Kit for extracting specific short fragment cfDNA in urine and application thereof

Abstract

The invention discloses a kit capable of being used for extracting specific short fragment cfDNA in urine and application of the kit. The kit comprises protease K, a lysis solution, a cleaning solution, an eluent and magnetic beads, wherein the magnetic beads comprise silicon hydroxyl magnetic microspheres and magnetic microspheres modified by streptavidin. According to the technical scheme, through matching of composition control of the magnetic beads and optimization of selection of the components, when the obtained kit is used for extracting free DNA in urine, the extraction efficiency of specific short fragments is greatly improved, and the detection accuracy is further improved.

Description

Technical Field

[0001] This invention relates to the field of DNA extraction technology. More specifically, it relates to a kit for the specific extraction of short cfDNA fragments from urine and its application. Background Technology

[0002] Cell-free DNA (cfDNA) in plasma originates from DNA released after cell damage and rupture. It is cleaved into fragments of approximately 190 bp by nucleases, and its production and clearance are a dynamic process (half-life approximately 5-150 minutes). This "global snapshot" capability makes it an ideal molecular biomarker for many diseases. Blood and urine are both important sources of human cfDNA samples. Compared to blood, urine samples are easier to collect, requiring no professional intervention; urine samples can be considered truly non-invasive; urine samples have a large volume, meeting the sample volume requirements of various tests; and longitudinal sampling at multiple time points is possible. It plays an important role in early tumor screening, disease diagnosis, and prognostic monitoring. However, due to glomerular filtration in the kidneys and the rapid degradation kinetics of all cfDNA in urine, cfDNA in urine is more fragmented. The distribution of cfDNA fragment lengths varies from sample to sample, but most urine cfDNA fragments are expected to be less than 100 bp. Recently, new single-stranded library preparation methods have shown that very short, previously undetectable fragments constitute a larger proportion in both plasmas. Reducing PCR amplicon length has been shown to improve clinical sensitivity and the detection concentration of urinary cfDNA. Traditional DNA extraction methods adsorb DNA onto silica on the surface of magnetic beads or adsorption columns. The driving force for DNA adsorption onto silica—namely, the hydrophobic interaction caused by dehydration of the silica and DNA surface and the hydrogen bonds between silica and the DNA backbone—is proportional to the DNA fragment length. Therefore, fragment recovery decreases as fragment length and concentration decrease, making it unsuitable for the extraction of short urinary cfDNA fragments (<100 bp).

[0003] Currently, many methods exist for enriching short DNA fragments after extraction, such as hybridization capture and single-strand library construction. However, these methods can only enrich the extracted short DNA fragments, leaving more short DNA fragments in the sample, which seriously affects the accuracy and sensitivity of the detection results. Therefore, there is an urgent need to develop a method that can efficiently extract short cfDNA fragments (<100bp). Summary of the Invention

[0004] Based on the above facts, the purpose of this invention is to provide a kit for the extraction of specific short fragments of cfDNA from urine and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A kit for the specific extraction of short cfDNA fragments from urine, the kit comprising:

[0007] Proteinase K, lysis buffer, washing buffer, elution buffer, and magnetic beads;

[0008] The magnetic beads include silanol magnetic microspheres and streptavidin-modified magnetic microspheres.

[0009] Furthermore, the mass ratio of the silanol magnetic microspheres to the streptavidin-modified magnetic microspheres is 1:1 to 5:1.

[0010] Furthermore, the structure of the silanol magnetic microspheres includes a core of iron oxide (Fe3O4) and a silicon dioxide layer covering the surface of the iron oxide.

[0011] Furthermore, the particle size of the silanol magnetic microspheres is 500-1000 nm.

[0012] Furthermore, the structure of the streptavidin-modified magnetic microspheres includes a core of iron oxide (Fe3O4) and APTS and streptavidin coated on the surface of the iron oxide.

[0013] Furthermore, the streptavidin-modified magnetic microspheres have a particle size of 100-400 nm.

[0014] Furthermore, the lysis buffer contains a guanidine salt, a chelating agent, a buffer solution, and a surfactant, wherein the guanidine salt contains guanidine isothiocyanate and guanidine hydrochloride.

[0015] Furthermore, the lysis buffer contains 3-5 mol / L guanidine isothiocyanate, 0.3-0.8 mol / L guanidine hydrochloride, 10-30 mmol / L chelating agent, 2-5.3 wt% surfactant, and 20-80 mmol / L buffer solution, and the pH of the lysis buffer is between 7.0 and 8.0.

[0016] Furthermore, the cleaning solution comprises a first cleaning solution and a second cleaning solution; wherein,

[0017] The first cleaning solution contains 1-3 mol / L guanidine isothiocyanate, 10-30 mmol / L Tris-HCl, 0.05 wt% surfactant (e.g., Triton X-100), 0.5-2 mol / L NaCl, and 30%-50% isopropanol by volume. The pH of the first cleaning solution is 7.0-8.0.

[0018] The second cleaning solution contains 60wt%-80wt% ethanol and the remainder is enzyme-free water.

[0019] Furthermore, the eluent contains 0-20 mmol / L Tris-EDTA and the remainder is enzyme-free water, and the pH of the eluent is between 7.0 and 8.0.

[0020] Furthermore, the preparation of the silanol magnetic microspheres includes the following steps:

[0021] Under a nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O were dissolved in water, heated, and oleic acid and ammonia were added in sequence. After mixing, oleic acid was added dropwise, mixed, and kept warm to obtain a black precipitate, which was denoted as precursor 1.

[0022] The precursor 1, silane coupling agent, surfactant and water were ultrasonically mixed to obtain a brown emulsion.

[0023] The brown emulsion was mixed with a dispersant and a catalyst, and the reaction was carried out in a closed system to obtain the silanol magnetic microspheres.

[0024] Furthermore, the preparation of the streptavidin-modified magnetic microspheres includes the following steps:

[0025] Under a nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O were dissolved in water and heated. Ammonia was then added and the reaction was maintained at this temperature to obtain a black precipitate, which was denoted as precursor 2.

[0026] After mixing the precursor 2 with ethylene glycol, APTS was added, and the mixture was subjected to ultrasonic reaction. Then, it was magnetically separated and dried sequentially to obtain the precursor 3.

[0027] Precursor 3 was dissolved in PBS buffer, then mixed with PBS buffer containing streptavidin and dispersed evenly. After magnetic separation and washing, the streptavidin-modified magnetic microspheres were obtained.

[0028] In another aspect, the present invention provides the application of the kit described above in the extraction of nucleic acids from urine.

[0029] Furthermore, the nucleic acid is a specific cfDNA.

[0030] Furthermore, the kit is used to extract specific cfDNA fragments from urine, and the minimum length of the specific cfDNA fragments is <100bp.

[0031] Furthermore, the application includes the following steps:

[0032] After removing impurities, the urine was mixed with the lysis buffer and proteinase K, and incubated at 60°C for 10 min. Then, biotin probe was added and mixed, silanol magnetic microspheres were added, mixed and allowed to stand, and then streptavidin-modified magnetic microspheres were added, mixed and allowed to stand. After centrifugation, the supernatant was removed to obtain the crude nucleic acid product.

[0033] The crude nucleic acid product was purified using a washing solution;

[0034] After mixing the purified crude nucleic acid product with the elution buffer, centrifuge and collect the supernatant, which is recorded as supernatant 1.

[0035] Add NaOH solution to the mixture after removing supernatant 1, mix well, centrifuge, and collect the supernatant, which is recorded as supernatant 2.

[0036] The extracted nucleic acid is contained in supernatant 1 and supernatant 2.

[0037] Furthermore, the method for purifying the crude nucleic acid product using a washing solution includes the following steps:

[0038] Add the first washing solution to the crude nucleic acid product, mix well, centrifuge, and remove the supernatant after standing.

[0039] Add the second washing solution, mix well, centrifuge, let stand, and remove the supernatant to obtain the purified crude nucleic acid product.

[0040] The beneficial effects of this invention are as follows:

[0041] The technical solution of the reagent kit provided in this invention, through the combination of magnetic beads and the optimization of the selection of each component, significantly improves the extraction efficiency of specific short fragments when the reagent kit is used to extract cell-free DNA from urine, and further improves the accuracy of detection. Attached Figure Description

[0042] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0043] Figure 1 The diagram illustrates the working principle of silanol magnetic microspheres and streptavidin-modified magnetic microspheres in some examples of the present invention.

[0044] Figure 2 A TEM image of the silanol magnetic microspheres in Example 1 is shown.

[0045] Figure 3 The image shows a TEM image of the streptavidin-modified magnetic microspheres from Example 2.

[0046] Figure 4 The image shows a quality inspection chart of urine sample 4200 from Example 4. Detailed Implementation

[0047] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0048] To effectively extract cfDNA fragments from urine, especially short cfDNA fragments (<100bp), to obtain more genetic information and thus improve the accuracy and sensitivity of nucleic acid detection results, a specific embodiment of the present invention provides a kit for extracting specific short cfDNA fragments from urine, the kit comprising:

[0049] Proteinase K, lysis buffer, washing buffer, elution buffer, and magnetic beads;

[0050] The magnetic beads include silanol magnetic microspheres and streptavidin-modified magnetic microspheres.

[0051] This technical solution effectively addresses the issue that some urine DNA extraction kits primarily extract DNA fragments between 100-200 bp in length, without any DNA fragments shorter than 100 bp.

[0052] It should be noted that the kit provided in this embodiment can not only effectively extract short cfDNA fragments (<100bp) from urine, but also effectively extract cfDNA fragments with a length of more than 100bp from urine.

[0053] In this embodiment, the extraction efficiency of short-fragment cfDNA from urine is effectively improved by using a mixture of two specific magnetic beads. In some examples, the structure of the silanol magnetic microspheres includes a core of magnetite (Fe3O4) and a silica layer coating the surface of the magnetite.

[0054] In some examples, the preferred particle size of the silanol magnetic microspheres is 500-1000 nm.

[0055] In some examples, the streptavidin-modified magnetic microspheres contain a core of iron(III) oxide and APTS and streptavidin coated on the surface of the iron(III) oxide.

[0056] In some examples, the streptavidin-modified magnetic microspheres have a particle size of 100-400 nm.

[0057] The working principle of the silanol-modified magnetic microspheres and the streptavidin-modified magnetic microspheres is as follows: Figure 1As shown. The specific working principle is that the probe modified on the magnetic microspheres specifically captures small fragments of cfDNA that have not been adsorbed by the silanol magnetic beads.

[0058] Generally, larger magnetic beads have greater magnetic responsiveness and a stronger ability to capture small nucleic acid fragments. Smaller magnetic beads have lower magnetic responsiveness and a stronger ability to capture larger nucleic acid fragments. However, for DNA, regardless of particle size, magnetic beads preferentially adsorb long DNA fragments. Even with large-diameter magnetic beads, many small DNA fragments cannot be adsorbed, thus requiring the use of biotin probes to bind to the DNA fragments to be detected. Furthermore, the amount of magnetic beads that can be added is limited; adding too many beads can cause them to aggregate and become unseparable, reducing the extraction efficiency. Therefore, in this embodiment, a mixture of appropriately sized silanol magnetic microspheres and streptavidin-modified magnetic microspheres is used for DNA extraction to ensure no genetic information is missed and to achieve higher accuracy.

[0059] In some preferred examples, the mass ratio of the silanol magnetic microspheres to the streptavidin-modified magnetic microspheres is 1:1-5:1, more preferably (1-3):1, 3:1, etc. This is more conducive to the effective extraction of short cfDNA fragments.

[0060] In this embodiment, there are no other requirements for the preparation of silanol magnetic microspheres. Some exemplary preferred methods for preparing silanol magnetic microspheres include the following steps:

[0061] Under a nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O were dissolved in water to obtain a mixed solution. The solution was heated, and oleic acid and ammonia were added in sequence. After mixing, oleic acid was added dropwise and mixed again. The solution was kept at a constant temperature to obtain a black precipitate, which was denoted as precursor 1.

[0062] The precursor 1, silane coupling agent, surfactant and water were ultrasonically mixed to obtain a brown emulsion.

[0063] The brown emulsion was mixed with a dispersant and a catalyst, and the reaction was carried out in a closed system to obtain the silanol magnetic microspheres.

[0064] In the preparation of the mixture, the preferred ratio of FeCl3·6H2O to water is (1.5-2) g / 100 ml, and the preferred ratio of FeCl2·4H2O to water is (0.5-1) g / 100 ml.

[0065] For example, the dispersant is selected from ethanol, and / or the catalyst is selected from ammonia. By dispersing this brown emulsion in the dispersant and catalyst, the reaction between the silane coupling agent and precursor 1 is promoted.

[0066] For example, the temperature after heating is 80°C.

[0067] For example, the mass ratio of oleic acid to FeCl3·6H2O is (0.08-0.12):1.

[0068] For example, the insulation temperature is 80°C and the time is 2-6 hours, preferably 4 hours.

[0069] For example, in the process of preparing the brown emulsion, the mixing ratio of precursor 1, tetraethyl orthosilicate, surfactant and water is (0.8-1.2)g:10ml:(0.8-1.2)g:125ml, preferably 1g:10ml:1g:125ml.

[0070] For example, the mixing ratio of the brown emulsion with the dispersant and catalyst is (100-125) ml:(100-125) ml:45 ml, preferably 125 ml:125 ml:45 ml.

[0071] For example, the temperature of the sealed reaction is 80°C and the time is 4-5 hours, preferably 4.5 hours.

[0072] In this example, the role of the silane coupling agent includes modifying the surface of precursor 1 with silanol groups. Exemplary silane coupling agents include, but are not limited to, tetraethyl orthosilicate (TEOS), 3-aminopropyltrimethoxysilane, etc. TEOS is preferred.

[0073] The silanol magnetic microspheres prepared by this method have a more suitable particle size and are more uniform in size.

[0074] In this embodiment, in some preferred examples, the preparation of the streptavidin-modified magnetic microspheres includes the following steps:

[0075] Under a nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O were dissolved in water and heated. Ammonia was then added and the reaction was maintained at this temperature to obtain a black precipitate, which was denoted as precursor 2.

[0076] After mixing the precursor 2 with ethylene glycol, 3-aminopropyltriethoxysilane (APTS) was added, and after ultrasonic reaction, the mixture was magnetically separated and dried to obtain precursor 3.

[0077] Precursor 3 was dissolved in PBS buffer, then mixed with PBS buffer containing streptavidin and dispersed evenly. After magnetic separation and washing, the streptavidin-modified magnetic microspheres were obtained.

[0078] Streptavidin can bind to biotin probes, thereby enabling more specific capture of small cfDNA fragments.

[0079] For example, the preferred ratio of FeCl3·6H2O to water is (1.5-2) g / 100 ml, and the preferred ratio of FeCl2·4H2O to water is (0.5-1) g / 100 ml.

[0080] For example, the temperature after heating is 80°C.

[0081] For example, the temperature of the heat preservation reaction is 80°C and the time is 2-6 hours, preferably 4 hours.

[0082] For example, the ratio of precursor 2 to APTS is 1:4.

[0083] For example, the temperature of the ultrasonic reaction is 20-50°C and the time is 2 hours.

[0084] For example, the pH of the PBS buffer is 9-9.5.

[0085] For example, the mass ratio of precursor 3 to streptavidin is 1:2.

[0086] In some preferred embodiments, the lysis buffer contains a guanidine salt, a chelating agent, a buffer solution, and a surfactant, wherein the guanidine salt contains guanidine isothiocyanate and guanidine hydrochloride. Preferably, the lysis buffer contains 3-5 mol / L guanidine isothiocyanate and 0.3-0.8 mol / L guanidine hydrochloride. Adding a small amount of guanidine hydrochloride and guanidine isothiocyanate to the lysis buffer can denature and inactivate proteins, and also inhibit nuclease activity, thereby reducing the degradation of urinary DNA and facilitating rapid rupture of the cell membrane to release nucleic acids.

[0087] In some examples, the lysis buffer contains 3-5 mol / L guanidine isothiocyanate, 0.3-0.8 mol / L guanidine hydrochloride, 10-30 mmol / L chelating agent, 2-5.3 wt% surfactant, and 20-80 mmol / L buffer solution, and the pH of the lysis buffer is between 7.0 and 8.0. In this case, the aforementioned effects are even better.

[0088] In some examples, the chelating agent is selected from ethylenediaminetetraacetic acid.

[0089] For example, the surfactant is selected from sodium dodecyl sulfate and sodium lauroyl sarcosinate.

[0090] For example, the buffer solution is selected from Tris-HCl.

[0091] By selecting and controlling the components in the lysis buffer, nucleic acid can be released more quickly.

[0092] In some examples, the cleaning solution comprises a first cleaning solution and a second cleaning solution; wherein,

[0093] The first cleaning solution contains 1-3 mol / L guanidine isothiocyanate, 10-30 mmol / L Tris-HCl, 0.05 wt% surfactant, 0.5-2 mol / L NaCl, and 30%-50% isopropanol by volume. The pH of the first cleaning solution is 7.0-8.0.

[0094] The second cleaning solution contains 60wt%-80wt% ethanol and the remainder is enzyme-free water.

[0095] The primary function of isopropanol in the washing solution is to dissolve proteins adsorbed on the magnetic beads and residual lysis buffer. However, because isopropanol is less volatile than ethanol, it needs to be dried and evaporated before subsequent elution; otherwise, the purity of the product will be affected. In this example, the first washing solution contains a lower concentration of isopropanol compared to publicly available techniques. Under these conditions, proteins are still dissolved well without significantly affecting subsequent elution. Furthermore, the final extracted DNA purity reaches above 1.9, higher than the approximately 1.8 purity of commercial kits.

[0096] In some preferred embodiments, the first cleaning solution contains 40% isopropanol by volume.

[0097] In this example, the second cleaning solution can also be supplemented with buffer solutions, such as Tris-HCl or EDTA, to maintain pH stability, depending on actual needs.

[0098] In some examples, the eluent contains 0-20 mmol / L Tris-EDTA and the remainder is enzyme-free water, and the pH of the eluent is between 7.0 and 8.0.

[0099] Furthermore, it is understood that the kit also contains a biotin probe. For example, the biotin probe can be selected according to the desired DNA fragment. Exemplary probe sequences include, but are not limited to, FAM-TCCCFACCCCTTCCGGGTCC-BHQ1.

[0100] In some examples, the ratio of proteinase K, lysis buffer, first washing buffer, second washing buffer, elution buffer, silanol magnetic microspheres, and streptavidin-modified magnetic microspheres in the kit is (20-30) μL:(300-350) μL:(600-700) μL:400 μL:(80-100) μL:(3-3.5) ml:(3-3.5) ml, preferably 20 μL:300 μL:600 μL:400 μL:80 μL:(3-3.5) ml:(3-3.5) ml. This kit exhibits superior extraction efficiency for short cfDNA fragments.

[0101] Another specific embodiment of the present invention provides the application of the kit described above in the extraction of nucleic acids from urine.

[0102] For example, the nucleic acid is a specific cfDNA.

[0103] For example, the kit is used to extract specific cfDNA fragments from urine, and the minimum length of the specific cfDNA fragments is <100 bp.

[0104] It is understood that when this kit is used to extract heterologous cfDNA from urine, it can effectively extract both short cfDNA fragments (<100bp) and cfDNA fragments longer than 100bp, such as cfDNA fragments of 100-200bp.

[0105] In some examples, the application includes the following steps:

[0106] After removing impurities, the urine was mixed with the lysis buffer and proteinase K, and incubated at 60°C for 10 min. Then, biotin probe was added and mixed, silanol magnetic microspheres were added, mixed and allowed to stand, and then streptavidin-modified magnetic microspheres were added, mixed and allowed to stand. After centrifugation, the supernatant was removed to obtain the crude nucleic acid product.

[0107] The crude nucleic acid product was purified using a washing solution;

[0108] After mixing the purified crude nucleic acid product with the elution buffer, centrifuge and collect the supernatant, which is recorded as supernatant 1.

[0109] Add NaOH solution to the mixture after removing supernatant 1, mix well, centrifuge, and collect the supernatant, which is recorded as supernatant 2.

[0110] The extracted nucleic acid is contained in supernatant 1 and supernatant 2.

[0111] Using the above extraction method, cfDNA fragments with a length of 100-200 bp can be obtained in supernatant 1; cfDNA fragments with a length of <100 bp, preferably cfDNA fragments of 40 to <100 bp, can be obtained in supernatant 2.

[0112] In some examples, the method for purifying the crude nucleic acid product using a washing solution includes the following steps:

[0113] Add the first washing solution to the crude nucleic acid product, mix well, centrifuge, and remove the supernatant after standing.

[0114] Add the second washing solution, mix well, centrifuge, let stand, and remove the supernatant to obtain the purified crude nucleic acid product.

[0115] The specific embodiments of the present invention will be described below with reference to some specific examples:

[0116] Example 1

[0117] The preparation method of silanol magnetic microspheres is as follows:

[0118] In a 500 mL three-necked flask, add 300 mL of distilled water and purge with N2 for 30 min, maintaining a nitrogen atmosphere thereafter. Then add 5 g of FeCl3·6H2O and stir until completely dissolved. Next, add 2 g of FeCl2·4H2O and continue stirring until completely dissolved. Then raise the temperature to 80 °C, add 500 mg of oleic acid (600 μL), and add 20 mL of 25% ammonia solution. After mixing thoroughly, add 0.2 g of oleic acid dropwise to the solution every 2 minutes for 10 minutes. Finally, maintain the reaction at 80 °C for 4 h. The resulting black precipitate is the precursor of magnetic bead 1.

[0119] The cleaned magnetic bead 1 precursor was placed in a test tube, and 10 ml of TEOS, 1 g of Triton X-100, and 125 ml of deionized water were added sequentially. After sonication for 10 min, a brown emulsion was obtained. Under vigorous mechanical stirring, the emulsion was transferred into a 500 ml round-bottom flask containing 125 ml of ethanol and 45 ml of ammonia, and reacted at 80 °C under sealed conditions for 4.5 h to obtain magnetic beads 1 with a particle size of 500-1000 nm, which are the silanol magnetic microspheres. TEM images of these microspheres are shown below. Figure 2 As shown.

[0120] Example 2

[0121] The preparation method of streptavidin-modified magnetic microspheres is as follows:

[0122] In a 500mL three-necked flask, add 300mL of distilled water and purge with N2 for 30min, then maintain a nitrogen atmosphere. Add 5g of FeCl3·6H2O and stir until completely dissolved. Then add 2g of FeCl2·4H2O and continue stirring until completely dissolved. Then raise the temperature to 80℃, add 20mL of 25% ammonia solution, and maintain the reaction at 80℃ for 4h. The resulting black precipitate is the magnetic bead 2 core.

[0123] Add 2g of magnetic bead 2 cores to 100ml of ethylene glycol solution, mix well, add 10g of APTS, sonicate at 20-50℃ for 2h, after magnetic separation, vacuum dry to obtain amino-modified magnetic bead 2 cores.

[0124] 4g of streptavidin was dissolved in 100ml of PBS buffer (pH 9-9.5). Then, 2g of amino-modified magnetic bead 2 cores were added to 50ml of PBS buffer. After ultrasonic dispersion, the mixture was added to the streptavidin-containing PBS buffer. The mixture was stirred at 37°C for 4-6 hours, followed by magnetic separation. After washing with PBS, magnetic bead 2, i.e., the streptavidin-modified magnetic microspheres, was obtained with a particle size of 200nm and stored in PBS solution. A TEM image of magnetic bead 2 is shown below. Figure 3 As shown.

[0125] Example 3

[0126] Extraction of human TERT promoter cfDNA from urine

[0127] This kit contains reagent components and consumable components, including:

[0128] The reagent components include:

[0129] The lysis buffer consists of 3-5 mol / L guanidine isothiocyanate, 0.3-0.8 mol / L guanidine hydrochloride, 10-30 mmol / L ethylenediaminetetraacetic acid, 1-3 wt% sodium dodecyl sulfate, 1-2 wt% sodium lauroyl sarcosinate, 20-80 mmol / L Tris-HCl, and 0.1-0.3 g of surfactant Triton X-100. The pH of the lysis buffer is adjusted to between 7.0 and 8.0.

[0130] Cleaning solution 1 consists of 1-3 mol / L guanidine isothiocyanate, 10-30 mmol / L Tris-HCl, 0.05 wt% surfactant Triton X-100, 0.5-2 mol / L NaCl, and 30%-50% isopropanol by volume. The pH of cleaning solution 1 is adjusted to between 7.0 and 8.0.

[0131] Cleaning solution 2: 60wt%-80wt% ethanol and the remainder enzyme-free water.

[0132] Eluent: 10 mmol / L Tris-EDTA, enzyme-free water, with a pH between 7.0 and 8.0.

[0133] Consumable components include:

[0134] Magnetic bead 1 in Example 1;

[0135] Magnetic bead 2 in Example 2; and

[0136] Proteinase K;

[0137] Biotin probe: Probe sequence: FAM-TCCCFACCCCTTCCGGGTCC-BHQ1.

[0138] The method for extracting human TERT promoter cfDNA from urine includes the following steps:

[0139] 1) DNA extraction:

[0140] First, take 200 μL of pretreated urine to remove impurities and place it in a 1.5 mL centrifuge tube. Then, add 300 μL of lysis buffer and 20 μL of proteinase K, vortex to mix, and incubate at 60 °C for 10 min. Next, add the biotin probe, vortex to mix, add 125 mL of 2.5% magnetic beads 1, vortex to mix, and let stand for 5 min. Then, add 125 mL of 2.5% magnetic beads 2, vortex to mix, and let stand for another 5 min. Finally, centrifuge at low speed, place the centrifuge tube on a magnetic rack, let it stand for two minutes, and then remove the supernatant.

[0141] 2) DNA purification:

[0142] Add 600 μL of cleaning solution 1 to the centrifuge tube, vortex to mix, let stand for two minutes, centrifuge at low speed, place on a magnetic rack, let stand for two minutes, and then remove the supernatant; then add 400 μL of cleaning solution 2, vortex to mix, let stand for two minutes, centrifuge at low speed, place on a magnetic rack, let stand for two minutes, and then remove the supernatant.

[0143] 3) DNA collection:

[0144] Add 80 μL of enzyme-free water to a centrifuge tube, vortex to mix, let stand for 2 min, centrifuge at low speed, place on a magnetic rack, and collect the supernatant (supernatant 1). Add 20 μL of 2M NaOH solution to the centrifuge tube, vortex to mix, let stand for 2 min, centrifuge at low speed, place on a magnetic rack, collect the supernatant (supernatant 2), and then add 20 μL of 2M HCl solution to neutralize. Supernatant 1 contains 100-200 bp cfDNA fragments, and supernatant 2 contains 40-100 bp cfDNA fragments. The concentrations of cfDNA fragments in supernatant 1 were 34.6 / 33.2 / 36.8 ng / μL (data from three experiments); the concentrations in supernatant 2 were 9.7 / 9.6 / 8.9 ng / μL (data from three experiments). This method yields high purity and concentration of both long and short cfDNA fragments.

[0145] Example 4

[0146] Extraction of short cfDNA fragments of the human TERT promoter from urine

[0147] This kit contains reagent components and consumable components. The reagent components include lysis buffer, washing buffer 1, washing buffer 2 and elution buffer. The consumable components include magnetic beads 2 (particle size 200nm) and proteinase K. Biotin probe: probe sequence: FAM-TCCCFACCCCTTCCGGGTCC-BHQ1.

[0148] The method for extracting human TERT promoter cfDNA from urine includes the following steps:

[0149] 1) DNA extraction:

[0150] First, take 200 μL of pretreated urine to remove impurities and place it in a 1.5 mL centrifuge tube. Then, add 20 μL of proteinase K, vortex to mix, and incubate at 60 °C for 10 min. Next, add the biotin probe, vortex to mix, and then add the bifunctional modified magnetic beads. Vortex to mix and let stand for 10 min. Finally, centrifuge at low speed, place the centrifuge tube on a magnetic rack, let it stand for two minutes, and then remove the supernatant.

[0151] 2) DNA purification:

[0152] Add 600 μL of cleaning solution 1 to the centrifuge tube, vortex to mix, let stand for two minutes, centrifuge at low speed, place on a magnetic rack, let stand for two minutes, and then remove the supernatant; then add 400 μL of cleaning solution 2, vortex to mix, let stand for two minutes, centrifuge at low speed, place on a magnetic rack, let stand for two minutes, and then remove the supernatant.

[0153] 3) DNA collection:

[0154] Add 80 μL of enzyme-free water to a centrifuge tube, vortex to mix, let stand for 2 minutes, centrifuge at low speed, place on a magnetic rack, and collect the supernatant (supernatant 1). Add 20 μL of 2M NaOH solution to the centrifuge tube, vortex to mix, let stand for 2 minutes, centrifuge at low speed, place on a magnetic rack, collect the supernatant (supernatant 2), and then add 20 μL of 2M HCl solution to neutralize. Supernatant 1 contains 100-200 bp cfDNA fragments, and supernatant 2 contains 40-100 bp cfDNA fragments. Use a 4200 micrometer to perform quality testing on supernatant 2; the results are as follows. Figure 4 As shown in the figure. The concentrations of supernatant 1 were 12.5 / 15.3 / 13.6 ng / uL (data from three experiments); the concentrations of supernatant 2 were 8.7 / 7.9 / 8.3 ng / uL (data from three experiments).

[0155] Example 5

[0156] Extraction of total cfDNA from urine

[0157] This kit contains reagent components and consumable components. The reagent components include lysis buffer, washing buffer 1, washing buffer 2 and elution buffer. The consumable components include the magnetic beads 1 (particle size of 500-1000nm) and proteinase K.

[0158] 1) DNA extraction:

[0159] First, take 200 μL of pretreated urine to remove impurities and place it in a 1.5 mL centrifuge tube. Then, add 300 μL of lysis buffer and 20 μL of proteinase K, vortex to mix, and incubate at 60 °C for 10 min. Next, add the magnetic beads 1, vortex to mix, and let stand for 10 min. Finally, centrifuge at low speed, place the centrifuge tube on a magnetic rack, let it stand for two minutes, and then remove the supernatant.

[0160] 2) DNA purification:

[0161] Add 600 μL of cleaning solution 1 to the centrifuge tube, vortex to mix, let stand for two minutes, centrifuge at low speed, place on a magnetic rack, let stand for two minutes, and then remove the supernatant; then add 400 μL of cleaning solution 2, vortex to mix, let stand for two minutes, centrifuge at low speed, place on a magnetic rack, let stand for two minutes, and then remove the supernatant.

[0162] 3) DNA collection:

[0163] Add 80 μL of enzyme-free water to a centrifuge tube, vortex to mix, let stand for 2 minutes, centrifuge at low speed, place on a magnetic rack, aspirate and save the supernatant. The obtained supernatant contains 100-200 bp of cfDNA.

[0164] This method makes it difficult to detect cfDNA fragments of 40-100bp (possibly because the concentration is too low to be detected).

[0165] The extraction rates of this kit and the commercial kit were compared using nanodrop (repeated three times), and the results are shown in Table 1 below.

[0166] Table 1

[0167] A260 / A280 Example 5 Reagent Kit 1.92 / 1.91 / 1.89 Commercial reagent kits 1.78 / 1.82 / 1.76

[0168] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A kit for extracting specific short fragment cfDNA from urine, characterized in that, The kit contains: Proteinase K, lysis buffer, washing buffer, elution buffer, and magnetic beads; The magnetic beads include silanol magnetic microspheres and streptavidin-modified magnetic microspheres.

2. The reagent kit according to claim 1, characterized in that, The mass ratio of the silanol magnetic microspheres to the streptavidin-modified magnetic microspheres is 1:1 to 5:

1.

3. The reagent kit according to claim 1, characterized in that, The structure of the silanol magnetic microspheres includes a core of magnetite (Fe3O4) and a silicon dioxide layer coating the surface of the magnetite; and / or The particle size of the silanol magnetic microspheres is 500-1000 nm.

4. The reagent kit according to claim 1, characterized in that, The streptavidin-modified magnetic microspheres contain a core of magnetite (Fe3O4) and APTS and streptavidin coated on the surface of the magnetite; and / or The streptavidin-modified magnetic microspheres have a particle size of 100-400 nm.

5. The reagent kit according to claim 1, characterized in that, The lysis buffer contains a guanidine salt, a chelating agent, a buffer solution, and a surfactant, wherein the guanidine salt contains guanidine isothiocyanate and guanidine hydrochloride; and / or The lysis buffer contains 3-5 mol / L guanidine isothiocyanate, 0.3-0.8 mol / L guanidine hydrochloride, 10-30 mmol / L chelating agent, 2-5.3 wt% surfactant, and 20-80 mmol / L buffer solution, and the pH of the lysis buffer is between 7.0 and 8.

0.

6. The reagent kit according to claim 1, characterized in that, The cleaning solution comprises a first cleaning solution and a second cleaning solution; wherein... The first cleaning solution contains 1-3 mol / L guanidine isothiocyanate, 10-30 mmol / L Tris-HCl, 0.05 wt% surfactant, 0.5-2 mol / L NaCl, and 30%-50% isopropanol by volume. The pH of the first cleaning solution is 7.0-8.

0. The second cleaning solution contains 60wt%-80wt% ethanol and the remainder is enzyme-free water.

7. The kit according to claim 1, characterized in that, The eluent contains 0-20 mmol / L Tris-EDTA and the remainder is enzyme-free water, and the pH of the eluent is between 7.0 and 8.

0.

8. The kit according to claim 1 or 3, characterized in that, The preparation of the silanol magnetic microspheres includes the following steps: Under a nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O were dissolved in water, heated, and oleic acid and ammonia were added in sequence. After mixing, oleic acid was added dropwise, mixed, and kept warm to obtain a black precipitate, which was denoted as precursor 1. The precursor 1, silane coupling agent, surfactant and water were ultrasonically mixed to obtain a brown emulsion. The brown emulsion was mixed with a dispersant and a catalyst, and the reaction was carried out in a sealed environment to obtain the silanol magnetic microspheres.

9. The kit according to claim 1 or 3, characterized in that, The preparation of the streptavidin-modified magnetic microspheres includes the following steps: Under a nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O were dissolved in water and heated. Ammonia was then added and the reaction was maintained at this temperature to obtain a black precipitate, which was denoted as precursor 2. After mixing the precursor 2 with ethylene glycol, APTS was added, and the mixture was subjected to ultrasonic reaction. Then, it was magnetically separated and dried sequentially to obtain the precursor 3. Precursor 3 was dissolved in PBS buffer, then mixed with PBS buffer containing streptavidin and dispersed evenly. After magnetic separation and washing, the streptavidin-modified magnetic microspheres were obtained.

10. The use of the kit according to any one of claims 1-9 in the extraction of nucleic acids from urine.

11. The application according to claim 10, characterized in that, The nucleic acid is a specific cfDNA.

12. The application according to claim 11, characterized in that, The kit is used to extract specific cfDNA fragments from urine, and the minimum length of the specific cfDNA fragment is <100 bp.

13. The application according to claim 10, characterized in that, Includes the following steps: After the urine was cleaned and mixed with the lysis buffer and proteinase K, it was incubated at 60°C for 10 min. Then, biotin probe was added and mixed. Silyl hydroxyl magnetic microspheres were added, mixed and allowed to stand. Then, streptavidin-modified magnetic microspheres were added, mixed and allowed to stand. After centrifugation, the supernatant was removed to obtain the crude nucleic acid product. The crude nucleic acid product was purified using a washing solution; After mixing the purified crude nucleic acid product with the elution buffer, centrifuge and collect the supernatant, which is recorded as supernatant 1. Add NaOH solution to the mixture after removing supernatant 1, mix well, centrifuge, and collect the supernatant, which is recorded as supernatant 2. The extracted nucleic acid is contained in supernatant 1 and supernatant 2.

14. The application according to claim 13, characterized in that, The method for purifying the crude nucleic acid product using a washing solution includes the following steps: Add the first washing solution to the crude nucleic acid product, mix well, centrifuge, and remove the supernatant after standing. Add the second washing solution, mix well, centrifuge, let stand, and remove the supernatant to obtain the purified crude nucleic acid product.

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