous metabolic disorder products (such as abnormally elevated inflammatory mediators). These substances have diverse chemical properties and complex inhibitory mechanisms, making them difficult to systematically remove through traditional lysis and purification processes. (2) High heterogeneity and complexity of sample matrices: In addition to conventional components, such samples often contain exogenous pathogen-related substances, abnormal cellular components, a large number of denatured proteins, and various metabolites. This highly heterogeneous and complex matrix easily leads to non-specific co-precipitation of nucleic acids and impurities, resulting in severe competitive binding during subsequent solid-phase extraction, which seriously affects the yield and purity of RNA. (3) Extremely stringent requirements for RNA integrity: In-depth analysis based on transcriptomes, such as studies of the host's global response gene profile, often relies on information from long transcript fragments. This requires the entire extraction process to have stronger RNase inhibition and physical protection capabilities than usual in order to obtain RNA with high integrity.

[0004] Eutectic solvents, as an emerging green solvent, have shown potential in the field of nucleic acid extraction. Existing technologies (such as the published patent CN114525275B) show that DES based on choline chloride, etc., can selectively enrich nucleic acids or impurities by disrupting cell membranes and denaturing proteins, and by utilizing the separation of hydrophobic and hydrophilic phases formed by them, and have been successfully applied to DNA extraction. However, there are still obvious shortcomings in directly applying the existing DES system to the extraction of RNA from complex whole blood: (1) Insufficient targeted inhibition of refractory RNases: Existing DES designs focus on their liquid separation effect and phase separation ability, but lack targeted inhibition efficiency optimization for specific, active, and refractory RNases (such as eosinophil-derived neurotoxins) in whole blood. Thoroughly inhibiting such RNases is the key to protecting unstable RNA molecules in complex samples. (2) Limited removal capacity for specific inhibitors in complex samples: Conventional DES systems have limited removal efficiency for complex inhibitor groups present in high concentrations in complex samples such as hemoglobin, lipopolysaccharide, and small molecule drug metabolites, which is difficult to meet the requirements of downstream high-sensitivity detection for RNA purity. Theoretically, introducing natural compounds with RNase inhibitory functions as DES components is a potential direction for solving the above-mentioned RNA integrity problem. Literature reports suggest that certain pentacyclic triterpenoids (such as ursolic acid and lupeol) may have inhibitory potential against specific RNases. However, converting common components of existing DES systems (such as choline chloride, hexafluoroisopropanol, decanoic acid, glycerol, etc.) into practically usable DES formulations faces significant technical obstacles. The specific analysis is as follows: (1) Limitations of the physicochemical properties of active ingredients: Compounds such as ursolic acid and lupeol, which have potential inhibitory activity, are high-melting-point solids at room temperature and have extremely low solubility in water and common organic solvents. Their complex molecular structure and scarcity determine that they are difficult to use as basic reagents for large-scale, stable, and low-cost DES formulation. (2) Application defects of conventional hydrophobic components: Other conventional components that may form hydrophobic DES also have inherent defects. For example, undecyl alcohol may cause excessively high DES viscosity, resulting in slow phase separation and RNA loss; acetic acid, due to its strong acidity and volatility, is prone to RNA hydrolysis and has poor system reproducibility.

[0005] Therefore, developing a dedicated RNA extraction system capable of simultaneously achieving efficient lysis, strong inhibition of complex sample-specific RNases, and deep removal of complex small molecule inhibitors is of great significance for advancing the accurate diagnosis and research of complex samples. Based on this, it is necessary to provide RNA purification pretreatment reagents, methods, and kits for complex whole blood samples to address the shortcomings of existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a pretreatment reagent, method, and kit for RNA purification from whole blood samples. By optimizing the pretreatment reagent, hydrophobic DESs are introduced as the core pretreatment component. These are first mixed with the whole blood sample. Utilizing their unique physicochemical properties, the DESs form an independent phase layer after centrifugation, selectively extracting lipid-soluble drug components, inflammatory proteins, and pigments into the hydrophobic DESs phase and initially purifying the RNA. The upper aqueous phase, purified by this pretreatment, is then collected, and a lysis buffer containing lysis salt is added to fully release the nucleic acid. RNase-free DNase I is then introduced for digestion, allowing DNase I to completely degrade gDNA under optimal conditions. This invention combines hydrophobic DESs pretreatment with DNase I digestion, and uses PVP (targeted adsorption of residual pigments and phenols). This process significantly reduces the impurity load of the lysis system through front-end pretreatment, creating a low-interference, high-efficiency purification environment for subsequent magnetic bead-specific RNA binding, forming a multi-level, synergistic removal network for complex sample interferences. In particular, it provides a multi-level, synergistic removal network for interferences in whole blood samples from patients with bloodstream infections.

[0007] To achieve the above objectives, embodiments of the present invention provide a pretreatment reagent for RNA purification from whole blood samples, comprising a hydrophobic eutectic solvent DESs and polyvinylpyrrolidone (PVP); wherein the hydrogen bond donor in the hydrophobic eutectic solvent is thymol.

[0008] Preferably, the hydrogen bond acceptor HBA in the hydrophobic eutectic solvent includes choline chloride.

[0009] Preferably, the hydrogen bond donor in the hydrophobic eutectic solvent further includes at least one of menthol and geraniol.

[0010] In this invention, the unique physicochemical properties of hydrophobic DESs are fully utilized to achieve dual purification: (1) Phase separation extraction: Hydrophobic DESs form a clear interface with the aqueous phase, selectively extracting inhibitors such as denatured proteins, lipids, hemoglobin, and lipid-soluble drugs into the hydrophobic DESs phase. (2) Activity protection: The hydrophobic DESs formed by the natural monoterpene composition containing thymol can efficiently lyse cells due to the membrane interference properties of thymol, menthol, and geraniol in the natural monoterpene composition. It has natural antibacterial properties and synergistic liquid-free effect with the hydrophobic DESs, strongly inhibiting RNase and providing "barrier-like" protection for fragile RNA.

[0011] Preferably, the molar ratio of hydrogen bond acceptors to hydrogen bond donors in the hydrophobic eutectic solvent is 1:2 to 1:4. More preferably, the molar ratio of hydrogen bond acceptors to hydrogen bond donors in the hydrophobic eutectic solvent is 1:3.

[0012] Preferably, the mass concentration of the polyvinylpyrrolidone is 1%-1.5%. More preferably, the mass concentration of the polyvinylpyrrolidone is 1.5%.

[0013] The present invention also aims to provide a pretreatment method for RNA purification from whole blood samples, comprising the following steps:

[0014] S1: Take a whole blood sample from a patient with bloodstream infection, add the above pretreatment reagent, immediately vortex and mix vigorously to obtain a homogeneous emulsion, and then incubate at room temperature.

[0015] S2: Centrifuge the whole blood sample processed in step S1 to obtain the supernatant, add the lysis extraction buffer containing the lysis salt and nucleic acid extraction magnetic beads, mix thoroughly to lyse the residual cells and release the nucleic acid;

[0016] S3: After the treatment in step S2, add RNase-free DNase I, gently mix by blowing, and incubate at a certain temperature.

[0017] Preferably, the preparation process of the pretreatment reagent in step S1 specifically includes the following steps:

[0018] S1.1: Mix hydrogen bond acceptors and hydrogen bond donors in a molar ratio, stir at a heating temperature to obtain a homogeneous and clear liquid, and cool to room temperature to obtain hydrophobic DESs;

[0019] S1.2: While stirring, slowly add PVP to the buffer solution until fully dissolved, then add hydrophobic DESs and stir until homogeneous to obtain the pretreatment reagent.

[0020] Preferably, the volume concentration of hydrophobic DESs in the pretreatment reagent is 15%-25%. More preferably, the volume concentration of hydrophobic DESs in the pretreatment reagent is 20%.

[0021] Preferably, the lysis extract comprises 4 M guanidine isothiocyanate or guanidine hydrochloride, 20 mM-50 mM Tris-HCl, 1 mM-5 mM EDTA, N-lauroyl sarcosinate sodium with a mass concentration of 0.5%-1%, and RNase-free water.

[0022] Another object of the present invention is to provide a kit for RNA purification from whole blood samples, which uses the above-mentioned pretreatment reagents to treat patients with bloodstream infections.

[0023] Preferably, the kit includes a negative control and a positive control.

[0024] Reaction mechanism

[0025] This invention creatively selects choline chloride as a hydrogen bond acceptor (HBA) and natural monoterpenes such as thymol, menthol, and geraniol as hydrogen bond donors (HBD) to construct a functionalized hydrophobic eutectic solvent (DES). This design fully utilizes the physical properties of these monoterpenoid HBDs, which are liquid or low-melting-point solids at room temperature (e.g., thymol has a melting point of 49-51°C), enabling them to readily form a homogeneous and stable liquid hydrophobic DES phase with choline chloride. This hydrophobic DES phase combines the advantages of inexpensive and readily available raw materials, allowing for large-scale industrial production, with the function of extracting and separating impurities through its hydrophobicity and intermolecular forces. To overcome the interference of specific impurities (such as hemoglobin metabolites) in complex whole blood samples (patients with bloodstream infections), this invention introduces polyvinylpyrrolidone (PVP) into the pretreatment process. The carbonyl group on the PVP molecular chain can form insoluble complexes with impurities in the sample, such as phenols and pigments (e.g., the porphyrin ring of hemoglobin), through hydrogen bonding and hydrophobic interactions, thereby being specifically removed in subsequent centrifugation or phase separation steps. This mechanism directly and significantly improves the purity of RNA products (specifically, by optimizing the A260 / A230 ratio), fundamentally ensuring the accuracy and sensitivity of downstream detection methods such as qRT-PCR.

[0026] The above-described solution of the present invention has the following beneficial effects:

[0027] This invention utilizes choline chloride to provide the basis for ion interactions and hydrogen bonding networks, thymol to provide strong hydrophobic driving forces, protein binding forces, and auxiliary RNase inhibition capabilities, and PVP to specifically adsorb special impurities such as pigments. When these three components are mixed in a specific ratio, a "contaminant collection phase" with suitable viscosity and good phase separation behavior is formed. Ultimately, this achieves:

[0028] (1) The efficient co-purification and complete preservation of total RNA in whole blood samples from patients with complex bloodstream infections provides a high-quality nucleic acid template for qRT-PCR;

[0029] (2) Simultaneous deep removal of multiple impurities such as proteins, endotoxins, lipids and small molecule drug inhibitors;

[0030] (3) While performing strong lysis, it effectively protects the integrity and activity of RNA, meeting the high-sensitivity detection requirements of downstream qRT-PCR, sequencing and other processes.

[0031] Compared to existing technologies that use forward nucleic acid capture, this invention systematically overcomes the core technical bottlenecks of easy RNA degradation and difficult inhibitor removal in complex (bloodstream infection patients) whole blood samples through the front-end design of hydrophobic DES "reverse impurity removal", achieving a synergistic purification effect of 1+1>2. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 These are the RNA amplification curves of Example 1 and Comparative Example 1 of the present invention; wherein, the dotted line is Comparative Example 1 and the solid line is Example 1.

[0034] Figure 2 These are the RNA amplification curves of Example 1 and Comparative Example 2 of the present invention; wherein, the dotted line is Comparative Example 2 and the solid line is Example 1.

[0035] Figure 3 These are the RNA amplification curves of Examples 1, 5, and 6 of the present invention; wherein, the solid line represents Example 1, the dotted line represents Example 5, and the dashed line represents Example 6;

[0036] Figure 4 These are RNA amplification curves for Examples 1 and 7, Comparative Examples 2 and 3 of the present invention; wherein, the solid line represents Example 7; the dotted line represents Example 1; the triangular line represents Comparative Example 2; and the dashed line represents Comparative Example 3. Figure 4 (a) Amplification curves of STOM target RNA in Examples 1 and 7, Comparative Examples 2 and 3; Figure 4 (b) are the amplification curves of the internal control RNA in Examples 1 and 7, Comparative Example 2, and Comparative Example 3; Figure 4 (c) Amplification curves of ABHD14B target RNA for Examples 1 and 7, Comparative Examples 2 and 3; Figure 4 (d) shows the FLT3LG target RNA amplification curves of Examples 1 and 7, Comparative Examples 2 and 3. Detailed Implementation

[0037] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0038] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0039] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0040] In this document, "%" specifically refers to mass concentration or volume fraction; in the following tables, "S1" is the sample of Example 1, "S2" is the sample of Example 2, "S3" is the sample of Example 3, "S4" is the sample of Example 4, "S5" is the sample of Example 5, "S6" is the sample of Example 6, and "S7" is the sample of Example 7; in the following tables, "D1" is the sample of Comparative Example 1, "D2" is the sample of Comparative Example 2, "D3" is the sample of Comparative Example 3, "D4" is the sample of Comparative Example 4, "D5" is the sample of Comparative Example 5, "D6" is the sample of Comparative Example 6, "D7" is the sample of Comparative Example 7, "D8" is the sample of Comparative Example 8, "D9" is the sample of Comparative Example 9, "D10" is the sample of Comparative Example 10, "D11" is the sample of Comparative Example 11, "D12" is the sample of Comparative Example 12, and "D13" is the sample of Comparative Example 13.

[0041] This invention addresses existing problems by providing a pretreatment reagent, method, and kit for RNA purification from whole blood samples.

[0042] This invention utilizes choline chloride to provide the basis for ion interactions and hydrogen bonding networks, thymol to provide strong hydrophobic driving force, protein binding force, and auxiliary RNase inhibition ability, and PVP to specifically adsorb special impurities such as pigments. The three components are mixed in a specific ratio to form a "contaminant collection phase" with suitable viscosity and good phase separation behavior.

[0043] Compared to existing technologies that use forward nucleic acid capture, this invention systematically overcomes the core technical bottlenecks of easy RNA degradation and difficult inhibitor removal in complex (bloodstream infection patients) whole blood samples through the front-end design of hydrophobic DES "reverse impurity removal", achieving a synergistic purification effect of 1+1>2.

[0044] The following will be explained through specific embodiments and comparative examples.

[0045] Example 1

[0046] This embodiment provides a pretreatment method for purifying RNA from whole blood samples of patients with bloodstream infections, specifically including the following steps:

[0047] S1: Take 100μL of whole blood sample from a patient with bloodstream infection and add 300μL of hydrophobic DES final treatment working solution. Immediately vortex mix vigorously for 15s to obtain a uniform emulsion. Then let it stand at room temperature for 5min to allow the hydrophobic DES phase to fully bind with impurities such as proteins and lipids.

[0048] S2: Centrifuge the whole blood sample processed in step S1 at 12000×g for 5min to remove impurities such as denatured proteins and peptides, small molecule organic matter and drugs, lipids and microbial membrane components.

[0049] S3: Take the supernatant (clear or slightly turbid aqueous phase containing nucleic acid) after centrifugation in step S2 and add it to the lysis extraction buffer containing the lysis salt (4M guanidine isothiocyanate or guanidine hydrochloride, 20mM-50mM Tris-HCl, 1mM-5mM EDTA, N-lauroyl sarcosinate sodium with a mass concentration of 0.5%-1%, RNase-free water), mix thoroughly to lyse residual cells and release nucleic acids;

[0050] S4: After the treatment in step S3, add 5-10U of RNase-free DNase I, gently mix by pipetting, and incubate at 37°C for 15 minutes to remove genomic DNA;

[0051] S5: RNA was washed and eluted using conventional magnetic bead method to obtain RNA; this RNA can be used as a template for subsequent PCR.

[0052] The preparation process of the hydrophobic DESs final treatment working solution specifically includes the following steps:

[0053] S1.1: Prepare basic hydrophobic DES (choline chloride: thymol molar ratio 1:3)

[0054] a. Weigh out 13.96g of choline chloride (0.1mol) and 45.07g of thymol (0.3mol);

[0055] b. Place the weighed choline chloride and thymol in a dry flask and stir magnetically at 70-80℃ until a homogeneous, clear, particle-free transparent liquid is formed.

[0056] c. Cooling and storage: Cool to room temperature to obtain a pale yellow, oily, hydrophobic base DES. Store in a sealed container away from light.

[0057] S1.2: Prepare the DES final treatment working solution (20% (v / v) basic hydrophobic DES + 1% (w / v) PVP)

[0058] a. Weigh out 20 mL of basic hydrophobic DES and 1.0 g of PVP;

[0059] b. While stirring, slowly add 1.0 g of PVP to about 50 mL of pH-neutral buffer (50 mM Tris-HCl, pH 7.0) until fully dissolved. Then add 20 mL of basic hydrophobic DES and continue stirring to homogenize the system. Finally, add the above buffer to make up to 100 mL to obtain the final treatment working solution of hydrophobic DESs. Store at 4 °C for later use.

[0060] Choline chloride as a hydrogen bond acceptor: Choline chloride is a quaternary ammonium salt cationic compound. Its quaternary ammonium cation can transiently bind to negatively charged nucleic acid phosphate backbones, endotoxin polysaccharide chains, and cell membrane phospholipids through electrostatic interactions, effectively disrupting membrane structures and promoting the release of nucleic acids from protein complexes. Its hydroxyl groups can form an extended hydrogen bond network with hydrogen bond donors (such as the phenolic hydroxyl group of thymol), constituting a polar matrix of the eutectic solvent and endowing the system with unique solvation capabilities. Thymol as a hydrogen bond donor: Thymol is a monoterpene phenol with an aromatic ring and phenolic hydroxyl group, exhibiting strong hydrophobicity. Its hydrophobic aromatic ring can strongly bind to denatured proteins (especially hydrophobic residues exposed under inflammatory conditions) and lipids through π-π stacking and hydrophobic interactions, promoting the aggregation and precipitation of such impurities. Simultaneously, its phenolic hydroxyl group possesses both antioxidant and weak acidic properties, locally inhibiting RNase activity, thereby protecting RNA integrity and playing a role in bioprotection. Furthermore, the high hydrophobicity of thymol is a core factor driving the separation of the eutectic solvent phase from the aqueous phase. Furthermore, the synergistic mechanism of the eutectic solvent: the hydrophobic eutectic solvent formed by the combination of choline chloride and thymol can serve as a "contaminant collection phase." This system, through multiple interactions including hydrogen bonding, ions, and hydrophobicity, efficiently extracts impurities such as proteins, lipids, most endotoxins, and small molecule inhibitors from whole blood samples of patients with bloodstream infections from the aqueous phase and distributes them to the lower DES phase, achieving preliminary separation of impurities from target nucleic acids. System advantages: Compared to pure ionic liquids, this eutectic solvent system has a moderate viscosity, ensuring both the speed of phase separation and the efficient diffusion and transfer of RNA released into the aqueous phase. Simultaneously, the low-water-activity environment it creates helps indirectly stabilize the secondary structure of RNA. The synergistic mechanism of the auxiliary additive PVP: To address the complex pathological impurities (such as hemoglobin derivatives) unique to whole blood samples of patients with bloodstream infections, the water-soluble polymer polyvinylpyrrolidone is introduced into the hydrophobic DES system. PVP can form hydrogen bonds or hydrophobic interactions with specific impurities in the sample, such as phenols and pigments (e.g., the porphyrin ring of hemoglobin), through the carbonyl group on its molecular chain, generating insoluble complexes that are then specifically removed during centrifugation or phase separation. This significantly improves the purity of the final RNA product (specifically, by optimizing the A260 / A230 ratio), thus effectively ensuring the accuracy and reliability of downstream high-sensitivity detection methods such as qRT-PCR.

[0061] Example 2

[0062] The difference from Example 1 is that 0.3 mol of thymol is replaced with 0.15 mol of thymol and 0.15 mol of menthol. All other steps and parameters are the same as in Example 1.

[0063] Example 3

[0064] The difference from Example 1 is that 0.3 mol of thymol is replaced with 0.15 mol of thymol and 0.15 mol of geraniol, while the other steps and parameters are the same as in Example 1.

[0065] Example 4

[0066] The difference from Example 1 is that 0.3 mol thymol is replaced with 0.1 mol thymol, 0.1 mol menthol and 0.1 mol geraniol, while the other steps and parameters are the same as in Example 1.

[0067] Example 5

[0068] The difference from Example 1 is that 0.3 mol of thymol is replaced with 0.2 mol of thymol, while the other steps and parameters are the same as in Example 1.

[0069] Example 6

[0070] The difference from Example 1 is that 0.3 mol of thymol is replaced with 0.4 mol of thymol, while the other steps and parameters are the same as in Example 1.

[0071] Example 7

[0072] The difference from Example 1 is that 1.0g of PVP is replaced with 1.5g of PVP, while the other steps and parameters are the same as in Example 1.

[0073] Comparative Example 1

[0074] RNA was extracted using a conventional magnetic bead method kit, without hydrophobic DES and DNase I digestion.

[0075] Comparative Example 2

[0076] The difference from Example 1 is that PVP is not added.

[0077] Comparative Example 3

[0078] The difference from Example 1 is that 1.0g of PVP is replaced with 2.5g of PVP.

[0079] Comparative Example 4

[0080] The difference from Example 1 is that thymol is replaced with menthol.

[0081] Comparative Example 5

[0082] The difference from Example 1 is that thymol is replaced with geraniol.

[0083] Comparative Example 6

[0084] The difference from Example 1 is that thymol is replaced with carvacrol.

[0085] Comparative Example 7

[0086] The difference from Example 1 is that 0.3 mol thymol is replaced with 0.15 mol menthol and 0.15 mol carvacrol.

[0087] Comparative Example 8

[0088] The difference from Example 1 is that 0.3 mol thymol is replaced with 0.15 mol geraniol and 0.15 mol carvacrol.

[0089] Comparative Example 9

[0090] The difference from Example 1 is that 0.3 mol thymol is replaced with 0.15 mol menthol and 0.15 mol geraniol.

[0091] Comparative Example 10

[0092] The difference from Example 1 is that 0.3 mol thymol is replaced with 0.1 mol carvacrol, 0.1 mol menthol and 0.1 mol geraniol, while the other steps and parameters are the same as in Example 1.

[0093] Comparative Example 11

[0094] The difference from Example 1 is that choline chloride and thymol are replaced with betaine and hexafluoroisopropanol.

[0095] Comparative Example 12

[0096] The difference from Example 1 is that thymol is replaced with decanoic acid.

[0097] Comparative Example 13

[0098] The difference from Example 1 is that choline chloride and thymol are replaced with betaine and decanoic acid.

[0099] Five standardized simulated RNA samples extracted from the above-described examples and comparative examples were tested using a UV spectrophotometer. The A260 / A230 test results are shown in Table 1 below.

[0100] Table 1

[0101]

[0102] As shown in Table 1 above, the RNA purity (A260 / A230) of all experimental groups containing thymol (Examples 1 to 4) remained stable in the high range of 2.02 to 2.11, proving that thymol is an essential core substance for extracting high-purity RNA from whole blood samples of patients with complex bloodstream infections.

[0103] In the comparative examples without thymol, the systems containing carvacrol (also phenols) (Comparative Examples 6, 7, 8, and 10) generally had higher purity (1.83-1.92) than the pure alcohol systems (Comparative Examples 4, 5, and 9, purity 1.72-1.78). This indicates that the phenolic hydroxyl structure has certain common advantages in impurity removal.

[0104] However, Comparative Example 6 (single carvacrol) was less effective than Example 1 (single thymol), demonstrating that not all phenols are equivalent. The smaller hydroxyl group (-OH) of thymol allows it to form a stronger, tighter, and more regular hydrogen bond network with choline chloride, resulting in a more homogeneous DES phase and more stable viscosity. Furthermore, its moderate hydrophobicity ensures that the DES phase can effectively bind impurities while also rapidly and thoroughly separating from the aqueous phase after centrifugation, avoiding RNA entrainment and loss due to excessive hydrophobicity. Carvacrol DES may lead to incomplete phase separation or unclear interfaces. The specific structure and hydrophobic balance of thymol are the unique reasons for its superior performance.

[0105] Even using the most complex alternative combination, Comparative Example 10 (menthol + geraniol + carvacrol), its optimal purity was still significantly lower than that of the simplest core combination, Example 1 (choline chloride: thymol). This demonstrates that, without including the core component (thymol) of this invention, no simple mixture or complex combination of any other monoterpenoids can achieve or surpass the technical effects of this invention.

[0106] The advantage of this invention stems from the inherent properties of specific components, rather than from the simple summation of components:

[0107] Choline chloride and thymol in a 1:3 molar ratio (Example 1) represent the performance baseline and optimal solution of this invention. Introducing menthol and / or geraniol into thymol (Examples 2 to 4) results in a slight but stable increase in purity (to approximately 2.11).

[0108] Comparative Example 4: Choline chloride:carvacrol combination, where carvacrol is the methoxy isomer of thymol. The methoxy group (-OCH3) is larger than the hydroxyl group (-OH), which may hinder the formation of a tight and stable hydrogen bond network with choline chloride. This results in slightly inferior thermal stability or solution behavior of the hydrophobic DES itself, thus affecting its uniformity and reliability as a "purifying phase".

[0109] Betaine and hexafluoroisopropanol are a combination of these compounds. Hexafluoroisopropanol is a highly polar fluorinated alcohol, and the resulting DES is too hydrophilic to drive the clear separation of the aqueous and hydrophobic phases. Its strong electron-withdrawing property may interfere with nucleic acid stability. Furthermore, it completely lacks bioactive molecules (such as phenolic RNase inhibitors) and cannot protect RNA in the early stages of lysis.

[0110] Choline chloride: Decanoic acid combination. Decanoic acid is a long-chain fatty acid, and the resulting DES has excessively high viscosity, leading to extremely slow phase separation. RNA is easily encapsulated in the high-viscosity DES phase and is difficult to release, resulting in a sharp drop in recovery rate and operational difficulties. Betaine: Decanoic acid combination. The carboxylic acid inner salt structure of betaine is less efficient as a hydrogen bond acceptor than quaternary ammonium salts, and the introduction of decanoic acid leads to high viscosity. This system performs poorly in phase separation, impurity removal, and operation.

[0111] Discoveries regarding the optimization of the hydrogen bond donor (HBD) ratio

[0112] In the construction of the eutectic solvent (DES) system, the ratio of hydrogen bond donor (HBD) to hydrogen bond acceptor (HBA) is a key parameter affecting nucleic acid purification efficiency. Experiments show that when the HBD ratio is low (e.g., a molar ratio of 1:2), the thymol content in the system is relatively insufficient. This results in the overall hydrophobicity of the DES and its key binding ability to proteins and other impurities in the sample not reaching optimal levels, thus failing to achieve optimal purification results for complex matrices.

[0113] Conversely, when the HBD ratio is too high (e.g., a molar ratio of 1:4), the viscosity of the DES system will increase significantly. Excessive viscosity may have the following adverse consequences: firstly, it increases the physical adsorption or retention loss of RNA during the process; secondly, in subsequent phase separation and aqueous phase transfer steps, trace amounts of DES phase may be entrained into the final product, potentially introducing inhibitors and thus affecting the purity and recovery rate of the extracted RNA.

[0114] Limitations on the working concentration of eutectic solvent (DES)

[0115] Furthermore, the overall working concentration of DES in the lysis and purification system is also crucial. If the concentration of DES is too low (e.g., less than 20% by volume), it means insufficient effective functional components. In this case, the DES system may not be able to adequately handle the large amounts of coexisting high concentrations of inhibitors (such as hemoglobin, inflammatory proteins, and drug metabolites) in complex whole blood samples (patients with bloodstream infections), resulting in incomplete removal of impurities and failing to meet the purity requirements of downstream high-sensitivity molecular detection for nucleic acids.

[0116] Optimization of polyvinylpyrrolidone (PVP) mass concentration

[0117] Experiments revealed that the mass concentration of the auxiliary additive polyvinylpyrrolidone (PVP) has a significant and non-linear effect on the purity of the final RNA product. RNA purity initially increases and then decreases with increasing PVP concentration, peaking at 1.5% (w / v). This phenomenon reveals a dual mechanism of PVP's effect: when the PVP concentration is insufficient, its molecules cannot adequately form insoluble complexes (such as phenols and pigments) with complex impurities in whole blood samples (from patients with bloodstream infections), resulting in insufficient impurity adsorption and a lower baseline RNA purity. Conversely, when the PVP concentration exceeds the optimal threshold, the inherent thickening effect of PVP polymers adversely alters the overall working fluid viscosity. This change in physical property triggers a series of operational problems, such as reduced liquid-phase mass transfer efficiency, unclear phase separation interfaces, or entrainment of trace viscous components. These process disturbances ultimately interfere with the purification process, leading to a decrease in RNA extraction purity. Therefore, precisely limiting the PVP concentration to 1.5% (w / v) is one of the key process parameters optimized and established in this invention to overcome the unique interferences in complex whole blood samples (patients with bloodstream infections) and achieve high-purity RNA extraction. This specific concentration achieves the optimal balance between impurity removal and maintaining operational feasibility in this complex sample system.

[0118] The above embodiments combine hydrophobic DESs pretreatment with DNase I digestion and PVP. This process significantly reduces the impurity load of the lysis system through pretreatment, creating a low-interference, high-efficiency purification environment for subsequent magnetic bead-specific RNA binding, forming a multi-level, synergistic removal network for interfering substances in whole blood samples from patients with bloodstream infections. Furthermore, the RNA obtained in the above embodiments and comparative examples is used as a template for PCR, specifically including the following steps:

[0119] (1) The PCR primer and probe sequences are shown in Table 2 below.

[0120] Table 2

[0121]

[0122] (2) The PCR reaction system is shown in Table 3 below.

[0123] Table 3

[0124]

[0125] (3) The PCR reaction procedure is shown in Table 4 below.

[0126] Table 4

[0127]

[0128] Amplification results as follows Figures 1 to 4As shown, blue: STOM; green: internal control gene; orange: ABHD14B; red: FLT3LG; Figure 1 The images show the RNA amplification curves for Example 1 and Comparative Example 1. The Ct value of Example 1 is significantly lower than that of Comparative Example 1, which demonstrates that pretreatment with hydrophobic DES and DNase I improves RNA yield. Figure 2 These are the RNA amplification curves of Example 1 and Comparative Example 2 of the present invention. The introduction of PVP significantly improved RNA purity and resulted in better performance of downstream PCR experiments. Figure 3 The RNA amplification curves of Examples 1, 5, and 6 of this invention show that the RNA purification effect is optimal when the molar ratio of HBA to HBD is 1:3. Figure 4 These are the RNA amplification curves of Examples 1 and 7, Comparative Example 2 and Comparative Example 3 of the present invention. The RNA purity was highest when the PVP mass concentration was 1.5%.

[0129] The above description represents the 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 principles 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 pretreatment reagent for RNA purification from whole blood samples, characterized in that, The mixture comprises a hydrophobic eutectic solvent and polyvinylpyrrolidone; the hydrogen bond donor in the hydrophobic eutectic solvent includes thymol; the hydrogen bond acceptor in the hydrophobic eutectic solvent includes choline chloride; the molar ratio of hydrogen bond acceptor to hydrogen bond donor in the hydrophobic eutectic solvent is 1:2 to 1:4; the mass concentration of the polyvinylpyrrolidone is 1%-1.5%; and the volume concentration of the hydrophobic eutectic solvent is 15%-25%.

2. The pretreatment reagent for RNA purification from whole blood samples according to claim 1, characterized in that, The hydrogen bond donors in the hydrophobic eutectic solvent also include at least one of menthol and geraniol.

3. The pretreatment reagent for RNA purification from whole blood samples according to claim 1, characterized in that, The molar ratio of hydrogen bond acceptor to hydrogen bond donor in the hydrophobic eutectic solvent is 1:3; the mass concentration of the polyvinylpyrrolidone is 1.5%.

4. A pretreatment method for RNA purification from whole blood samples, characterized in that, Includes the following steps: S1: Take a whole blood sample from a patient with bloodstream infection, add the pretreatment reagent as described in claim 1, immediately vortex mix vigorously to obtain a homogeneous emulsion, and then incubate at room temperature. S2: Centrifuge the whole blood sample processed in step S1 to obtain the supernatant, add the lysis extraction buffer containing the lysis salt and nucleic acid extraction magnetic beads, mix thoroughly to lyse the residual cells and release the nucleic acid; S3: After the treatment in step S2, add RNase-free DNase I, gently mix by blowing, and incubate at a certain temperature.

5. The pretreatment method for RNA purification from whole blood samples according to claim 4, characterized in that, The preparation process of the pretreatment reagent in step S1 specifically includes the following steps: S1.1: Mix hydrogen bond acceptors and hydrogen bond donors in a molar ratio, stir at a heating temperature to obtain a homogeneous and clear liquid, and cool to room temperature to obtain a hydrophobic eutectic solvent. S1.2: While stirring, slowly add polyvinylpyrrolidone to the buffer solution until fully dissolved, then add a hydrophobic eutectic solvent and stir until homogeneous to obtain the pretreatment reagent.

6. The pretreatment method for RNA purification from whole blood samples according to claim 4, characterized in that, The lysis extract includes 4M guanidine isothiocyanate or guanidine hydrochloride, 20mM-50mM Tris-HCl, 1mM-5mM EDTA, 0.5%-1% N-lauroyl sarcosinate sodium, and RNase-free water.

7. A kit for RNA purification from whole blood samples, characterized in that, Includes the pretreatment reagent for RNA purification of whole blood samples as described in claim 1.

8. A kit for RNA purification from whole blood samples according to claim 7, characterized in that, The kit includes a positive control and a negative control.

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