Method and kit for enriching low concentration pathogen nucleic acid in water body
Patent Information
- Application Number
- CN202610872490.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-16
- Publication Date
- 2026-08-28
AI Technical Summary
[0006]本发明旨在克服现有对低浓度靶标富集效率低的不足，提供一种水体中低浓度病原体核酸的富集方法及试剂盒，以实现无需复杂前处理的现场快速富集，显著提升低载量样本的检测灵敏度与结果可靠性
本发明通过特定配比构建的富集裂解液（异硫氰酸胍、醋酸钠、吐温-20）与洗脱液（Tris-HCl、异丙醇、Triton X-100）产生协同作用，高效裂解病原体并促进靶标核酸定向吸附于磁珠，富集效率与检测灵敏度显著提升，使原本无法检出或呈弱阳性的低载量样本实现稳定检出，大幅降低假阴性率。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of pathogen nucleic acid enrichment technology. More specifically, it relates to a method and kit for enriching low concentrations of pathogen nucleic acids in water. Background Technology
[0002] African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and various wild boars (such as African wild boar and European wild boar). The World Organisation for Animal Health (OIE) lists it as a notifiable animal disease, and my country also classifies it as a Class A animal disease requiring special attention. The disease has a short course and rapid onset; the mortality rate in the most acute and acute forms can reach 100%. The main clinical manifestations are high fever (40-42℃), rapid heartbeat, and rapid breathing. Some affected pigs also cough, and there is serous or mucopurulent discharge from the eyes and nose. Cyanosis of the skin is present, and necropsy reveals significant hemorrhage in the lymph nodes, kidneys, and gastrointestinal mucosa. Because the clinical symptoms of ASF are highly similar to those of classical swine fever (CSF), clinical observation alone is insufficient for differentiation; definitive diagnosis requires laboratory testing.
[0003] African swine fever (ASFV) pathogens are commonly transmitted over long distances through contaminated water, excrement, and environmental media. Therefore, monitoring pathogen nucleic acid in environmental samples such as water bodies is crucial for early warning, interrupting transmission chains, and implementing precise control measures. Currently, real-time quantitative PCR (qPCR) technology is recognized by the World Organisation for Animal Health (OIE) as the "gold standard" for pathogen nucleic acid detection due to its high specificity and sensitivity. However, actual environmental water samples are typically large in volume, have extremely low pathogen concentrations, and contain large amounts of humic acid, metal ions, organic impurities, and PCR inhibitors. Direct extraction and detection of these samples can easily dilute or inhibit the target nucleic acid, leading to a significant decrease in detection sensitivity. Low-load samples frequently produce false negative results, severely delaying early warning and response to outbreaks.
[0004] To improve the detection rate of low-concentration samples, water enrichment and concentration are usually required before nucleic acid extraction. Existing enrichment technologies mainly include polyethylene glycol (PEG) precipitation, ultrafiltration membrane concentration, and conventional magnetic bead adsorption. Among these, PEG precipitation is time-consuming, involves complex procedures, and is prone to introducing interference from impurities; ultrafiltration membrane concentration relies on dedicated negative pressure or centrifugation equipment, and the membrane pores are easily clogged, with nucleic acid recovery rates greatly affected by water turbidity; while commercially available conventional nucleic acid extraction kits are mostly designed for small-volume clinical samples, and when directly used in large-volume, low-concentration water samples, magnetic bead binding efficiency is low, elution losses are significant, and most systems are not optimized for interference resistance in complex water matrices. Furthermore, the above methods generally lack field adaptability, making it difficult to meet the urgent need for "rapid, simple, and highly sensitive" enrichment technologies in grassroots farms and field monitoring sites.
[0005] Therefore, it is of great significance to develop a method and kit for the enrichment of pathogens and nucleic acids that is easy to operate, has high enrichment efficiency, strong resistance to interference from water matrix, and good stability, so as to overcome the technical bottleneck of high false negative rate in the detection of low-concentration environmental samples. Summary of the Invention
[0006] This invention aims to overcome the shortcomings of existing methods for enriching low-concentration targets with low efficiency, and provides a method and kit for enriching low-concentration pathogen nucleic acids in water, so as to achieve rapid on-site enrichment without complicated pretreatment, and significantly improve the detection sensitivity and reliability of low-load samples.
[0007] The first objective of this invention is to provide a kit for enriching the nucleic acids of pathogens.
[0008] A second objective of this invention is to provide applications of the above-described reagent kit.
[0009] A third objective of this invention is to provide a method for enriching low-concentration pathogen nucleic acids in water.
[0010] A fourth objective of this invention is to provide the application of the above-described kit or enrichment method in the detection of African swine fever virus in water.
[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a kit for enriching pathogen nucleic acids, comprising an enrichment lysis buffer and an enrichment elution buffer; the enrichment lysis buffer contains guanidine isothiocyanate, sodium acetate, and Tween-20; the enrichment elution buffer contains tris(hydroxymethyl)aminomethane, isopropanol, and Triton X-100.
[0012] Preferably, in the above-mentioned kit, the enrichment lysis buffer comprises the following components: 300-1000 mg / mL guanidine isothiocyanate, 75-225 μmol / mL sodium acetate, and 10-15% v / v Tween-20.
[0013] More preferably, in the above kit, the enrichment lysis buffer comprises the following components: 300-500 mg / mL guanidine isothiocyanate, 75-150 μmol / mL sodium acetate, and 12.5-15% v / v Tween-20.
[0014] More preferably, in the above-mentioned kit, the enrichment lysis buffer comprises the following components: 300 mg / mL guanidine isothiocyanate, 75 μmol / mL sodium acetate, and 12.5% v / v Tween-20.
[0015] Preferably, in the above kit, the enrichment eluent comprises the following components: 5-15 μmol / mL tris(hydroxymethyl)aminomethane, 10-20% v / v isopropanol and 0.5-2% v / v Triton X-100.
[0016] More preferably, in the above kit, the enrichment eluent comprises the following components: 5-15 μmol / mL tris(hydroxymethyl)aminomethane, 10-20% v / v isopropanol and 1-2% v / v Triton X-100.
[0017] More preferably, in the above kit, the enrichment eluent comprises the following components: 10 μmol / mL tris(hydroxymethyl)aminomethane, 20% v / v isopropanol and 1% v / v Triton X-100.
[0018] Preferably, the kit further includes magnetic beads, the surface of which is modified with at least one of carboxyl, amino, or silanol groups.
[0019] Specifically, the pathogen mentioned above is a virus (preferably African swine fever virus).
[0020] The application of the above-mentioned kit in enriching viral nucleic acids should also be within the scope of protection of this invention.
[0021] Specifically, the virus in question is the African swine fever virus.
[0022] This invention provides a method for enriching low-concentration pathogen nucleic acids in water, using the aforementioned kit.
[0023] As an alternative specific implementation, the above enrichment method includes the following steps: S1. Mix the enriched lysis buffer with the water sample and shake well to obtain the lysis buffer; S2. Add magnetic beads to the lysis solution obtained in S1, mix well and incubate; S3. Magnetic beads are attracted under the action of a magnetic field, and the supernatant is discarded; S4. Add enrichment elution buffer to the magnetic beads, resuspend, centrifuge and collect the supernatant to obtain the enriched sample.
[0024] As an alternative implementation, in step S1, the volume ratio of the enrichment lysis buffer to the water sample is 1:(3~6); in step S4, the amount of enrichment elution buffer added is 300~500 μL.
[0025] Optionally, in step S1, the volume ratio of the enrichment lysis buffer to the water sample is 1:4; in step S4, the amount of enrichment elution buffer added is 400 μL.
[0026] As an alternative implementation, in step S2, the incubation time is 3 to 6 minutes.
[0027] As an alternative implementation, in step S3, the adsorption time is 8-10 min.
[0028] As an alternative implementation, in step S4, the centrifugation time is 40~60s.
[0029] Optionally, in the above enrichment method, the water sample in S1 undergoes pretreatment, which is to remove impurities from the water sample.
[0030] This invention provides the application of the above-described kit or enrichment method in the detection of African swine fever virus in water.
[0031] The present invention has the following beneficial effects: This invention utilizes a specific ratio of enrichment lysis buffer (guanidine isothiocyanate, sodium acetate, Tween-20) and elution buffer (Tris-HCl, isopropanol, Triton X-100) to achieve synergistic effects, efficiently lysing pathogens and promoting the targeted adsorption of target nucleic acids onto magnetic beads. This significantly improves enrichment efficiency and detection sensitivity, enabling stable detection of low-load samples that were previously undetectable or weakly positive, and greatly reducing the false negative rate.
[0032] The enrichment reagent provided by this invention is simple to prepare, requiring no complex pretreatment of water samples, and enabling integrated sample processing and enrichment operations on-site. This method demonstrates excellent performance in terms of resistance to matrix interference, streamlined process, reagent conservation, rapid operation, and on-site applicability, maximizing sample authenticity and timeliness, and significantly improving the efficiency and reliability of on-site testing. This method effectively overcomes the technical bottlenecks of low pathogen load and the susceptibility of nucleic acids to false negatives, helping aquaculture farms to implement timely disease control measures, and has excellent application prospects and value. Detailed Implementation
[0033] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.
[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.
[0035] The ASFV fake virus was purchased from Shenggong Biotechnology, product number M591238-0001.
[0036] The magnetic beads were purchased from Suzhou Beaver Biotechnology, model SC300, item number TTLTLB0109.
[0037] Example 1: Preparation of enriched lysis buffer and enriched eluent I. Preparation of enriched pyrolysis solution Take 62.5 mL of Tween 20, 150 g of guanidine isothiocyanate and 12.5 mL of 3 M sodium acetate buffer (pH 5.2), place them in a volumetric flask, dilute to 500 mL with purified water, and heat in a 65°C water bath until completely dissolved to obtain the enriched lysis buffer.
[0038] The preparation method of 3M sodium acetate buffer (pH 5.2) is as follows: Weigh 204g of sodium acetate trihydrate (NaOAc•3H2O) and place it in a 1L beaker. Add about 200 mL of deionized water and stir to dissolve. Adjust the pH value to 5.2 with glacial acetic acid, add deionized water to make up to 500 mL, and autoclave at 121℃ for 20 min. Store at room temperature.
[0039] II. Preparation of enrichment eluent Measure 10 mL of Tris-HCl buffer (100 mM, pH 7.5), 20 mL of isopropanol, and 1 mL of Triton X-100, mix them, and then add ultrapure water to bring the volume to 100 mL. Adjust the pH to 10.5 with NaOH solution, mix thoroughly, and the enrichment eluent is obtained.
[0040] The preparation method for 100 mM Tris-HCl is as follows: Weigh 6.06 g of Tris base (tris(hydroxymethyl)aminomethane), add 400 mL of double-distilled water (ddH2O), and stir until completely dissolved. Monitor the pH with a pH meter, and add 1 M HCl dropwise to adjust the pH to 7.5. Transfer the solution to a 500 mL volumetric flask, rinse the beaker with ddH2O and top up to the mark, mix well, and autoclave (121℃, 15-20 minutes). Store at room temperature.
[0041] Example 2: Method for Enriching Pathogen Nucleic Acids in Water Samples A method for enriching pathogen nucleic acids in water samples includes the following steps: (1) Sample pretreatment: Remove suspended particles and visible impurities from the water sample using a 0.45 μm filter membrane or centrifugation (skip this step if there are no impurities). (2) Lysis: Add 10 mL of the enriched lysis buffer from Example 1 to a 50 mL centrifuge tube, add the mixed water sample to 50 mL, shake well, and obtain the lysis buffer; (3) Magnetic bead enrichment: Add 300 μL of magnetic beads (Beaver SC300, TTLTLB0109) to the lysis buffer and shake well for 5 min by inverting; (4) Recovering magnetic beads: Place the lysis buffer in a magnetic rack for 8-10 minutes to adsorb the magnetic beads. After the magnetic beads are completely adsorbed, discard the supernatant and remove the bottom residue with a pipette. (5) Elution: Add 400 μL of the enrichment elution buffer from Example 1 to the centrifuge tube, rinse the magnetic beads on the tube wall to the bottom, and transfer the fully resuspended sample to a 2 mL centrifuge tube; gently shake the 2 mL centrifuge tube containing the resuspended magnetic beads to mix them, and then centrifuge the centrifuge tube for 50 s. The supernatant is the enriched sample, which is used for subsequent experiments. (6) Nucleic acid extraction: Nucleic acid was extracted from the enriched samples using a commercial nucleic acid extraction kit (Bori, BSC86T1C) to obtain nucleic acid samples; (7) qPCR detection: Fluorescent PCR detection is performed on nucleic acid samples to obtain the detection results.
[0042] Experiment Example 1: Effect of Guanidine Isothiocyanate Content in Enrichment Buffer on Enrichment Efficiency I. Preparation of enriched lysis buffers of guanidine isothiocyanate at different concentrations The enrichment lysis buffer was prepared according to the method in Example 1, with the amounts of Tween-20 (62.5 mL) and 3 M sodium acetate buffer (pH 5.2, 12.5 mL) fixed, and only the amount of guanidine isothiocyanate was adjusted. Based on 500 mL of lysis buffer, the amounts of guanidine isothiocyanate added were 150 g, 250 g, and 500 g, resulting in three enrichment lysis buffers with different concentrations of guanidine isothiocyanate.
[0043] II. Experimental Methods (1) Sample preparation: Add a random amount of ASFV pseudovirus (Sangon Biotech, catalog number M591238-0001) to 100 mL of DEPC water, mix well and use it as the sample to be enriched (blind sample). (2) Lysis: Take 10 mL of enrichment lysis buffer containing different concentrations of guanidine isothiocyanate, add it to the sample to be enriched to 50 mL, shake well, and obtain the lysis buffer; (3) Magnetic bead enrichment: Add 300 μL of magnetic beads to the lysis solution and shake well for 5 min by inverting; (4) Recovering magnetic beads: Place the lysis buffer in a magnetic rack for 8-10 minutes to adsorb the magnetic beads. After the magnetic beads are completely adsorbed, discard the supernatant and remove the bottom residue with a pipette. (5) Elution: Add 400 μL of the enrichment eluent prepared in Example 1 to each tube, rinse the magnetic beads on the tube wall to the bottom, and transfer them to a 2 mL centrifuge tube after full resuspension; gently shake the 2 mL centrifuge tube containing the resuspension magnetic beads to mix them, and then centrifuge the centrifuge tube in a handheld centrifuge for 50 s. The supernatant is the enriched sample, which is used for subsequent experiments. (6) Nucleic acid extraction: Nucleic acid was extracted from the enriched samples using a commercial nucleic acid extraction kit (BorgBSC86T1C) to obtain nucleic acid samples; (7) qPCR detection: The nucleic acid samples were detected by fluorescent PCR using the OIE standard for ASFV virus (each sample was repeated 3 times) to obtain the detection results. The primer and probe sequences for qPCR are shown in Table 1, and the reaction system and qPCR amplification program are shown in Table 2 and Table 3, respectively.
[0044] Table 1 Primers and Probes
[0045] Table 2 Reaction System
[0046] Table 3 qPCR amplification program
[0047] III. Experimental Results The experimental results are shown in Table 4. The results indicate that the guanidine isothiocyanate content in the enrichment lysis buffer has a significant impact on the enrichment effect of ASFV pseudovirus: when the guanidine isothiocyanate content is 150 g / 500 mL, the average Ct value increases by 6.52; at 250 g / 500 mL, it increases by 5.97; and at 500 g / 500 mL, it increases by 5.25. A larger ΔCt value indicates higher enrichment efficiency; therefore, 150 g / 500 mL was selected as the optimal addition amount of guanidine isothiocyanate in the lysis buffer.
[0048] Table 4. Enrichment effect of lysis buffers with different guanidine isothiocyanate contents
[0049] Experimental Example 2: Effect of Tween-20 Content in Enrichment Buffer on Enrichment Efficiency I. Preparation of enriched lysis buffers with different Tween-20 contents The enrichment lysis buffer was prepared according to the method in Example 1. The amounts of guanidine isothiocyanate (150 g) and 3 M sodium acetate buffer (pH 5.2, 12.5 mL) were fixed, and only the amount of Tween-20 was adjusted. Based on a 500 mL lysis buffer system, Tween-20 gradients of 50 mL, 62.5 mL, and 75 mL were set to obtain three enrichment lysis buffers with different concentrations of Tween-20.
[0050] II. Experimental Methods The steps are the same as in Experimental Example 1, except that the lysis buffer in step (2) is replaced with enriched lysis buffers with different Tween-20 contents prepared in this experimental example. The remaining steps are the same as in Experimental Example 1. The specific steps are as follows: (1) Sample preparation: Add a random amount of ASFV pseudovirus to 100 mL of DEPC water, mix well and use as the enrichment sample (blind sample). (2) Lysis: Take 10 mL of lysis buffer containing different concentrations of Tween-20, add it to the sample to be enriched to 50 mL, shake well, and obtain the lysis buffer; (3) Magnetic bead enrichment: Add 300 μL of magnetic beads to the lysis solution and shake well for 5 min by inverting; (4) Recovering magnetic beads: Place the lysis buffer in a magnetic rack for 8-10 minutes to adsorb the magnetic beads. After the magnetic beads are completely adsorbed, discard the supernatant and remove the bottom residue with a pipette. (5) Elution: Add 400 μL of the enrichment eluent prepared in Example 1 to each tube, rinse the magnetic beads on the tube wall to the bottom, and transfer them to a 2 mL centrifuge tube after full resuspension. Gently shake the 2 mL centrifuge tube containing the resuspension magnetic beads to mix them, and then centrifuge the centrifuge tube for 50 s. The supernatant is the enriched sample, which is used for subsequent experiments. (6) Nucleic acid extraction: Nucleic acid was extracted from the enriched sample using a commercially available nucleic acid extraction kit to obtain nucleic acid samples; (7) qPCR detection: The nucleic acid samples were tested by fluorescent PCR using the OIE standard for ASFV virus (each sample was tested in triplicate) to obtain the detection results. The specific method for qPCR detection is the same as in Experiment 1.
[0051] III. Experimental Results The experimental results are shown in Table 5. The results indicate that different Tween-20 contents in the enrichment lysis buffer resulted in different enrichment effects for ASFV virus. The average CT value increased by 5.24 for enrichment lysis buffer containing 50 mL of Tween-20; by 5.43 for containing 62.5 mL of Tween-20; and by 5.27 for containing 75 mL of Tween-20. The preferred Tween-20 content in the enrichment lysis buffer is 62.5 mL / 500 mL.
[0052] Table 5. Enrichment effect of lysis buffers with different Tween-20 contents
[0053] Example 3: Effect of 3M sodium acetate buffer content in enrichment lysis buffer on enrichment effect I. Preparation of enrichment and lysis buffers with different 3 M sodium acetate buffer contents The enrichment lysis buffer was prepared according to the method in Example 1, with the amounts of guanidine isothiocyanate (150 g) and Tween-20 (62.5 mL) fixed, and only the amount of 3 M sodium acetate buffer (pH 5.2) was adjusted. Based on a 500 mL lysis buffer system, three enrichment lysis buffers with different sodium acetate contents were prepared by setting gradients of 12.5 mL, 25 mL, and 37.5 mL.
[0054] II. Experimental Methods The steps are the same as in Experiment 1, except that the lysis buffer in step (2) is replaced with enrichment lysis buffers prepared in this experiment with different concentrations of 3 M sodium acetate buffer. The remaining steps are the same as in Experiment 1. The specific steps are as follows: (1) Add a random amount of ASFV pseudovirus to 100 mL of DEPC water, mix well and use as an enrichment sample (blind sample). (2) Lysis: Take 10 mL of enrichment lysis buffer with different sodium acetate contents, add it to the sample to be enriched to 50 mL, shake well, and obtain the lysis buffer; (3) Magnetic bead enrichment: Add 300 μL of magnetic beads to the lysis solution and shake well for 5 min by inverting; (4) Recovering magnetic beads: Place the lysis buffer in a magnetic rack for 8-10 minutes to adsorb the magnetic beads. After the magnetic beads are completely adsorbed, discard the supernatant and remove the bottom residue with a pipette. (5) Elution: Add 400 μL of the enrichment eluent prepared in Example 1 to each tube, rinse the magnetic beads on the tube wall to the bottom, and transfer them to a 2 mL centrifuge tube after full resuspension. Gently shake the 2 mL centrifuge tube containing the resuspension magnetic beads to mix them, and then centrifuge the centrifuge tube for 50 s. The supernatant is the enriched sample, which is used for subsequent experiments. (6) Nucleic acid extraction: Nucleic acid was extracted from the enriched sample using a commercially available nucleic acid extraction kit to obtain nucleic acid samples; (7) qPCR detection: The nucleic acid samples were tested by fluorescent PCR using the OIE standard for ASFV virus (each sample was tested in triplicate) to obtain the detection results. The specific method for qPCR detection is the same as in Experiment 1.
[0055] III. Experimental Results The experimental results are shown in Table 6. The results indicate that different concentrations of 3M sodium acetate buffer in the enrichment lysis buffer resulted in different enrichment effects for ASFV virus: the average CT value increased by 6.51 with 12.5 mL of 3M sodium acetate buffer; by 5.72 with 25 mL of 3M sodium acetate buffer; and by 5.38 with 37.5 mL of 3M sodium acetate buffer. The optimal concentration of 3M sodium acetate buffer in the enrichment lysis buffer is 12.5 mL / 500 mL.
[0056] Table 6. Enrichment effect of lysis buffers with different 3 M sodium acetate buffer contents
[0057] Experiment Example 4: Effect of Isopropanol Content in Enrichment Eluent on Enrichment Efficiency I. Preparation of enrichment eluents with different isopropanol contents The enrichment eluent was prepared according to the method in Example 1. The amounts of Tris-HCl buffer (100 mM, pH 7.5, 10 mL) and Triton X-100 (1 mL) were fixed, and only the amount of isopropanol was adjusted. Based on a 100 mL eluent system, isopropanol gradients of 0 mL, 10 mL, and 20 mL were set to obtain three enrichment eluents with different concentrations of isopropanol.
[0058] II. Experimental Methods The steps are the same as in Experiment 1, except that the enrichment eluent in step (5) is replaced with enrichment eluents with different isopropanol contents prepared in this experiment. The remaining steps are the same as in Experiment 1. The specific steps are as follows: (1) Add a random amount of ASFV pseudovirus to 100 mL of DEPC water, mix well and use as an enrichment sample (blind sample). (2) Lysis: Take 10 mL of the enrichment lysis buffer prepared in Example 1, add it to the sample to be enriched to 50 mL, shake well, and obtain the lysis buffer; (3) Magnetic bead enrichment: Add 300 μL of magnetic beads to the lysis solution and shake well for 5 min by inverting; (4) Recovering magnetic beads: Place the lysis buffer in a magnetic rack for 8-10 minutes to adsorb the magnetic beads. After the magnetic beads are completely adsorbed, discard the supernatant and remove the bottom residue with a pipette. (5) Elution: Add 400 μL of enrichment eluent with different isopropanol contents to each tube, rinse the magnetic beads on the tube wall to the bottom, resuspend them fully and transfer them to a 2 mL centrifuge tube, gently shake the 2 mL centrifuge tube containing the resuspended magnetic beads to mix them, and then centrifuge the centrifuge tube for 50 s. The supernatant is the enriched sample. The enriched sample is used for subsequent experiments. (6) Nucleic acid extraction: Nucleic acid was extracted from the enriched sample using a commercially available nucleic acid extraction kit to obtain nucleic acid samples; (7) qPCR detection: The nucleic acid samples were tested by fluorescent PCR using the OIE standard for ASFV virus (each sample was tested in triplicate) to obtain the detection results. The specific method for qPCR detection is the same as in Experiment 1.
[0059] III. Experimental Results The experimental results are shown in Table 7. The results indicate that different isopropanol contents in the enrichment eluent resulted in different enrichment effects for ASFV virus: the average CT value increased by 5.35 for the enrichment eluent containing 0 mL of isopropanol; by 5.81 for the enrichment eluent containing 10 mL of isopropanol; and by 5.905 for the enrichment eluent containing 20 mL of isopropanol. The preferred isopropanol content in the enrichment eluent is 20 mL / 100 mL.
[0060] Table 7. Enrichment effect of eluents with different isopropanol contents
[0061] Experiment Example 5: Effect of Triton X-100 content in the enrichment eluent on the enrichment effect I. Preparation of enrichment eluents with different Triton X-100 contents The enrichment eluent was prepared according to the method in Example 1. The amounts of Tris-HCl buffer (100 mM, pH 7.5, 10 mL) and isopropanol (20 mL) were fixed, and only the amount of Triton X-100 was adjusted. Based on a 100 mL eluent system, enrichment eluents with concentrations of 0.5 mL, 1 mL, and 2 mL were prepared to obtain three different concentrations of Triton X-100.
[0062] II. Experimental Methods The steps are the same as in Experiment 1, except that the enrichment eluent in step (5) is replaced with enrichment eluents of different Triton X-100 concentrations prepared in this experiment. The remaining steps are the same as in Experiment 1. The specific steps are as follows: (1) Add a random amount of ASFV pseudovirus to 100 mL of DEPC water, mix well and use it as a sample to be enriched (blind sample). (2) Lysis: Take 10 mL of the enrichment lysis buffer prepared in Example 1, add it to the sample to be enriched to 50 mL, shake well, and obtain the lysis buffer; (3) Magnetic bead enrichment: Add 300 μL of magnetic beads to the lysis solution and shake well for 5 min by inverting; (4) Recovering magnetic beads: Place the lysis buffer in a magnetic rack for 8-10 minutes to adsorb the magnetic beads. After the magnetic beads are completely adsorbed, discard the supernatant and remove the bottom residue with a pipette. (5) Elution: Add 400 μL of enrichment eluent with different Triton X-100 contents to each tube, rinse the magnetic beads on the tube wall to the bottom, resuspend them fully and transfer them to a 2 mL centrifuge tube, gently shake the 2 mL centrifuge tube containing the resuspended magnetic beads to mix them, and then centrifuge the centrifuge tube for 50 s. The supernatant is the enriched sample. The enriched sample is used for subsequent experiments. (6) Nucleic acid extraction: Nucleic acid was extracted from the enriched sample using a commercially available nucleic acid extraction kit to obtain nucleic acid samples; (7) qPCR detection: The nucleic acid samples were tested by fluorescent PCR using the OIE standard for ASFV virus (each sample was tested in triplicate) to obtain the detection results. The specific method for qPCR detection is the same as in Experiment 1.
[0063] III. Experimental Results The experimental results are shown in Table 8. The results indicate that different Triton X-100 contents in the enrichment eluent resulted in different enrichment effects for ASFV virus: the average CT value increased by 5.49 for 0.5 mL Triton X-100, by 5.705 for 1 mL Triton X-100, and by 5.64 for 2 mL Triton X-100. The optimal Triton X-100 addition amount in the enrichment eluent is 1 mL / 100 mL.
[0064] Table 8. Enrichment effect of eluents with different Triton X-100 contents
[0065] Experimental Example 6: Reagent Kit Sensitivity Evaluation I. Experimental Methods (1) Sample preparation: Prepare ASFV pseudovirus stock solution (qPCR detection Ct value of about 25), and use “20 mL stock solution + 180 mL DEPC water” to perform 10-fold serial dilution to obtain a series of samples with medium and high concentrations (samples 1~3) and low concentrations (samples 4~8). The theoretical Ct values of each concentration are shown in Table 9.
[0066] Table 9 Theoretical Ct values for gradient dilution samples
[0067] (2) Lysis: Take 10 mL of the enriched lysis buffer prepared in Example 1, add each concentration sample (sample 1~8) to 50 mL, shake well, and obtain the lysis buffer; (3) Magnetic bead enrichment: Add 300 μL of magnetic beads to the lysis solution and shake well for 5 min by inverting; (4) Recovering magnetic beads: Place the lysis buffer in a magnetic rack for 8-10 minutes to adsorb the magnetic beads. After the magnetic beads are completely adsorbed, discard the supernatant and remove the bottom residue with a pipette. (5) Elution: Add 400 μL of the enrichment eluent prepared in Example 1 to each tube, rinse the magnetic beads on the tube wall to the bottom, and transfer them to a 2 mL centrifuge tube after full resuspension. Gently shake the 2 mL centrifuge tube containing the resuspension magnetic beads to mix them, and then centrifuge the centrifuge tube for 50 s. The supernatant is the enriched sample, which is used for subsequent experiments. (6) Nucleic acid extraction: Nucleic acid was extracted from the enriched sample using a commercially available nucleic acid extraction kit to obtain nucleic acid samples; (7) qPCR detection: The nucleic acid samples were tested by fluorescent PCR using the OIE standard for ASFV virus (each sample was tested in triplicate) to obtain the detection results. The specific method for qPCR detection is the same as in Experiment 1.
[0068] II. Experimental Results The results are shown in Table 10. For medium-to-high concentration samples (Ct 24-30 range), the average increase in CT value was 5.175 due to the saturation of magnetic bead binding sites. For low concentration samples (Ct>35 range), the enrichment system significantly concentrated the target nucleic acid, with an average increase in CT value of 7.286, enabling stable detection of low-load samples that were previously undetectable or weakly positive (such as samples 6 and 7). Considering the entire concentration range, the kit of this invention achieved an average increase in CT value of 6.381, effectively extending the detection limit by 3-4 orders of magnitude, fully meeting the enrichment requirements for low-concentration pathogen nucleic acids in aquatic environments.
[0069] Table 10 Evaluation of enrichment effect of samples at different concentrations
[0070] Note: 41.3, 44.6, and 47.9 are theoretical values for gradient dilution. Experiment 7: Stability Evaluation of the Reagent Kit The prepared enrichment system (including lysis buffer, eluent and magnetic beads) was stored in the dark at 4 ℃, 25 ℃ and 37 ℃ for 1 month.
[0071] I. Experimental Methods (1) Sample preparation: An unknown amount of ASFV pseudovirus was added to 100 mL of DEPC water. After quantitative fluorescence detection, the CT value of the stock solution was determined to be 29.15. The homogenized test sample was obtained by diluting the stock solution with 8 mL of stock solution and 300 mL of DEPC water. Subsequently, the sample was enriched using enrichment systems that had been stored at different temperatures for one month. (2) Lysis: Take 10 mL of lysis buffer, add it to 50 mL of the mixed water sample, and shake well; (3) Magnetic bead enrichment: Add 300 μL of magnetic beads to the lysis solution and shake well for 5 min by inverting; (4) Recovering magnetic beads: Place the lysis buffer in a magnetic rack for 8-10 minutes to adsorb the magnetic beads. After the magnetic beads are completely adsorbed, discard the supernatant and remove the bottom residue with a pipette. (5) Elution: Add 400 μL of elution buffer to each tube, rinse the magnetic beads on the tube wall to the bottom, resuspend them fully and transfer them to a 2 mL centrifuge tube. Gently shake the 2 mL centrifuge tube containing the resuspended magnetic beads to mix them. Then centrifuge the centrifuge tube for 50 seconds. The supernatant is the enriched sample. Use the enriched sample for subsequent experiments. (6) Nucleic acid extraction: A commercial nucleic acid extraction kit was used for extraction, and the extracted nucleic acid was used for subsequent experiments; (7) PCR detection: The nucleic acid samples were tested by fluorescent PCR using the OIE standard for ASFV virus (each sample was tested in triplicate) to obtain the detection results. The specific method for qPCR detection is the same as in Experiment 1.
[0072] II. Experimental Results The results are shown in Table 11. For the enrichment system stored at different temperatures for one month, it still maintained good enrichment effect under storage conditions of 4℃ and 25℃, while under storage conditions of 37℃, the efficiency decreased slightly but still had significant enrichment ability, proving that the system has good stability.
[0073] Table 11. Enrichment stability of the reagent kit at different storage temperatures
[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A kit for enriching pathogen nucleic acids, characterized in that, It includes an enrichment lysis buffer and an enrichment eluent; the enrichment lysis buffer contains guanidine isothiocyanate, sodium acetate, and Tween-20; the enrichment eluent contains tris(hydroxymethyl)aminomethane, isopropanol, and Triton X-100.
2. The reagent kit according to claim 1, characterized in that, The enrichment lysis buffer comprises the following components: 300-1000 mg / mL guanidine isothiocyanate, 75-225 μmol / mL sodium acetate, and 10-15% v / v Tween-20.
3. The reagent kit according to claim 2, characterized in that, The enrichment lysis buffer comprises the following components: 300-500 mg / mL guanidine isothiocyanate, 75-150 μmol / mL sodium acetate, and 12.5-15% v / v Tween-20.
4. The kit according to claim 1, characterized in that, The enrichment eluent comprises the following components: 5-15 μmol / mL tris(hydroxymethyl)aminomethane, 10-20% v / v isopropanol, and 0.5-2% v / v Triton X-100.
5. The reagent kit according to claim 1, characterized in that, The kit also includes magnetic beads, the surface of which is modified with at least one of carboxyl, amino, or silanol groups.
6. The use of the kit described in any one of claims 1 to 5 in the enrichment of viral nucleic acids.
7. A method for enriching low-concentration pathogen nucleic acids in water, characterized in that, Enrichment was performed using the kit described in any one of claims 1 to 5.
8. The enrichment method according to claim 7, characterized in that, Includes the following steps: S1. Take the enriched lysis buffer and mix it with the water sample, then shake well to obtain the lysis buffer; S2. Add magnetic beads to the lysis solution obtained in S1, mix well and incubate; S3. Magnetic beads are attracted under the action of a magnetic field, and the supernatant is discarded; S4. Add enrichment elution buffer to the magnetic beads, resuspend, centrifuge and collect the supernatant to obtain the enriched sample.
9. The enrichment method according to claim 8, characterized in that, In step S1, the volume ratio of the enrichment lysis buffer to the water sample is 1:(3~6); in step S4, the amount of enrichment elution buffer added is 300~500 μL.
10. The application of the kit according to any one of claims 1 to 5 or the enrichment method according to any one of claims 7 to 9 in the detection of African swine fever virus in water.