An alcohol-free rapid nucleic acid extraction kit and an extraction method thereof

Abstract

The present application relates to the technical field of nucleic acid extraction, in particular to a non-alcohol nucleic acid rapid extraction kit and an extraction method thereof, the kit is composed of four components, namely, a lysis solution, a magnetic bead suspension, a non-alcohol washing solution and an eluent, and none of the four components contains ethanol and isopropyl alcohol, the non-alcohol washing solution contains 0.5 to 1.5 moles of imidazole or imidazole compounds per liter and 1.2 to 2.5 moles of inorganic salts per liter, and the pH is 6.5 to 7.5, through the competitive replacement effect of imidazole molecules on the non-specific adsorption of protein on the surface of magnetic beads, the protein impurities can be efficiently removed by single-step washing, the extraction method comprises three steps of lysis combination, single-step washing and elution, and the total operation time is not more than 15 minutes. 260 / A 280 The ratio reaches 1.85 to 1.95.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction technology, specifically to an alcohol-free rapid nucleic acid extraction kit and its extraction method. Background Technology

[0002] Nucleic acid extraction is a fundamental preliminary step in modern molecular diagnostics, infectious disease screening, tumor companion diagnostics, precision medicine, gene testing, and the construction of biobanks. The yield, purity, and downstream compatibility of the extracted product directly determine the sensitivity, specificity, and batch-to-batch stability of downstream detection methods such as real-time quantitative polymerase chain reaction (qPCR), reverse transcription polymerase chain reaction (RTP), digital polymerase chain reaction (DPCR), isothermal amplification (ARA), and next-generation sequencing (NGS). Currently, the mainstream approach used in clinical testing and molecular diagnostic laboratories is the magnetic bead-based nucleic acid extraction kit. This method utilizes the adsorption of nucleic acids by surface-modified superparamagnetic iron oxide microspheres in a high-ionic-strength environment of guanidine salt. An external magnetic field is applied to separate nucleic acids from biological background impurities. After multiple washing steps to remove impurities such as proteins, polysaccharides, and lipids, purified nucleic acids are obtained by elution with a low-salt buffer. This method, which does not require centrifugation and organic solvent extraction, is widely used in high-throughput screening, automated extraction, and point-of-care molecular diagnostics. Since Boom et al. proposed the principle of nucleic acid adsorption based on guanidine isothiocyanate and silica matrix in the 1990s, it has become the fourth generation of mainstream nucleic acid purification technology after phenol-chloroform extraction, salting out, and silica gel column centrifugation.

[0003] However, commercially available magnetic bead-based nucleic acid extraction kits still generally rely on anhydrous ethanol or isopropanol as organic solvents in their washing systems, which has led to three major technical challenges that restrict their clinical application.

[0004] First, the alcoholic organic solvents introduced in the washing step are difficult to completely remove during the elution stage, often leaving 0.5% to 2% ethanol or isopropanol residues in the extract. Existing studies have shown that residual ethanol concentrations exceeding 1% in the elution product can significantly reduce Taq polymerase activity, leading to decreased polymerase chain reaction (PCR) amplification efficiency or amplification failure. For real-time quantitative PCR, alcohol residues can cause Ct values ​​to drift by 1 to 3 cycles, severely affecting the detection limit for low-copy targets, particularly in low-viral-load samples. Furthermore, isopropanol has a stronger inhibitory effect on reverse transcriptase, making alcohol residues a major factor contributing to false-negative results in viral RNA extraction scenarios. In next-generation sequencing library construction, alcohol residues also affect adapter ligation efficiency and library amplification cycle number, resulting in data loss.

[0005] Secondly, traditional magnetic bead-based nucleic acid extraction kits typically involve three to four washing steps to thoroughly remove impurities such as proteins and polysaccharides and avoid interference from residual alcohol. Each step requires adding washing solution, mixing, magnetic adsorption, and discarding the supernatant. Some kits also require a room temperature drying step after the final wash to evaporate residual alcohol. The extraction time for a single batch is generally between 30 and 40 minutes. For example, QIAGEN's QIAamp DNA Blood Mini Kit has a total operation time of approximately 30 minutes, Tiangen Biotech's DP304 blood cell and tissue genomic DNA extraction kit has a total operation time of approximately 35 minutes, and Thermo Fisher's MagMAX series automated extraction kits take approximately 25 to 30 minutes on an automated extraction instrument. This time window restricts the clinical timeliness requirements for high-throughput screening, emergency sample testing, and large-volume sample processing in fever clinics. In emergency screening scenarios for emerging and re-emerging infectious disease outbreaks, reducing sample turnaround time by 10% can significantly improve screening throughput and early prevention and control capabilities.

[0006] Third, alcohol washing steps have poor compatibility with automated nucleic acid extractors. Ethanol and isopropanol are volatile organic compounds (VOCs), requiring dedicated sealed dispensing systems, anti-volatile caps, and VOC emission control devices in automated instrument packaging design, significantly increasing instrument costs and daily maintenance requirements. Furthermore, in high-throughput scenarios with 96-well or 384-well plates, volatile reagents exhibit liquid level drops and concentration drift during multiple transfers by the robotic arm, affecting batch-to-batch consistency. Alcohol reagents also present flammable and explosive safety management requirements during dispensing and transportation, increasing cold chain and storage costs. Given the increasingly stringent VOC emission controls in medical institutions, alcohol-free solutions place a significantly lower burden on laboratory ventilation and waste management.

[0007] In recent years, some studies have attempted to replace ethanol or isopropanol with components such as polyethylene glycol, sodium citrate, glycol compounds, and nonionic surfactants in magnetic bead washing systems. For example, US Patent Application No. US20240368581A1 discloses an alcohol-free magnetic bead nucleic acid extraction system based on guanidine isothiocyanate, polyethylene glycol 8000, sodium chloride, and nonionic surfactants. This system employs a two-step washing process, and the impurity removal mechanism follows the traditional pathway of polyethylene glycol precipitation-assisted retention and nonionic surfactant elution. US Patent Application No. US20110224419A1 systematically studied the application of ethanol-substituted chemicals in the purification of nucleic acids from silicon-based membranes and silicon-coated magnetic microspheres. The substitutes tested in this application were all polyols and ethers, and the mechanism still relied on low dielectric constant simulation. None of the above solutions address the real bottleneck of protein nonspecific adsorption on the magnetic bead surface, which remains a problem of impurity residue. The number of washing steps and total operation time have not been significantly improved.

[0008] In summary, achieving efficient removal of protein and polysaccharide impurities in a single washing step without introducing any alcohol-based organic solvents, while maintaining the electrostatic adsorption stability of nucleic acids and magnetic beads, and ensuring that the washing product can be directly used in automated nucleic acid extractors and downstream polymerase chain reaction amplification, fundamentally eliminating the inhibition of downstream enzyme reactions by alcohol residues and significantly shortening the extraction operation time, remains a key technical challenge to be overcome in the field of nucleic acid extraction kits. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides an alcohol-free nucleic acid rapid extraction kit and its extraction method.

[0010] The technical principle of this invention is based on the competitive displacement effect of imidazole molecules on non-specifically adsorbed proteins on the surface of magnetic beads. It is generally believed by those skilled in the art that the washing step in magnetic bead nucleic acid extraction must rely on the low dielectric constant environment of alcohol solvents such as ethanol or isopropanol to maintain nucleic acids on the magnetic bead surface through dehydration and precipitation while removing water-soluble protein impurities. This understanding has been solidified by all mainstream commercial kits since Boom et al. proposed the principle of silica-based nucleic acid adsorption in 1990. However, the inventors discovered in extensive experimental research that the electrostatic adsorption and hydrogen bonding adsorption between nucleic acids and the magnetic bead surface can be stably maintained in aqueous solutions with sufficiently high inorganic salt ionic strength. An aqueous environment with a sodium chloride concentration of not less than 1.5 mol / L is sufficient to ensure that nucleic acids do not desorb from the magnetic bead surface. The real technical bottleneck for impurity residue lies not in the dehydration and precipitation effect of alcohols but in the non-specific adsorption of proteins on the magnetic bead surface.

[0011] Hydrophobic amino acid residues in proteins, including aromatic side chains of tryptophan, phenylalanine, and tyrosine, and fatty side chains of leucine and isoleucine, form multiple weak bonds with the surface of magnetic beads through π-π stacking, van der Waals forces, hydrogen bonds, and electrostatic interactions. Traditional methods use ethanol or isopropanol to break the hydration layer of proteins and indirectly weaken the adsorption of proteins to the magnetic bead surface, which is inefficient and requires three to four steps of gradient concentration alcohol washing to achieve the required cleanliness of nucleic acid extraction products. This invention introduces imidazole or imidazole compounds as small molecule competitive displacement agents for proteins. The imidazole ring of the imidazole molecule has an electron-rich aromatic structure and dual capabilities as a hydrogen bond donor and hydrogen bond acceptor. When the imidazole concentration in the washing solution reaches 0.5 to 1.5 mol / L, it can occupy the non-specific binding sites of proteins on the magnetic bead surface at a sufficiently high local concentration, forcing the proteins to desorb from the magnetic bead surface into the solution, achieving efficient removal of protein impurities in a single washing step. Meanwhile, the high concentration of inorganic salts (1.2 to 2.5 mol / L) provides sufficient ionic strength shielding for the electrostatic interaction between nucleic acids and magnetic beads, maintaining the stability of nucleic acid adsorption and preventing nucleic acid loss. The weakly acidic to neutral environment (pH 6.5 to 7.5) with an imidazole pKa of approximately 6.95 ensures that imidazole exists in a dual form: an unprotonated neutral molecule and a protonated cation. This retains the competitive ability of the imidazole ring for protein binding sites while also maintaining high ionic strength through the cation form.

[0012] Based on the above technical principles, the first aspect of this invention provides an alcohol-free rapid nucleic acid extraction kit, comprising four components: a lysis buffer, a magnetic bead suspension, an alcohol-free washing buffer, and an elution buffer, all of which are free of ethanol and isopropanol. The lysis buffer contains guanidine salt at a concentration of 3.5 to 5.5 mol / L and an anionic surfactant at a mass percentage of 0.3% to 1.0%. The magnetic bead suspension contains superparamagnetic iron oxide microspheres modified with carboxyl or silicon groups, the microspheres having a particle size of 100 to 400 nm, a saturation magnetization of not less than 60 electromagnetic units per gram, and a nucleic acid binding capacity of not less than 5 micrograms of nucleic acid bound per milligram of microsphere. The alcohol-free washing buffer contains imidazole or an imidazole-like compound at a concentration of 0.5 to 1.5 mol / L and an inorganic salt at a concentration of 1.2 to 2.5 mol / L, the pH of which is 6.5 to 7.5. The imidazole or imidazole-like compound acts as a protein competitive displacement agent to remove non-specifically adsorbed protein impurities from the surface of the magnetic beads. The elution buffer is a low-salt Tris-EDTA buffer with a pH of 8.0 to 8.5.

[0013] Further, the imidazole or imidazole compound is selected from at least one of imidazole, 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole, and the concentration of the imidazole or imidazole compound is preferably 0.8 to 1.2 mol / L; the inorganic salt is selected from at least one of sodium chloride, potassium chloride, and ammonium sulfate, and the concentration of the inorganic salt is preferably 1.5 to 2.0 mol / L; the pH of the alcohol-free washing solution is preferably 6.8 to 7.2; the guanidine salt is selected from guanidine hydrochloride or guanidine isothiocyanate, and the concentration of the guanidine salt is preferably 4.0 to 5.0 mol / L; the anionic surfactant is preferably sodium dodecyl sulfate, and the mass percentage is preferably 0.4% to 0.6%; the particle size of the superparamagnetic iron oxide microspheres is preferably 200 to 300 nm, and the saturation magnetization is preferably 60 to 90 electromagnetic units per gram; the eluent contains 5 to 15 mmol / L Tris-HCl and 0.05 to 0.2 mmol / L ethylenediaminetetraacetic acid.

[0014] A second aspect of the present invention provides a rapid nucleic acid extraction method using the above-described kit, the method comprising three steps: lysis binding, single-step washing, and elution. In the lysis binding step, the sample is mixed with the lysis buffer and incubated at 50-60°C for 3-6 minutes. The magnetic bead suspension is added, and the mixture is stirred and bound at room temperature for 2-4 minutes. The supernatant is then magnetically separated and discarded. In the single-step washing step, the magnetic beads are resuspended in the alcohol-free washing buffer, stirred at room temperature for 1-2 minutes, and the supernatant is magnetically separated and discarded. This single-step washing process does not include any alcohol-containing washing steps. In the elution step, the magnetic beads are resuspended in the elution buffer preheated to 60-70°C, incubated for 2-3 minutes, and the supernatant is magnetically separated and collected to obtain the extracted nucleic acid. The total operation time of the method from sample addition to obtaining the extracted nucleic acid does not exceed 15 minutes. Furthermore, in step one of the method, the incubation temperature is preferably 56°C and the incubation time is preferably 4 minutes; in step two, the volume of the alcohol-free washing solution is preferably 1 to 2 times the sample volume; in step three, the preheating temperature of the elution solution is preferably 65°C and the incubation time is preferably 2 to 3 minutes.

[0015] The beneficial effects of this invention include the following five aspects.

[0016] First, the residual concentrations of ethanol and isopropanol in the extracted products are below the detection limits. High-performance liquid chromatography (HPLC) was used to detect the extracted products from this invention, and the residual concentrations of both ethanol and isopropanol were below the detection limits of 0.01%. This fundamentally eliminates the inhibition of downstream polymerase chain reaction (PCR), reverse transcription PCR, digital polymerase chain reaction (DPCR), isothermal amplification, and next-generation sequencing library construction by residual alcohols, significantly reducing the risk of false negatives in samples with low viral loads.

[0017] Second, the extraction process is compressed into a three-step closed loop of lysis binding, single-step washing, and elution, with a total operation time of no more than 15 minutes, which is about 50% to 60% shorter than the traditional three- to four-step alcohol washing scheme of 30 to 40 minutes. This time efficiency advantage significantly improves clinical testing throughput and early prevention and control capabilities in high-throughput scenarios such as emergency screening for sudden infectious diseases, emergency sample testing, and large-scale sample processing in fever clinics.

[0018] Third, the nucleic acid yield is 18% to 23% higher than QIAGEN products, 15% to 20% higher than Tiangen products, and 10% to 15% higher than ThermoFisher products; A 260 / A 280 The ratio reached 1.85 to 1.95, A 260 / A 230 The ratios reached 2.05 to 2.15, which are superior to mainstream commercial reagent kits in the industry, demonstrating significant advantages in both overall yield and purity. This yield advantage is attributed to the avoidance of partial loss of small nucleic acid fragments during the alcohol precipitation stage under alcohol-free aqueous phase conditions, and the thorough removal of protein impurities by imidazole displacement washing.

[0019] Fourth, the intra-batch Ct value variation coefficient of the extracts from the four types of clinical samples—whole blood genomic DNA, pharyngeal swab influenza virus DNA, pharyngeal swab adenovirus DNA, and formalin-fixed paraffin-embedded tissue DNA—is less than 1.5%, and the standard deviation of the Ct value is less than 0.4 cycles. The inter-batch stability meets the requirements of clinical laboratory quality control for internal laboratory precision and inter-laboratory reproducibility.

[0020] Fifth, the reagent components are all aqueous with low volatility, with an evaporation rate of less than 0.01% per hour within the room temperature range of 15 to 30°C. This ensures compatibility with high-throughput automated nucleic acid extractors for 96-well and 384-well plates, facilitating reagent dispensing and robotic pipetting operations while eliminating the burden of volatile organic compound emissions and flammable / explosive safety management. The kit has a shelf life of no less than 12 months under sealed storage conditions at 4°C, significantly reducing transportation and cold chain costs. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the extraction process of the alcohol-free nucleic acid rapid extraction kit of the present invention; Figure 2 This invention's reagent kit, compared to those from QIAGEN, Tiangen, and Thermo Fisher, demonstrates superior performance in nucleic acid yield and A... 260 / A 280 A bar chart comparing four key indicators: ratio, coefficient of variation of Ct value, and total operation time; Figure 3This is a schematic diagram of agarose gel electrophoresis of nucleic acid products extracted from four types of samples using the kit of the present invention: whole blood genomic DNA, pharyngeal swab influenza virus RNA, pharyngeal swab adenovirus DNA, and FFPE paraffin tissue DNA. Figure 4 This is a schematic diagram showing the fragment size distribution of DNA extracted from FFPE paraffin tissue using the kit of the present invention. Detailed Embodiments

[0022] The following is in conjunction with the appendix Figure 1-4 The present invention will be further described in detail with reference to specific embodiments. These embodiments are intended to illustrate the technical solutions and implementation effects of the present invention, and the scope of protection of the present invention is not limited to the following embodiments. All reagents used in the following embodiments are analytical grade reagents, and the magnetic beads used are commercially available carboxylated superparamagnetic iron oxide microspheres or carboxylated iron oxide microspheres prepared according to conventional methods in the art. The particle size, saturation magnetization, and nucleic acid binding capacity of the microspheres were determined using standard methods with a dynamic light scattering particle size analyzer, a vibrating sample magnetometer, and a UV-Vis spectrophotometer. All clinical samples used in the following embodiments were collected with informed consent, and the experiments were approved by the ethics review committee of the clinical institution.

[0023] Example 1: Extraction of whole blood genomic DNA.

[0024] This kit consists of four independent components: a lysis buffer bottle, a magnetic bead suspension bottle, an alcohol-free wash buffer bottle, and an elution buffer bottle. None of the four components contain ethanol or isopropanol.

[0025] Preparation of lysis buffer: Weigh 4.5 mol / L guanidine hydrochloride, 0.5% sodium dodecyl sulfate, and 10 mmol / L Tris-HCl, make up to volume with ultrapure water, adjust the pH to 7.5 with hydrochloric acid, filter through a 0.22-micron filter membrane, and dispense into sterile storage bottles.

[0026] Preparation of magnetic bead suspension: Commercially available carboxylated Fe3O4 magnetic beads with a particle size of 220 nm, a saturation magnetization of 68 electromagnetic units per gram, and a nucleic acid binding capacity of 6.5 micrograms per milligram were resuspended in an aqueous solution containing 10 mmol / L Tris-HCl to a concentration of 30 mg / mL. The suspension was then ultrasonically dispersed until uniform. The polydispersity index was measured to be 0.12 using a Malvern dynamic light scattering particle size analyzer, meeting the uniformity requirements for separation in automated extractors.

[0027] Preparation of alcohol-free washing solution: Weigh 1.0 mol / L imidazole and 1.8 mol / L sodium chloride, make up to volume with ultrapure water, slowly adjust the pH to 7.0 with concentrated hydrochloric acid, filter through a 0.22-micron filter membrane and dispense.

[0028] Eluent preparation: Weigh 10 mmol / L Tris-HCl and 0.1 mmol / L ethylenediaminetetraacetic acid, make up to volume with ultrapure water, adjust pH to 8.2 with sodium hydroxide, filter through a 0.22 micron filter membrane and dispense.

[0029] The four reagent components were tested using a standard high-performance liquid chromatography method, and the residual concentrations of ethanol and isopropanol were both below the detection limit of 0.01%. All components of the kit were stored sealed at 4°C, and initial batch stability studies showed no significant changes in performance over 12 months.

[0030] Whole blood anticoagulated with EDTA was collected from 10 healthy volunteers. 200 μL of whole blood sample was collected from each volunteer and placed in a 2 mL microcentrifuge tube. A total of 12 parallel samples were prepared, including 2 control samples for repeated operation. All samples were collected with informed consent and the experiment was approved by the ethics review committee of the institution.

[0031] like Figure 1 As shown, the extraction process of the alcohol-free nucleic acid rapid extraction kit of the present invention includes four consecutive steps: lysis binding, magnetic adsorption, single-step alcohol-free washing and elution. The total operation time from adding the sample to obtaining the extracted nucleic acid does not exceed 15 minutes.

[0032] Step 1: Ligation and Binding: Add 600 μL of lysis buffer preheated to room temperature to each 200 μL whole blood sample, vortex to mix for 10 seconds, and incubate in a 56°C constant temperature metal bath for 4 minutes; after incubation, add 30 μL of magnetic bead suspension, mix by pipetting, and let it stand at room temperature (25°C) for 3 minutes to bind, manually inverting and mixing twice during the process to ensure that the magnetic beads and lysis products are in full contact.

[0033] Step 2: Magnetic Adsorption: Place the centrifuge tube in an eight-link magnetic rack and allow it to adsorb at room temperature for 2 minutes. Once the solution is clear and the magnetic beads have completely gathered on the tube wall, carefully discard the supernatant with a pipette to avoid aspirating the magnetic bead layer.

[0034] Step 3: Single-step alcohol-free washing: Add 300 μL of alcohol-free washing solution, remove the centrifuge tube from the magnetic rack, pipette and resuspend the magnetic beads 10 times to ensure that the magnetic beads are evenly dispersed in the washing solution without visible aggregation, mix at room temperature for 1.5 minutes; place back on the magnetic rack, adsorb for 1 minute, discard the supernatant. The single-step alcohol-free washing process does not include any alcohol-containing washing steps.

[0035] Step 4: Elution: Add 80 μL of elution buffer preheated to 65°C, pipette to resuspend the magnetic beads, and incubate at room temperature for 2.5 minutes to promote complete dissociation of nucleic acids from the surface of the magnetic beads; place on a magnetic rack for 1 minute to adsorb, and transfer the supernatant to a new 1.5 mL centrifuge tube. This is the purified whole blood genomic DNA sample, which can be directly used for downstream applications such as polymerase chain reaction amplification, enzyme digestion, and library construction, or stored at -20°C.

[0036] The total operation time, from sample addition to obtaining purified nucleic acid, was 14 minutes, a 53% reduction compared to the 30 minutes of the QIAGEN QIAamp DNA Blood Mini Kit reference protocol. Extraction products from 12 parallel samples were measured using a NanoDrop One UV-Vis spectrophotometer, with an average deoxyribonucleic acid yield of 6.5 μg ± 0.4 μg, a 20% increase compared to the 5.4 μg ± 0.5 μg of the QIAamp DNA Blood Mini Kit reference protocol; A 260 / A 280 The ratio is 1.91 plus or minus 0.02, A 260 / A 230 The ratio was 2.10 ± 0.05, indicating excellent purity. The extracted product was analyzed using an Agilent 2100 bioanalyzer with a Genomic DNA chip; the main peak molecular weight was greater than 20,000 base pairs, the DIN value was greater than 7.5, and the product exhibited good integrity, making it suitable for downstream applications such as next-generation sequencing, enzyme digestion, and Southern hybridization.

[0037] The extracted product was used for real-time quantitative polymerase chain reaction (PCR) amplification of the β-actin internal reference gene. The reaction system contained 5 μL of extracted nucleic acid, 10 μL of 2x SYBR Green Master Mix, and 0.4 μmol / L each of forward and reverse primers, for a total volume of 20 μL. The amplification conditions were 95°C pre-denaturation for 30 seconds, 95°C denaturation for 5 seconds, and 60°C annealing extension for 30 seconds, for a total of 40 cycles. The average Ct value of 12 parallel samples was 28.5 ± 0.4, with an intra-assay coefficient of variation of 1.4%, significantly lower than the 2.1% intra-assay coefficient of variation of the QIAamp reference protocol. Ethanol and isopropanol residues in the extracted product were detected by high-performance liquid chromatography (HPLC), both below the detection limit of 0.01%, and had no inhibitory effect on the downstream polymerase chain reaction. The competitive displacement of non-specifically adsorbed proteins by imidazole molecules on the magnetic bead surface is the core mechanism of this invention for achieving single-step alcohol-free washing.

[0038] This embodiment verifies the yield, purity, timeliness, and compatibility with downstream polymerase chain reaction of the kit of the present invention in the scenario of whole blood genomic DNA extraction. The single-step washing system of imidazole 1.0 mol / L, sodium chloride 1.8 mol / L, and pH 7.0 is superior to the QIAamp three-step alcohol washing, completely removing protein impurities. (A) 260 / A 280The system boasts advantages in both ratio and intra-batch Ct value coefficient of variation; the anionic cleavage system with 4.5 mol / L guanidine salt and 0.5% sodium dodecyl sulfate, combined with imidazole displacement washing, forms a tandem mechanism, where the anionic environment introduced by sodium dodecyl sulfate is removed during the imidazole washing step, avoiding inhibition of downstream polymerase chain reaction; the 65°C preheated elution buffer combined with 2.5 minutes of incubation effectively promotes the complete dissociation of nucleic acids from the surface of carboxyl magnetic beads, achieving an elution efficiency of over 95%.

[0039] Example 2: Extraction of influenza virus ribonucleic acid from pharyngeal swabs.

[0040] The formulations of the lysis buffer, magnetic bead suspension, alcohol-free washing buffer, and elution buffer were exactly the same as in Example 1. In addition, 40 units per milliliter of ribonuclease inhibitor were added to the lysis buffer to protect the integrity of viral ribonucleic acid.

[0041] Sample preparation: Representative clinical strains covering four subtypes of influenza A virus (H1N1 and H3N2) and influenza B virus (Yamagata and Victoria lineages) were selected from pharyngeal swab specimens of clinically diagnosed influenza patients. Five parallel samples were collected for each subtype, for a total of 20 samples. Pharyngeal swab samples were placed in virus preservation solution in a 3 ml virus sampling tube, vortexed for 30 seconds, and 200 μL of the sample solution was used for extraction. All sample collection and experimental procedures were approved by the ethics review committee of the clinical institution.

[0042] Add 600 μL of lysis buffer (preheated to room temperature, containing ribonuclease inhibitor) to each 200 μL sample solution, vortex mix for 15 seconds, and incubate in a 56°C constant temperature metal bath for 4 minutes to fully lyse virus particles and release ribonucleic acid; add 30 μL of magnetic bead suspension, mix at room temperature for 3 minutes, manually inverting and mixing twice during the process.

[0043] The subsequent magnetic adsorption, single-step alcohol-free washing, and elution steps were exactly the same as in Example 1: magnetic adsorption for 2 minutes, supernatant discarded; 300 μL of alcohol-free washing buffer added for resuspending; incubation at room temperature for 1.5 minutes; magnetic adsorption for 1 minute, supernatant discarded; 80 μL of elution buffer preheated to 65°C added; incubation for 2.5 minutes; magnetic adsorption for 1 minute; supernatant collected. The extracted product was used for downstream reverse transcription polymerase chain reaction or stored at -80°C.

[0044] The total operation time, from sample addition to obtaining purified nucleic acid, was 14 minutes, approximately 50% shorter than the 25-30 minutes of the QIAamp Viral RNA MiniKit reference protocol. Viral ribonucleic acid was detected from 20 samples using reverse transcription real-time quantitative polymerase chain reaction (RT-PCR). The reaction system employed a one-step reverse transcription premix, with amplification primers targeting conserved regions of the M or HA genes for each subtype. Amplification conditions included 40 cycles: 50°C reverse transcription for 15 minutes, 95°C pre-denaturation for 30 seconds, 95°C denaturation for 10 seconds, and 60°C annealing extension for 30 seconds.

[0045] The Ct values ​​of the four subtypes of viral ribonucleic acid obtained by real-time quantitative polymerase chain reaction are as follows: H1N1 subtype Ct value was 24.8 ± 0.3; H3N2 subtype Ct value was 25.1 ± 0.4; Yamagata subtype B Ct value was 25.6 ± 0.4; and Victoria subtype B Ct value was 25.9 ± 0.3. The average intra-assay Ct value standard deviation for all four subtypes was less than 0.4 cycles, and the intra-assay coefficient of variation was less than 1.6%. Compared with parallel tests using a traditional three-step alcohol-wash kit, the Ct value of the extracted product using the kit of this invention was obtained on average 0.8 to 1.2 cycles earlier, indicating an improved viral ribonucleic acid yield. This is attributed to the fact that the present invention avoids partial loss of small fragments of ribonucleic acid during the alcohol precipitation stage under alcohol-free aqueous phase conditions.

[0046] The extracted products were analyzed using the Qubit RNA HS quantitative kit, and the average ribonucleic acid concentration was 18.5 nanograms per microliter ± 2.1 nanograms per microliter. The total ribonucleic acid yield corresponding to 200 microliters of sample solution was 1.48 micrograms ± 0.17 micrograms. 260 / A 280 The ratio was 1.94 ± 0.03. The extracted product was analyzed using the Agilent 2100 Bioanalyzer RNA 6000 Pico chip. The average RIN value (ribonucleic acid integrity number) was 7.2 ± 0.4, with an 18S / 28S ribosomal RNA ratio of approximately 1.8, indicating good ribonucleic acid integrity. This results in a product suitable for downstream applications such as reverse transcription polymerase chain reaction, multiplexing, and next-generation sequencing library construction.

[0047] This embodiment verifies the multi-subtype compatibility, Ct value stability, and ribonucleic acid integrity protection capability of the kit for influenza virus ribonucleic acid extraction from pharyngeal swabs. The imidazole alcohol-free washing solution avoids partial loss of small ribonucleic acid fragments during the ethanol precipitation stage, and the high ionic strength environment of 1.8 mol / L sodium chloride provides sufficient and stable electrostatic shielding for the adsorption of ribonucleic acid and magnetic beads. The integrity of the viral ribonucleic acid product meets the technical requirements of downstream one-step reverse transcription polymerase chain reaction, multiplexing, and high-throughput screening. In the pharyngeal swab sample extraction scenario, the viral load dynamic range spans 3 to 4 orders of magnitude. The batch-to-batch consistency of the kit meets the requirements of clinical laboratory quality control, significantly reducing the false negative risk of low-copy viral load samples. In the emergency screening scenario of emerging infectious disease outbreaks, the dual advantages of timeliness and yield of the kit facilitate rapid processing and accurate diagnosis of large batches of samples per day in fever clinics.

[0048] Pharyngeal swab samples have a highly complex matrix, typically containing not only the target viral nucleic acid but also human host cells, oral resident flora, mucopolysaccharides, salivary amylase, and food residues. Host proteins and mucopolysaccharides in this background are typical interfering substances that are difficult to completely remove using traditional multi-step alcohol washing processes. Residual proteins may non-specifically bind to reverse transcriptase or Taq polymerase in the downstream one-step reverse transcription polymerase chain reaction, inhibiting enzyme activity. Residual mucopolysaccharides may cause background drift during the quantitative fluorescence signal acquisition stage. The imidazole displacement washing system of this invention simultaneously treats protein and mucopolysaccharide impurities under aqueous conditions. The hydrogen bond competition of imidazole molecules for mucopolysaccharides and their π-π stacking competition for proteins work in parallel, simultaneously reducing protein and mucopolysaccharide residues in the washing product below the detection limit. This technical principle is particularly effective against the complex background of pharyngeal swab samples. Example verification showed no non-linear shift in Ct values ​​or abnormal amplification curves in 20 pharyngeal swab samples, indicating that the kit of this invention maintains stable amplification efficiency and quantitative accuracy even in complex matrix samples such as pharyngeal swabs.

[0049] Example 3: Extraction of adenovirus deoxyribonucleic acid from pharyngeal swabs.

[0050] The four-component formulation of the kit is exactly the same as in Example 1, requiring no additional components. Adenovirus is a non-enveloped double-stranded deoxyribonucleic acid virus that can be efficiently lysed in 4.5 mol / L guanidine hydrochloride lysis buffer.

[0051] Sample preparation: Pharyngeal swab samples were collected from children clinically diagnosed with adenovirus infection, covering two clinically prevalent subtypes: human adenovirus type 3 (a representative clinical strain of double-stranded deoxyribonucleic acid virus for respiratory infections) and human adenovirus type 7 (a common pathogen causing severe adenovirus pneumonia). Eight parallel samples were collected for each subtype, for a total of 16 samples. The pharyngeal swabs were placed in the virus preservation solution in the virus sampling tube, vortexed for 30 seconds, and then 200 μL of the sample solution was used for extraction.

[0052] Add 600 μL of preheated lysis buffer to each 200 μL sample solution, vortex for 10 seconds, and incubate in a 56°C constant temperature metal bath for 4 minutes to fully lyse adenovirus particles and release genomic DNA; add 30 μL of magnetic bead suspension, mix at room temperature for 3 minutes, and manually invert twice during the process.

[0053] The subsequent magnetic adsorption, single-step alcohol-free washing, and elution steps were exactly the same as in Example 1: magnetic rack adsorption for 2 minutes, discard the supernatant, add 300 μL of alcohol-free washing solution to resuspend, room temperature for 1.5 minutes, magnetic rack adsorption for 1 minute, discard the supernatant; add 80 μL of elution solution preheated to 65°C, incubate for 2.5 minutes, magnetic rack adsorption for 1 minute, and collect the supernatant.

[0054] The total operation time, from sample addition to obtaining purified nucleic acid, was 14 minutes. Adenovirus deoxyribonucleic acid was detected from the extracted products of 16 samples using real-time quantitative polymerase chain reaction (qPCR). The amplification primers targeted the conserved region of the adenovirus hexagonal gene, and the amplification conditions included 40 cycles of 95°C pre-denaturation for 5 minutes, 95°C denaturation for 10 seconds, and 60°C annealing extension for 30 seconds.

[0055] The mean Ct value for the human adenovirus type 3 sample group was 22.4 ± 0.3, with an intra-assay coefficient of variation of 1.3%; the mean Ct value for the human adenovirus type 7 sample group was 23.1 ± 0.2, with an intra-assay coefficient of variation of 0.9%. The standard deviation of the mean Ct values ​​for both subtypes was less than 0.3 cycles, which is better than the standard deviation of Ct values ​​of 0.6 to 0.8 cycles in the parallel test groups of the QIAamp DSP Virus Kit.

[0056] The extracted product, measured using a NanoDrop One UV-Vis spectrophotometer, showed an average deoxyribonucleic acid (DNA) concentration of 45.6 nanograms per microliter ± 5.2 nanograms per microliter. A 260 / A 280 The ratio is 1.89 plus or minus 0.02, A 260 / A 230 The ratio is 2.08 plus or minus 0.06. For example... Figure 3As shown, the extracted product was loaded onto a 1% agarose gel for electrophoresis at a voltage of 5 volts per centimeter for 40 minutes. After ethidium bromide staining, it was observed under a gel imaging system. Clear bands were displayed in the molecular weight range of approximately 36,000 base pairs in the adenovirus genome, with no obvious degradation or smearing. The integrity met the requirements for downstream next-generation sequencing library construction and targeted gene detection. Figure 3 The invention also demonstrates the electrophoretic band characteristics of the extracted products from four types of samples: whole blood genomic DNA, pharyngeal swab influenza virus RNA, adenovirus DNA, and FFPE paraffin tissue DNA, reflecting the differences in the integrity of the matrix nucleic acids in different samples.

[0057] To further verify the effect of imidazole concentration on the extraction performance of the kit of this invention, adenovirus type 3 samples were used as templates, and imidazole concentration gradients of 0.3, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 mol / L were set. Other reagent components and operating conditions remained consistent, and the nucleic acid yield and A were tested in parallel. 260 / A 280 ratio.

[0058] The results showed that when the imidazole concentration was below 0.5 mol / L, the protein competitive substitution efficiency was insufficient. 260 / A 280 A ratio below 1.75 indicates substandard purity. At imidazole concentrations of 0.8 to 1.2 mol / L, both yield and purity are within the optimal range. When the imidazole concentration exceeds 1.5 mol / L, the yield begins to decrease slightly, by approximately 5% to 10%, presumably due to competitive interference from high-concentration imidazole on the electrostatic adsorption of nucleic acids onto magnetic beads. At an imidazole concentration of 2.0 mol / L, the yield decreases significantly by approximately 15%. Based on the above parameter optimization results, this invention determines the preferred imidazole concentration range to be 0.8 to 1.2 mol / L. This embodiment verifies the high yield and high reproducibility of the kit of this invention in the extraction of adenovirus double-stranded deoxyribonucleic acid from pharyngeal swabs. Adenovirus is a non-enveloped double-stranded deoxyribonucleic acid virus. In guanidine salt lysis buffer, the dissociation efficiency of the capsid protein from the genomic DNA depends on the synergistic effect of the guanidine salt concentration and the anionic surfactant. The lysis system of this invention, consisting of 4.5 mol / L guanidine hydrochloride and 0.5% sodium dodecyl sulfate, can completely lyse adenovirus within 4 minutes at 56°C. A single-step wash with 1.0 mol / L imidazole can thoroughly remove the non-specific adsorption of the capsid protein to host cell proteins, maintaining good integrity of the elution products. The standard deviation of the Ct value in this embodiment is within 0.3 cycles, which is superior to the industry control of 0.6 to 0.8 cycles, reflecting the batch-to-batch consistency advantage of this kit in high-throughput clinical screening scenarios. This embodiment also determined the optimal range of 0.8 to 1.2 mol / L through imidazole concentration gradient optimization experiments.

[0059] Example 4: Extraction of deoxyribonucleic acid from formalin-fixed paraffin-embedded tissue.

[0060] The four-component formulation of the kit is basically the same as that in Example 1. The final concentration of proteinase K in the lysis buffer is adjusted to 0.4 mg / mL to improve the enzymatic digestion efficiency of protein cross-linking structures in paraffin-embedded tissues.

[0061] Sample preparation: Fifteen archived formalin-fixed paraffin-embedded tissue samples were collected, with paraffin embedding times ranging from 1 to 5 years, covering four tissue sources: lung, liver, intestine, and breast. Four 10-micrometer-thick paraffin sections were cut from each sample and placed in 2 ml microcentrifuge tubes. All samples were approved by the clinical institution's ethics review committee for use in this study.

[0062] Pre-dewaxing treatment: Add 1 mL of xylene, vortex for 30 seconds, let stand at room temperature for 5 minutes, centrifuge at 12000 rpm for 2 minutes, and discard the supernatant; repeat the dewaxing step twice, then add 1 mL of anhydrous ethanol and vortex wash twice to remove residual xylene, and vacuum dry for 5 minutes. The pre-dewaxing treatment is a standard pretreatment step for paraffin tissue samples in the art. The anhydrous ethanol is only used for xylene elution. After the vacuum drying step, the ethanol completely evaporates and does not enter the subsequent magnetic bead extraction process. Therefore, the main extraction process of this invention's kit remains alcohol-free.

[0063] Add 600 μL of lysis buffer containing 0.4 mg / mL proteinase K to the dewaxed tissue sample, vortex to mix, and incubate in a 56°C constant temperature metal bath for 60 minutes to fully enzymatically dissolve the protein cross-links in the paraffin tissue; then heat to 80°C to inactivate proteinase K for 10 minutes. This step also helps to remove formalin cross-links and improve the efficiency of deoxyribonucleic acid release.

[0064] After incubation, the tube wall condensate was collected by short-term centrifugation, and 30 μL of magnetic bead suspension was added. The mixture was stirred at room temperature for 3 minutes. The subsequent magnetic adsorption, single-step alcohol-free washing, and elution steps were exactly the same as in Example 1. The volume of the elution solution in the elution step was adjusted to 100 μL to improve the elution efficiency of paraffin-embedded tissue samples.

[0065] Extracts from 15 paraffin-embedded tissue samples were analyzed using the Qubit dsDNA HS quantitative kit. The average yield of double-stranded deoxyribonucleic acid (DDNA) was 850 nm ± 180 nm. 260 / A 280 The ratio is 1.86 plus or minus 0.04, A 260 / A 230 The ratio is 1.95 plus or minus 0.08. For example... Figure 4As shown, the fragment size distribution of the extracted products was detected by an Agilent 2100 bioanalyzer. The main peak was distributed in the range of 150 to 300 base pairs, and the DIN value, i.e., the DNA integrity index, was 3.2 plus or minus 0.5 on average. This meets the basic input requirements for paraffin-embedded tissue DNA in the construction of next-generation sequencing libraries, namely, the main peak is greater than 150 base pairs and the DIN value is greater than 2.0.

[0066] The extracted products were used for next-generation sequencing library construction. The library preparation was carried out using a commercial library preparation kit. Libraries were successfully constructed from all 15 samples. The average library concentration was 25 nanomoles plus or minus 8 nanomoles, which met the requirements for sequencing. The sequencing data quality Q30 was greater than 90%, the alignment rate was greater than 95%, and the detection of key hotspot mutations met expectations. The results were consistent with the performance of the libraries extracted from fresh frozen tissue.

[0067] To further verify the compatibility of the kit for samples with different paraffin embedding times, 15 samples were divided into three subgroups according to their embedding time: 5 samples from 1-2 years, 5 samples from 2-3 years, and 5 samples from 3-5 years. Nucleic acid yield and fragmentation degree were compared in each subgroup. The results showed that the average deoxyribonucleic acid yield was 1050 nanograms (DIN) in the 1-2 year group (DIN value 3.6); 920 nanograms (DIN value 3.3) in the 2-3 year group; and 680 nanograms (DIN value 2.9) in the 3-5 year group. All three groups successfully completed next-generation sequencing library construction. Although the yield was slightly lower in the long embedding time group, it still met the basic requirements for downstream applications, reflecting the good compatibility of the kit for long-term archived paraffin tissue samples.

[0068] This embodiment verifies the compatibility of the kit of the present invention with long-term embedded samples in the extraction of deoxyribonucleic acid (DNA) from formalin-fixed paraffin-embedded tissues. Samples with paraffin embedding times ranging from 1 to 5 years can consistently yield DNA fragments with a main peak of 150 to 300 base pairs, meeting the needs of downstream clinical applications such as next-generation sequencing, targeted gene detection, and tumor hotspot mutation screening. The single-step alcohol-free washing method of the present invention is particularly advantageous in the extraction of paraffin-embedded tissues because these samples have a high degree of protein cross-linking. Traditional multi-step alcohol washing easily leads to further loss of small DNA fragments, while the imidazole-displacement washing method of the present invention avoids the loss of fragmented DNA during the ethanol precipitation stage under aqueous conditions, thus protecting both yield and fragment quality. This embodiment provides data support for the application of the kit of the present invention in the fields of precision oncology diagnosis and treatment and biobank archived tissue detection.

[0069] Example 5: Parallel comparative experiment with three competing products: QIAGEN, Tiangen, and Thermo Fisher.

[0070] To verify the comprehensive performance advantages of the kit of this invention compared with mainstream commercial magnetic bead-based nucleic acid extraction kits in the industry, the QIAamp DNA Blood Mini Kit (product number 51106) from QIAGEN, the DP304 blood cell tissue genomic DNA extraction kit from Tiangen Biotech, and the MagMAX DNA Multi-Sample Ultra 2.0 Kit from Thermo Fisher Scientific were selected as control kits. Parallel comparative tests were conducted using whole blood genomic DNA extraction as the baseline sample.

[0071] Sample preparation: Ethylenediaminetetraacetic acid (EDTA) anticoagulated whole blood was collected from 8 healthy volunteers. The whole blood from each volunteer was aliquoted into 4 equal parts, each 200 μL. Parallel extraction was performed using the kit of this invention, the QIAGEN kit, the Tiangen kit, and the Thermo Fisher kit. All extraction operations were strictly performed according to the standard instructions of each kit. Extraction and detection were completed by the same operator on the same day to eliminate inter-operator variability.

[0072] like Figure 2 As shown, the reagent kit of this invention, compared with those of three mainstream commercial reagent kits in the industry—QIAGEN, Tiangen, and Thermo Fisher—in terms of nucleic acid yield, A... 260 / A 280 The comparative results of four key indicators—ratio, Ct value, intra-batch coefficient of variation, and total operation time—are presented intuitively, and the reagent kit of this invention has advantages in all four indicators.

[0073] Regarding nucleic acid yield, the average deoxyribonucleic acid yield of the kit of this invention is 6.5 μg ± 0.4 μg, compared to 5.3 μg ± 0.5 μg for QIAGEN, 5.5 μg ± 0.6 μg for Tiangen, and 5.8 μg ± 0.4 μg for Thermo Fisher. The yield of the kit of this invention is 22.6% higher than that of QIAGEN, 18.2% higher than that of Tiangen, and 12.1% higher than that of Thermo Fisher.

[0074] Regarding purity indicators, the reagent kit A of this invention... 260 / A 280 The ratios are 1.91 ± 0.02, QIAGEN is 1.83 ± 0.05, Tiangen is 1.79 ± 0.06, and Thermo Fisher is 1.88 ± 0.04; Reagent Kit A of this invention 260 / A 230 The ratios were 2.10 ± 0.05, QIAGEN 1.95 ± 0.08, Tiangen 1.88 ± 0.09, and Thermo Fisher 2.05 ± 0.06. The purity of this invention's kit is superior to all three control kits.

[0075] Eight samples were used for real-time quantitative polymerase chain reaction (qPCR) amplification of the β-actin internal reference gene. The amplification conditions were the same as in Example 1. The coefficient of variation (Ct) of the intra-batch Ct values ​​was 1.4% for the kit of this invention, 2.1% for QIAGEN, 2.5% for Tiangen, and 1.8% for Thermo Fisher. The Ct stability of the kit of this invention is comparable to or better than that of the three control kits.

[0076] Regarding the total operation time, the reagent kit of this invention takes 14 minutes, compared to 30 minutes for QIAGEN, 35 minutes for Tiangen, and 28 minutes for Thermo Fisher. The operation time of the reagent kit of this invention is reduced by 53.3% compared to QIAGEN, 60.0% compared to Tiangen, and 50.0% compared to Thermo Fisher.

[0077] Regarding the detection of alcohol residues, the extracts from the kit of this invention were detected by high performance liquid chromatography, and the residual concentrations of ethanol and isopropanol were both below the detection limit of 0.01%; the extracts from the QIAGEN and Tiangen kits showed ethanol residues of 0.3% to 0.8%; and the extracts from the Thermo Fisher kit showed isopropanol residues of 0.2% to 0.5%.

[0078] The comparative data of the four key indicators are summarized below. Regarding yield, the average deoxyribonucleic acid yields of this invention, QIAGEN, Tiangen, and Thermo Fisher were 6.5, 5.3, 5.5, and 5.8 μg, respectively, with standard deviations of 0.4, 0.5, 0.6, and 0.4 μg, respectively. One-way ANOVA showed that the yield differences between this invention and the three control kits were statistically significant (p-value less than 0.001). 260 / A 280 Regarding the purity ratio, the 1.91 of this invention is significantly better than Tiangen's 1.79 and Qiagen's 1.83, and while the difference from Thermo Fisher's 1.88 is small, it is still statistically significant (p < 0.01). Regarding the intra-batch Ct coefficient of variation, the 1.4% of this invention is significantly better than Qiagen's 2.1% and Tiangen's 2.5%, and comparable to Thermo Fisher's 1.8%. Regarding the total operating time, the 14 minutes of this invention is more than 50% shorter than Qiagen's 30 minutes, Tiangen's 35 minutes, and Thermo Fisher's 28 minutes, with extremely significant differences.

[0079] To further verify the performance stability of the kit under different initial sample volumes, parallel extractions were performed on whole blood samples with initial volumes of 100 μL, 200 μL, and 400 μL using the kit. The nucleic acid yield showed a good linear relationship with the initial volume, and the correlation coefficient of the linear regression equation was greater than 0.99, reflecting that the extraction efficiency of the kit was stable and consistent under different initial sample volumes, which is beneficial for the flexible application of the kit in clinical testing laboratories in both scenarios with limited or abundant sample volumes.

[0080] Based on the comparison of the four key indicators above, the reagent kit of this invention achieves advantages in four dimensions: nucleic acid yield, purity, downstream polymerase chain reaction stability, and total operation time. It is also the only kit to achieve complete alcohol residue-free extraction, demonstrating superior overall performance compared to mainstream commercially available kits. This reagent kit is particularly suitable for clinical molecular diagnostics and high-throughput screening scenarios requiring high sensitivity in downstream polymerase chain reactions and stringent time constraints. The comprehensive performance advantages of this invention are attributed to the highly efficient removal of protein impurities via imidazole displacement washing, the stable maintenance of nucleic acid-magnetic bead electrostatic adsorption by high saline solution, the significant reduction in operation time due to the single-step washing process, and the inherent compatibility of the all-aqueous, low-volatility system with automation. These four advantages represent a systematic breakthrough in the field of nucleic acid extraction based on the technical principles of this invention.

[0081] Example 6: Compatibility verification of automated nucleic acid extractor.

[0082] The four components of the reagent kit of this invention were aliquoted into 96-well and 384-well plates, with sufficient reagent added to each well (300 μL per well in the 96-well plate and 80 μL per well in the 384-well plate). The plates were then incubated at 25°C for 24 hours. The reagent mass change was measured every 2 hours, and the evaporation rate of each component was calculated. The results showed that the evaporation rate of the lysis buffer was 0.008% per hour, the evaporation rate of the magnetic bead suspension was 0.007% per hour, the evaporation rate of the alcohol-free wash buffer was 0.005% per hour, and the evaporation rate of the eluent was 0.006% per hour. All four evaporation rates were below the threshold of 0.01% per hour, meeting the reagent stability requirements for high-throughput automated scenarios using 96-well and 384-well plates.

[0083] The temperature range was extended to 15 to 30°C for testing, and the evaporation rate was less than 0.01% per hour, verifying the stability of the kit under different laboratory temperature conditions.

[0084] The reagent kit of this invention was loaded into a commercially available 96-throughput automated nucleic acid extractor. Using 96 pharyngeal swab samples as the extraction targets, batch extraction was performed according to a preset program. The program parameters included 4 minutes of incubation at 56°C, 2 minutes of magnetic adsorption, 1.5 minutes of single-step washing, 2.5 minutes of elution incubation, and the magnetic adsorption and liquid transfer steps. The total extraction program runtime was 18 minutes, including the time for the instrument's robotic arm. The average nucleic acid yield of the 96 samples was consistent with that of manual extraction. The extracted products were verified by real-time quantitative polymerase chain reaction (qPCR). The coefficient of variation (Ct) value of the 96 samples was 1.6%, and the batch-to-batch consistency met the quality control requirements for automated high-throughput clinical testing.

[0085] The reagent kit of this invention was loaded into a 384-throughput automated nucleic acid extractor and batch extraction was performed using 384 simulated samples for verification. No drop in liquid level or drift in reagent concentration was observed during multiple liquid transfers by the robotic arm. The coefficient of variation of Ct values ​​of the extracted products from the 384 samples was 1.8%, demonstrating the good compatibility of the reagent kit of this invention in ultra-high throughput automated scenarios.

[0086] The reagent kit was stored at 4°C in a sealed container, and the extraction performance stability was assessed at 1, 3, 6, 9, and 12 months. At each time point, the reagent was clear and free of precipitation, indicating good nucleic acid extraction performance, i.e., yield and A. 260 / A 280 The ratio and Ct value are consistent with the initial batch, the yield decay is less than 5%, the Ct value drift is less than 0.3 cycles, and the shelf life of the kit is not less than 12 months. The long-term storage stability meets the requirements of commercial production, cold chain transportation, and routine inventory management in clinical laboratories.

[0087] The four components of this reagent kit are formulated using commercially available raw materials commonly used in the field. The guanidine salt, sodium dodecyl sulfate, imidazole, sodium chloride, Tris-HCl, and ethylenediaminetetraacetic acid are all industrially produced reagents that meet the analytical purity standards of the Chinese Pharmacopoeia. The carboxylated superparamagnetic iron oxide microspheres are commercially available raw materials that can be stably supplied by multiple domestic and imported suppliers, eliminating raw material supply risks during mass production of the kit. The kit manufacturing process is completed in a Class 10,000 cleanroom environment. The guanidine salt lysis buffer and magnetic bead suspension are prepared using 0.22-micron terminal filtration to control microbial load. The manufacturing process complies with Good Manufacturing Practices (GMP) requirements for in vitro diagnostic reagents.

[0088] The overall kit complies with the requirements for classification management and registration of in vitro diagnostic reagents. Because it does not contain volatile organic compounds (VOCs), there are no flammable or explosive hazardous material control requirements throughout the entire production, storage, and transportation chain, significantly reducing compliance costs in production and distribution. Against the backdrop of increasingly stringent VOC emission controls in medical institutions, this kit provides clinical testing laboratories with a nucleic acid extraction solution that aligns with the development of green chemistry.

Claims

1. An alcohol-free rapid nucleic acid extraction kit, characterized in that, The kit consists of four components: lysis buffer, magnetic bead suspension, alcohol-free washing buffer, and elution buffer. None of these four components contain ethanol or isopropanol. The lysis buffer contains a guanidine salt at a concentration of 3.5 to 5.5 mol / L and an anionic surfactant at a mass percentage of 0.3% to 1.0%. The guanidine salt is used to lyse cells or virus particles in biological samples to release nucleic acids, and the anionic surfactant is used to promote membrane rupture and protein denaturation. The magnetic bead suspension contains superparamagnetic iron oxide microspheres with carboxyl or silicon-based surface modifications. The microspheres have a particle size of 100 to 400 nanometers and a saturation magnetization of not less than 60 Ω·cm. Each magnetic unit per gram has a nucleic acid binding capacity of not less than 5 micrograms of nucleic acid bound per milligram of microsphere, and the concentration of the microspheres in the magnetic bead suspension is 20 to 40 milligrams per milliliter; the alcohol-free washing solution contains imidazole or imidazole compounds at a concentration of 0.5 to 1.5 mol / L and inorganic salts at a concentration of 1.2 to 2.5 mol / L, and the pH of the alcohol-free washing solution is 6.5 to 7.

5. The imidazole or imidazole compounds act as protein competitive displacement agents to remove non-specifically adsorbed protein impurities on the surface of the magnetic beads, and the inorganic salts are used to maintain the electrostatic adsorption stability between the nucleic acid and the magnetic beads; the elution solution is a low-salt Tris-EDTA buffer at pH 8.0 to 8.5, used to dissociate and collect the nucleic acid from the surface of the magnetic beads.

2. The alcohol-free nucleic acid rapid extraction kit according to claim 1, characterized in that, The imidazole or imidazole compound is selected from at least one of imidazole, 1-methylimidazolium, 2-methylimidazolium, and 4-methylimidazolium.

3. The alcohol-free nucleic acid rapid extraction kit according to claim 1, characterized in that, The inorganic salt is selected from at least one of sodium chloride, potassium chloride, and ammonium sulfate, and the concentration of the inorganic salt is 1.5 to 2.0 mol per liter. The pH of the alcohol-free washing solution is 6.8 to 7.

2.

4. The alcohol-free nucleic acid rapid extraction kit according to claim 1, characterized in that, The guanidine salt is selected from guanidine hydrochloride or guanidine isothiocyanate, and the concentration of the guanidine salt is 4.0 to 5.0 mol per liter; the anionic surfactant is sodium dodecyl sulfate, and the mass percentage of sodium dodecyl sulfate is 0.4% to 0.6%.

5. The alcohol-free nucleic acid rapid extraction kit according to claim 1, characterized in that, The superparamagnetic iron oxide microspheres have a particle size of 200 to 300 nanometers and a saturation magnetization of 60 to 90 electromagnetic units per gram; the eluent contains 5 to 15 mmol / L Tris-HCl and 0.05 to 0.2 mmol / L ethylenediaminetetraacetic acid.

6. A method for rapid nucleic acid extraction using the alcohol-free nucleic acid rapid extraction kit according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: Step 1: Lysis and binding. The sample and the lysis buffer are mixed at a volume ratio of 1:2 to 1:4, vortexed, and incubated at 50 to 60°C for 3 to 6 minutes to fully lyse the cells or virus particles in the sample. After incubation, the magnetic bead suspension is added and mixed at room temperature for 2 to 4 minutes to promote the full binding of nucleic acids and magnetic beads. The supernatant is then discarded after magnetic separation. Step 2: Single-step washing. The magnetic beads are resuspended in the alcohol-free washing buffer to ensure uniform dispersion in the washing buffer. The mixture is mixed at room temperature for 1 to 2 minutes to... The method involves competitively displacing non-specifically adsorbed proteins with imidazole or imidazole compounds, followed by magnetic separation and discarding of the supernatant. This single-step washing process does not include any alcohol-containing washing steps, nor does it include washing steps using polyethylene glycol precipitants or polyol substitutes. Step three involves elution, resuspending the magnetic beads in the eluent preheated to 60 to 70°C, and incubating for 2 to 3 minutes to promote the dissociation of nucleic acids from the surface of the magnetic beads. The supernatant is then collected by magnetic separation to obtain the extracted nucleic acid. The total operation time from sample addition to obtaining the extracted nucleic acid does not exceed 15 minutes.

7. The rapid nucleic acid extraction method according to claim 6, characterized in that, In step one, the volume ratio of the sample to the lysis buffer is 1:2 to 1:4, the incubation temperature is 56°C, and the incubation time is 4 minutes.

8. The rapid nucleic acid extraction method according to claim 6, characterized in that, In step two, the volume of the alcohol-free washing solution is 1 to 2 times the sample volume, and the mixing time at room temperature is 1 to 2 minutes.

9. The rapid nucleic acid extraction method according to claim 6, characterized in that, In step three, the volume of the elution buffer is 0.3 to 0.5 times the sample volume, the preheating temperature is 65°C, and the incubation time is 2 to 3 minutes.

10. The rapid nucleic acid extraction method according to claim 6, characterized in that, The four components of the alcohol-free rapid nucleic acid extraction kit have an evaporation rate of less than 0.01% per hour in a temperature range of 15 to 30°C, and the method is compatible with automated nucleic acid extractors in 96-well or 384-well plate formats.

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