A kit suitable for nucleic acid drug metabolism extraction and detection and a method for extracting nucleic acid drugs using the kit

Abstract

The present application relates to the technical field of biochemistry and molecular biology, and specifically to a kit suitable for nucleic acid drug metabolism extraction and detection and a method for nucleic acid drug extraction using the kit. The kit includes: a lysate, a binding liquid enhancer, a magnetic bead suspension, a deproteinized liquid, a rinse solution and an eluent; the extraction method includes: first, the sample to be tested is fully mixed with the lysate, then the binding liquid enhancer and the magnetic bead suspension are added, and then the deproteinized liquid and the rinse solution are rinsed in turn, and finally the eluent is eluted to obtain the nucleic acid drug in the sample to be tested. The kit and extraction method provided by the present application are used in combination with a high-throughput nucleic acid extractor to complete automated extraction, which is simple and fast to operate and reduces manual operation. In addition, the kit provided by the present application can also be used to simultaneously extract and purify DNA and RNA molecular drugs, greatly improving the detection efficiency of nucleic acid drugs.

Description

Technical Field

[0001] The present application relates to the technical field of biochemistry and molecular biology, and in particular to a kit suitable for nucleic acid drug metabolism extraction and detection and a method for extracting nucleic acid drugs using the kit. Background Art

[0002] As a new type of treatment, nucleic acid drugs intervene at the root of inhibiting the expression of disease-related genes. They have made great progress in research and application and have become one of the hot topics in biomedical research.

[0003] Nucleic acid drug research and development is mainly divided into two parts: preclinical drug metabolism evaluation and clinical safety and efficacy evaluation. Due to the large number of experimental models and samples required for preclinical drug metabolism evaluation, coupled with the easy degradation and trace nature of nucleic acid drugs, the extraction and detection of nucleic acid drug metabolism poses many challenges.

[0004] Traditional nucleic acid drug metabolism detection methods are time-consuming and prone to missed detection for low-concentration or small molecule samples. Therefore, it is necessary to develop a new nucleic acid drug metabolism detection method. Summary of the invention

[0005] The present application provides a kit suitable for nucleic acid drug metabolism extraction detection and a method for nucleic acid drug extraction using the kit. The kit and extraction method provided by the present application can fundamentally solve the problems of easy degradation and missed detection in the process of nucleic acid drug metabolism extraction detection. At the same time, it can be used with a high-throughput nucleic acid extractor to complete automated extraction, which is easy and fast to operate and reduces manual operation. In addition, the kit provided by the present application can also be used to simultaneously extract and purify DNA and RNA molecular drugs, greatly improving the detection efficiency of nucleic acid drugs.

[0006] In the first aspect, the present application provides a kit suitable for nucleic acid drug metabolism extraction and detection, which adopts the following technical scheme:

[0007] A kit suitable for nucleic acid drug metabolism extraction and detection, characterized in that the kit comprises: a lysate, a binding solution enhancer, a magnetic bead suspension, a deproteinization solution, a rinsing solution and an elution solution;

[0008] The binding fluid enhancer is composed of one or more alcohols or alcohol polymers of different polarities and salts;

[0009] The deproteinization solution is composed of salt, buffer and enhancer; the salt is any one or more of guanidine hydrochloride, guanidine isothiocyanate and lithium chloride; the buffer is a 50-100mM pH7.5-9.0 buffer system; the enhancer is composed of alcohols of different polarities or high molecular polymers of alcohols and salt;

[0010] Among them, in the combination liquid enhancer and the deproteinized liquid, the alcohol is a monohydric alcohol, a dihydric alcohol or a polyhydric alcohol; and the salt is any one or more of lithium chloride, sodium chloride and potassium chloride.

[0011] The biological samples extracted by the kit provided in this application include but are not limited to: whole blood, serum, plasma, tissue, swabs, etc. These samples contain abundant nucleases, and improper storage after ex vivo can easily cause degradation of the nucleic acid to be detected. Therefore, after sampling, a lysis solution is immediately added to the sample to be tested, which inhibits nucleases while lysing the sample and protects the nucleic acid to be detected.

[0012] In order to improve the yield of low-concentration samples, this application integrates a large amount of experimental data and proposes a new binding fluid enhancer.

[0013] Specifically, the binding solution enhancer includes one or more alcohols or alcohol polymers of different polarities and salts of a certain concentration, which are used to extract and recover nucleic acids of different concentrations and different molecular weights. The binding solution enhancer helps nucleic acid molecules of different sizes to bind to the solid support by increasing the hydrophobic effect, thereby achieving the purpose of separation and purification.

[0014] The alcohols of different polarities involved include monohydric alcohols, dihydric alcohols and polyhydric alcohols. Monohydric alcohols, such as anhydrous ethanol and isopropanol, have strong polarity. After being added to the system, they will immediately destroy the hydration layer around the nucleic acid molecules. At a certain dosage, they can promote the adsorption of nucleic acids of different molecular weights on the solid phase medium. At the same time, they will also cause a large number of impurities to bind to the solid phase matrix, resulting in low nucleic acid purity. Polyhydric alcohols or high molecular polymers of alcohols selectively adsorb nucleic acid molecules at a certain dosage, while not adsorbing impurities, which can improve nucleic acid purity. Therefore, according to the size and purity requirements of the nucleic acid molecules to be purified and recovered, selecting one or several alcohols as binding liquid enhancers can improve the yield and purity as a whole.

[0015] The salt involved can be any one or more of lithium chloride, sodium chloride and potassium chloride.

[0016] Optionally, the salt involved is sodium chloride.

[0017] In a specific embodiment, the binding fluid enhancer includes: 50-70% isopropanol, 10-30% ethylene glycol high molecular polymer (polymer molecular weight between 2000-6000), and 1.5-2M sodium chloride.

[0018] In a specific embodiment, the formula of the bonding fluid enhancer can be 50% isopropanol, 60% isopropanol or 70% isopropanol.

[0019] In a specific embodiment, the formula of the bonding fluid enhancer may contain 50-60% isopropanol or 60-70% isopropanol.

[0020] In a specific embodiment, the formula of the bonding fluid enhancer may contain 10% ethylene glycol high molecular polymer, 20% ethylene glycol high molecular polymer or 30% ethylene glycol high molecular polymer.

[0021] In a specific embodiment, the formula of the bonding fluid enhancer may contain 10-20% ethylene glycol high molecular polymer or 20-30% ethylene glycol high molecular polymer.

[0022] In a specific embodiment, the binding fluid enhancer may contain 1.5 M sodium chloride or 2 M sodium chloride.

[0023] Different molecular weight polymers have different effects on nucleic acid fixation and impurity removal. In the present application, ethylene glycol polymers with a molecular weight range of 2000-6000 are selected.

[0024] The solid phase matrix matched with the above-mentioned binding liquid enhancer involves magnetic particles that can adsorb nucleic acids of different molecular weights and do not adsorb or rarely adsorb impurities. Therefore, magnetic particles with different modified groups can be mixed and used to meet the needs of this application. The magnetic bead suspension is used as a solid phase support to adsorb nucleic acid molecule drugs of different sizes under the action of the binding liquid enhancer.

[0025] Optionally, in the magnetic bead suspension, the magnetic beads may be mixed magnetic beads with different modification groups, or may be mixed magnetic beads with different particle sizes.

[0026] Optionally, the particle size of the magnetic beads in the magnetic bead suspension is in the range of 0.1-1 μm.

[0027] Optionally, the magnetic bead suspension includes any one or more of hydroxyl-modified magnetic beads, carboxyl-modified magnetic beads, amino-modified magnetic beads, and polymer-coated magnetic beads.

[0028] Optionally, magnetic beads modified with silanol and carboxyl groups are selected.

[0029] At the same time, the step of rinsing with a deproteinizing solution is equally important. During the purification process, the nucleic acid drug is adsorbed on a solid support, and other impurities are removed by rinsing. In this application, the deproteinizing solution involved includes salt, buffer, and enhancer.

[0030] The salt is usually a 2-3 M chaotropic salt. Specifically, the chaotropic salt is guanidine hydrochloride, guanidine isothiocyanate, and lithium chloride. The salt can be a chaotropic salt of different strengths, so that the nucleic acid is adsorbed on the solid support without being rinsed away.

[0031] The buffer is usually a 50-100 mM pH 7.5-9.0 buffer system.

[0032] The enhancer is composed of alcohols or high molecular polymers of alcohols with different polarities and salts.

[0033] Usually, a combination of binding liquid enhancers with different polarities is selected to achieve the purpose of removing impurities and adsorbing nucleic acids. Optionally, the enhancer can be a mixed solution of ethanol or propanol combined with ethylene glycol high molecular polymer.

[0034] In a specific embodiment, the deproteinization solution includes: 2-3M guanidine thiocyanate, 50-100mM pH8.0 Tris-HCl, 5-10% ethylene glycol high molecular polymer, 0.2-1M sodium chloride, and 30-40% anhydrous ethanol.

[0035] Specifically, the lysis solution includes a buffer, a chaotropic salt, a surfactant, a chelating agent, and a reducing agent. Among them, the chaotropic salt and the surfactant are mainly used to lyse biological samples and denature proteins; the chelating agent is used to chelate metal ions and inhibit nuclease activity; the reducing agent is used to reduce disulfide bonds, inhibit nuclease, and protect the nucleic acid to be detected from being degraded.

[0036] Optionally, the reducing agent is a thiol reducing agent.

[0037] Alternatively, the thiol reducing agent may be mercaptoethanol, dithiothreitol or tris(2-carboxyethyl)phosphine.

[0038] In a specific embodiment, the lysate comprises: 2-4M guanidine thiocyanate, 1-4% mass concentration of 3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, 1-3% mass concentration of lithium dodecylsulfonate, 100-200mM pH 8.0 Tris-HCl buffer, 1-2M lithium chloride, and 4-6% reducing agent.

[0039] Optionally, the rinsing solution comprises: 10-20 mM pH 8.0 Tris-HCl, 50-70% anhydrous ethanol.

[0040] Optionally, the elution solution is nuclease-free water.

[0041] Optionally, the biological samples involved in the extraction of the kit include, but are not limited to: whole blood, serum, plasma or tissue containing nucleic acid drugs.

[0042] Optionally, the nucleic acid drug is a DNA drug or an RNA drug.

[0043] Optionally, the DNA is plasmid DNA or linear double-stranded DNA.

[0044] Optionally, the RNA is a small molecule RNA of less than 100 nt (nt, ribonucleotide) or a large molecule RNA of several hundred to several thousand nt.

[0045] In a specific embodiment, when the target is to recover a small fragment of 50 bp, the binding solution enhancer includes: 70% isopropanol, 10% ethylene glycol high molecular polymer (polymer molecular weight between 2000-6000), and 2M sodium chloride, and the recovery effect is better.

[0046] In a specific embodiment, when the goal is to extract nucleic acid drugs with larger molecules in blood, the binding solution enhancer includes: 60% isopropanol, 10% ethylene glycol high molecular polymer (polymer molecular weight between 2000-6000), and 2M sodium chloride, and the recovery effect is better.

[0047] In the second aspect, the present application provides a method for extracting nucleic acid drugs using the above-mentioned kit, using the following technical scheme:

[0048] A method for extracting nucleic acid drugs using the above kit, the method specifically comprising the following steps:

[0049] First, the sample to be tested is fully mixed with the lysis solution, and then a binding solution enhancer and a magnetic bead suspension are added, followed by rinsing with a deproteinizing solution and a rinsing solution, and finally elution with an elution solution to obtain the nucleic acid drug in the sample to be tested.

[0050] In summary, the present application includes at least one of the following beneficial technical effects:

[0051] 1. The kit and extraction method provided by this application can fundamentally solve the problems of easy degradation and missed detection in the process of nucleic acid drug metabolism extraction and detection. At the same time, it can be used with a high-throughput nucleic acid extractor to complete automated extraction, which is easy and fast to operate and reduces manual operation. In addition, the kit provided by this application can also be used to extract and purify DNA and RNA molecular drugs at the same time, greatly improving the detection efficiency of nucleic acid drugs.

[0052] 2. This application utilizes lysis solution, binding solution enhancer, magnetic bead suspension, deproteinization solution, rinse solution and elution solution to quickly and efficiently separate and purify nucleic acid drugs of different molecular weights in biological samples such as serum, plasma, whole blood and tissues, and can meet the detection and recovery needs of nucleic acid drugs of different abundances. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a peak diagram of 50bp (base pair) nucleic acid molecule drug extraction and detection (the left picture is the extraction result of the traditional method, and the right picture is the extraction result of this application).

[0054] Figure 2 This is the peak diagram of the 50bp nucleic acid molecule drug library. DETAILED DESCRIPTION

[0055] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0056] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0057] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0058] The present application provides a kit suitable for nucleic acid drug metabolism extraction and detection.

[0059] The kit comprises a lysis solution, a binding solution enhancer, a magnetic bead suspension, a protein removal solution, a rinsing solution and an elution solution.

[0060] The lysate comprises: 2-4M guanidine thiocyanate, 1-4% 3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, 1-3% lithium dodecylsulfonate, 100-200mM Tris-HCl buffer with pH 8.0, 1-2M lithium chloride, and 4-6% reducing agent.

[0061] The bonding liquid enhancer includes: 50-70% isopropanol, 10-30% ethylene glycol high molecular polymer (polymer molecular weight tends to be between 2000-6000), and 1.5-2M sodium chloride.

[0062] The magnetic bead suspension contains silanol- and carboxyl-modified magnetic bead particles with a particle size range of 0.1-1 μm and a concentration of 100 mg / ml.

[0063] The deproteinizing solution includes: 2-3M guanidine isothiocyanate, 50-100mM pH 8.0 Tris-HCl, 5-10% ethylene glycol high molecular polymer, 0.2-1M sodium chloride, and 30-40% anhydrous ethanol.

[0064] The washing solution includes: 10-20 mM pH 8.0 Tris-HCl, 50-70% anhydrous ethanol.

[0065] The elution solution is nuclease-free water.

[0066] The present application also provides a method for extracting nucleic acid drugs using the kit.

[0067] The method specifically comprises the following steps:

[0068] (1) Sample lysis: Take 200-300 μl of the sample to be tested, add lysis buffer and 20 μl of protease according to 2-4 times the volume of the sample, mix thoroughly, and incubate in a 65°C metal bath for 10 min.

[0069] Before sample lysis, the corresponding sample processing method needs to be selected according to the specific sample type. Specifically: when the sample is a liquid sample such as serum, plasma, and whole blood, no sample pre-treatment is required and the sample can be directly lysed; when the sample is a tissue sample, it needs to be transferred to a homogenizer tube for homogenization after lysis; when the sample is a viscous sputum sample, a reducing agent needs to be added for liquefaction before lysis.

[0070] (2) Nucleic acid binding: Continue to add 300 μl of binding solution enhancer and 15 μl of magnetic bead suspension for nucleic acid binding.

[0071] After adding lysis buffer to the sample to be tested, the nucleic acid in the sample is released. Under the conditions of appropriate binding buffer enhancer and magnetic bead particles, the nucleic acid is bound to the magnetic beads to complete the initial purification.

[0072] (3) Rinse with deproteinization solution: After the magnetic beads adsorb nucleic acid, place the sample tube on a magnetic rack for magnetic bead adsorption. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant solution, add 900 μl of deproteinization solution, vortex mix for 2 min, and thoroughly wash away the impurities adsorbed on the magnetic beads. Then, place the sample tube on a magnetic rack for magnetic bead adsorption. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant solution, and repeat this step once.

[0073] (4) Rinse with rinse solution: Add 900 μl of rinse solution and vortex mix for 2 min to thoroughly wash away impurities adsorbed on the magnetic beads. Then place the sample tube on a magnetic rack for magnetic bead adsorption. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant and repeat this step once.

[0074] (5) Elution: Leave the sample tube open to air dry for 5 min. Observe that the inclined surface of the magnetic beads is moist but not cracked. Add the elution buffer preheated at 56°C and mix thoroughly for 5 min. Place the tube on a magnetic rack and let stand for 2 min. Transfer the elution buffer to a new tube and freeze it at -20°C or directly use it for downstream applications.

[0075] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application are clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application.

[0076] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0077] The present application is further described in detail below in conjunction with embodiments, drawings, comparative examples and test experiments.

[0078] Example

[0079] Example 1

[0080] This embodiment provides a kit suitable for nucleic acid drug metabolism extraction and detection, and uses the kit to extract, purify and recover 50bp small molecule nucleic acid drugs.

[0081] The sample to be tested is 200 μl of whole blood or plasma, containing 400 ng of 50 bp nucleic acid molecule drug (double-stranded DNA, dsDNA).

[0082] The specific steps include:

[0083] (1) Sample lysis: Take 200 μl of the sample to be tested, add lysis buffer and 20 μl of protease according to 2-4 times the volume of the sample, mix thoroughly, and incubate in a 65°C metal bath for 10 min.

[0084] The lysis solution includes: 2M guanidine thiocyanate, 2% 3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, 2% lithium dodecylsulfonate, 150mM Tris-HCl buffer with pH 8.0, 1.5M lithium chloride, and 5% mercaptoethanol.

[0085] (2) Nucleic acid binding: Continue to add 300 μl of binding solution enhancer and 15 μl of magnetic bead suspension for nucleic acid binding.

[0086] The binding liquid enhancer includes: 70% isopropanol, 10% ethylene glycol high molecular polymer (polymer molecular weight is between 2000-6000), and 2M sodium chloride.

[0087] The magnetic bead suspension is silicon hydroxyl and carboxyl modified magnetic beads with a particle size range of 0.1-1 μm and a concentration of 100 mg / ml.

[0088] (3) Rinse with deproteinization solution: After the magnetic beads adsorb nucleic acid, place the sample tube on a magnetic rack for magnetic bead adsorption. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant solution, add 900 μl of deproteinization solution, vortex mix for 2 min, and thoroughly wash away the impurities adsorbed on the magnetic beads. Then, place the sample tube on a magnetic rack for magnetic bead adsorption. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant solution, and repeat this step once.

[0089] The deproteinization solution includes: 2M guanidine thiocyanate, 100mM pH 8.0 Tris-HCl, 10% ethylene glycol high molecular polymer, 1M sodium chloride, and 30% anhydrous ethanol.

[0090] (4) Rinse with rinse solution: Add 900 μl of rinse solution and vortex mix for 2 min to thoroughly wash away impurities adsorbed on the magnetic beads. Then place the sample tube on a magnetic rack for magnetic bead adsorption. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant and repeat this step once.

[0091] The washing solution includes: 10 mM Tris-HCl, pH 8.0, 50% anhydrous ethanol.

[0092] (5) Elution: Leave the sample tube open to air dry for 5 min. Observe that the inclined surface of the magnetic beads is moist but not cracked. Add the elution buffer preheated at 56°C and mix thoroughly for 5 min. Place the tube on a magnetic rack and let stand for 2 min. Transfer the elution buffer to a new tube and freeze it at -20°C or directly use it for downstream applications.

[0093] The elution buffer was nuclease-free water.

[0094] Example 2

[0095] This embodiment provides a kit suitable for nucleic acid drug metabolism extraction and detection. The kit is used to extract, purify and recover 50bp small molecule nucleic acid drugs. The sample to be tested is consistent with the sample in Example 1.

[0096] The difference between this embodiment and embodiment 1 is that the formula of the bonding liquid enhancer is different, as shown in Table 1, and the remaining operation steps are consistent with those of embodiment 1.

[0097] Comparative Example

[0098] Comparative Example 1

[0099] This comparative example uses a traditional method to extract the sample to be tested in Example 1. The difference between this method and Example 1 is that the formula of the binding liquid enhancer is different, as shown in Table 1, and the remaining operation steps are consistent with Example 1.

[0100] Comparative Examples 2-5

[0101] Comparative Examples 2-5 respectively provide a kit suitable for nucleic acid drug metabolism extraction and detection. The kit is used to extract, purify and recover a 50 bp small molecule nucleic acid drug. The sample to be tested is consistent with the sample in Example 1.

[0102] The difference between the comparative example and Example 1 is that the formula of the bonding liquid enhancer is different, as shown in Table 1, and the remaining operation steps are consistent with Example 1.

[0103] Test 1

[0104] 1 μl of the eluted products obtained in Example 1 and Comparative Examples 1-5 were taken respectively, and the extraction effect was detected using an Agilent high-sensitivity DNA chip. The detection results of Example 1 and Comparative Example 1 are shown in FIG. Figure 1 The extraction effects of Example 1 and Comparative Examples 1-5 are shown in Table 1.

[0105] Figure 1 This is the recovery effect of 50bp nucleic acid molecule drugs detected by Agilent high-sensitivity DNA chip. The horizontal axis represents different DNA fragment sizes (expressed in bp, the number of bases), and the vertical axis represents the signal value of different DNA fragments. Using a ladder with known fragment size, a standard curve of migration time and fragment size is generated, and the fragment size of the sample can be determined by different migration times. The sample is quantified by the ratio of the sample peak area to the area of ​​the marker or ladder of known concentration. The higher and wider the peak of the fragment to be detected, the higher the recovery rate.

[0106] Depend on Figure 1 It can be seen that the left figure is the extraction result of the traditional method, showing that the nucleic acid molecule drug peak at 50bp is very low and almost undetectable; the right figure is the extraction result of the kit of this embodiment, showing that the recovery rate of the nucleic acid molecule drug at 50bp is very high.

[0107] Table 1 Extraction effect of the embodiment and the comparative example

[0108]

[0109]

[0110] As can be seen from Table 1, the binding solution enhancement solution provided in Example 1 of the present application is the best solution for recovering 50 bp small fragments.

[0111] Example 3

[0112] In this example, the extraction product containing the nucleic acid drug obtained in Example 1 was used to construct a library for next-generation sequencing.

[0113] The library construction process is a common method in this field and will not be described in detail here.

[0114] After the library was constructed, it was tested by Agilent high-sensitivity DNA chip. The library peak diagram is as follows Figure 2 shown.

[0115] Figure 2 This is the peak diagram of the 50bp nucleic acid molecule drug library detected by Agilent's high-sensitivity DNA chip. The extracted product contains 50bp nucleic acid molecule drugs. After a series of second-generation sequencing library construction steps such as connector connection, sorting, and PCR (polymerase chain reaction) enrichment, the chip detection can be seen as follows Figure 2 The arrow in the middle shows the distribution of small molecule nucleic acids in the library. After library construction (connected to the 120bp second-generation sequencing adapter sequence), the 50bp small molecule nucleic acid fragment is finally presented in the library as a 170bp fragment.

[0116] From the above, we can see that after the extraction of small molecule nucleic acid drugs, they are successfully applied to the construction of downstream second-generation sequencing libraries, which has profound significance for the metabolism of the drugs themselves and the related macro-omics research.

[0117] Example 4

[0118] In this embodiment, nucleic acid molecule drug standard DNA or plasmid is injected into experimental animals, and DNA is extracted and detected from animal tissues and blood to determine the metabolic abundance of the nucleic acid drug, thereby conducting a preclinical evaluation of the drug.

[0119] In this example, mice were used as experimental animals to establish a nucleic acid molecule drug (DNA) spike recovery model. First, a standard curve was prepared, and the standard product was diluted in series and then subjected to the same subsequent operations as the sample.

[0120] The specific process is as follows:

[0121] (1) Sample lysis:

[0122] Take 100 μl of whole blood sample and mix it directly with 380 μl of lysis buffer, then add 20 μl of protease, mix thoroughly, and incubate in a 65°C metal bath for 10 minutes.

[0123] Take 5-100 mg of tissue homogenate from different parts (tissue samples need to be ground with 380 μl of lysis solution, grinding conditions: steel beads or zirconium beads of appropriate particle size, 55HZ, 2 cycles), then add 20 μl of protease, mix thoroughly, and incubate in a 65°C metal bath for 10 minutes.

[0124] The lysis solution includes: 2M guanidine thiocyanate, 2% 3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, 2% lithium dodecylsulfonate, 150mM Tris-HCl buffer with pH 8.0, 1.5M lithium chloride, and 5% mercaptoethanol.

[0125] (2) Nucleic acid binding: After cooling to room temperature, take 450 μl of the processed sample, add 300 μl of binding solution enhancer and 15 μl of magnetic bead suspension for nucleic acid binding. It can also be used with appropriate consumables for automated extraction. Two parallel runs are performed for each group.

[0126] The binding liquid enhancer includes: 60% isopropanol, 10% ethylene glycol high molecular polymer (polymer molecular weight 3000), and 2M sodium chloride.

[0127] The magnetic bead suspension is silicon hydroxyl and carboxyl modified magnetic beads with a particle size range of 0.1-1 μm and a concentration of 100 mg / ml.

[0128] (3) Rinse with deproteinization solution: After the magnetic beads adsorb nucleic acid, place the sample tube on a magnetic rack for magnetic bead adsorption. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant solution, add 900 μl of deproteinization solution, vortex mix for 2 min, and thoroughly wash away the impurities adsorbed on the magnetic beads. Then, place the sample tube on a magnetic rack for magnetic bead adsorption. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant solution, and repeat this step once.

[0129] The deproteinization solution includes: 2M guanidine thiocyanate, 100mM pH 8.0 Tris-HCl, 10% ethylene glycol high molecular polymer, 1M sodium chloride, and 30% anhydrous ethanol.

[0130] (4) Rinse with rinse solution: Add 900 μl of rinse solution and vortex mix for 2 min to thoroughly wash away impurities adsorbed on the magnetic beads. Then place the sample tube on a magnetic rack for magnetic bead adsorption. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant and repeat this step once.

[0131] The washing solution includes: 10 mM Tris-HCl, pH 8.0, 50% anhydrous ethanol.

[0132] (5) Elution: Leave the sample tube open to air dry for 5 min. Observe that the inclined surface of the magnetic beads is moist but not cracked. Add the elution buffer preheated at 56°C and mix thoroughly for 5 min. Place the tube on a magnetic rack and let stand for 2 min. Transfer the elution buffer to a new tube and freeze it at -20°C or directly use it for downstream applications.

[0133] The elution buffer was nuclease-free water.

[0134] The spike recovery results are shown in Table 2.

[0135] Table 2 DNA spike recovery

[0136] Sample Type Detection concentration 1 Detection concentration 2 Standards Recovery rate (%) liver 2.20E+07 2.32E+07 4.00E+07 113 Heart 2.35E+07 2.25E+07 4.00E+07 115 spleen 2.15E+07 2.19E+07 4.00E+07 108 kidney 2.45E+07 2.50E+07 4.00E+07 123 Whole blood 1.89E+07 2.00E+07 4.00E+07 97

[0137] Note: Recovery rate (%) = measured value of spiked sample × 2 / theoretical value of standard product × 100.

[0138] Since the final elution volume of this experiment is 100 μL and the spike volume is 50 μL, the actual value of the sample spike needs to be multiplied by 2.

[0139] The results of recovery of different tissues and blood standards of experimental mice show that the kit and extraction method involved in this application can achieve a high recovery efficiency and meet the needs of preclinical new drug development. The recovery rate is between 50-150%, which meets industry standards.

[0140] Since blood and tissue samples contain more impurities, in order to obtain nucleic acid drugs with better purity for downstream detection, the binding solution enhancer used in this embodiment is 60% isopropanol, 10% ethylene glycol polymer, and 2M sodium chloride.

[0141] Comparative Examples 6-7

[0142] Comparative Examples 6-7 Inject nucleic acid molecule drug standard DNA or plasmid into experimental animals, extract and detect animal tissue and blood DNA, determine the metabolic abundance of nucleic acid drugs, and then conduct preclinical evaluation of the drugs.

[0143] The sample to be tested is a whole blood sample. The difference between the comparative example and Example 4 is that the formula of the binding liquid enhancer is different, as shown in Table 3, and the other operation steps are consistent with Example 4.

[0144] Test 2

[0145] The eluted products obtained in Example 4 and Comparative Examples 6 and 7 were respectively taken and the concentration and purity were measured using Nanodrop 2000. The results are shown in Table 3.

[0146] Table 3 Extraction effect of Example 4 and Comparative Examples 6 and 7

[0147]

[0148] In Table 3, 260 / 280 reflects the residual amount of nucleic acid protein, and 260 / 230 reflects the residual amount of impurities. The higher the value, the higher the purity of the nucleic acid. From the test results in Table 3, it can be seen that the nucleic acid obtained by extraction using the kit with the binding liquid enhancer formula of the present application has the highest yield and the best purity. From the above, it can be seen that for the extraction of nucleic acid drugs with larger molecules in blood, the binding liquid enhancer provided in Example 4 of the present application has a better effect.

[0149] Comparative Examples 8-9

[0150] In Comparative Examples 8-9, standard DNA of nucleic acid molecule drugs was injected into experimental animals, and DNA of animal tissues and blood was extracted and detected to determine the metabolic abundance of nucleic acid drugs, and then preclinical evaluation of the drugs was performed.

[0151] The sample to be tested is a whole blood sample. The difference between the comparative example and Example 4 is that the formula of the protein removal solution is different, as shown in Table 4, and the other operation steps are consistent with Example 4.

[0152] Test 3

[0153] The eluted products obtained in Example 4 and Comparative Examples 8 and 9 were respectively taken and the concentration and purity were measured using Nanodrop 2000. The results are shown in Table 4.

[0154] Table 4 Extraction effect of Example 4 and Comparative Examples 8 and 9

[0155]

[0156] It can be seen from the test results in Table 4 that the nucleic acid obtained by extraction using the kit with the protein removal solution formula of the present application has the highest yield and the best purity.

[0157] Example 5

[0158] In this embodiment, nucleic acid molecule drug standard RNA or viral protein-encapsulated RNA is injected into experimental animals, and RNA extraction and detection of animal tissue and blood are performed regularly to detect the metabolic abundance of the drug and make an assessment of the drug development stage.

[0159] In this example, SD rats and rhesus monkeys were used as experimental animals to establish a spike recovery model for nucleic acid molecule drugs (RNA).

[0160] The specific process is as follows:

[0161] First, a standard curve was prepared by serially diluting the standard stock solution with SD rat liver and rhesus monkey blank whole blood RNA dilutions, respectively.

[0162] Secondly, prepare the sample to be tested and dilute the standard stock solution (2.00×10 9copies / μL) was formulated to be high (2.00×10 7 copies / μL), low (2.00×10 5 Two positive solutions with concentrations of 100 copies / μL were used as test samples, and then added into blank whole blood lysate of SD rats and rhesus monkeys (whole blood: lysate = 1:4) at a ratio of 1:4 to prepare high and low concentration extraction samples. Three sets of each concentration were prepared in parallel, and three analysis batches of RNA extraction were required for each set.

[0163] Take 200 μL of the standard and the sample to be tested, add 450 μL of lysis mixture and 20 μL of proteinase K to a 2.0 mL centrifuge tube or a 96-well deep-well plate (the experiment can be completed with an automated extractor), vortex to mix, and let stand at room temperature for 5 minutes. After lysis, take 500 μL for RNA extraction.

[0164] The extraction method and reagents are the same as those in Example 4.

[0165] The RT-PCR test was performed in the same analytical batch to obtain the extraction recovery rate. The recovery rate calculation formula is as follows:

[0166] Recovery rate (Recovery%) = [(measured concentration of extracted sample) × elution volume] / [(measured concentration of unextracted sample) × actual sample volume during extraction] × 100%. Acceptable standard for recovery rate: The precision of recovery rate (%CV) of quality control samples at each concentration should be ≤30.0%.

[0167] The recovery rate calculation is shown in Table 5.

[0168] Table 5 RNA spike recovery

[0169]

[0170] The recovery rate results in Table 5 show that in the RNA spike recovery model for nucleic acid molecule drug detection, the kit and extraction method involved in the present application can achieve the standard of efficient recovery of RNA drugs, and the recovery rates of standards of different concentrations can meet industry standards.

[0171] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0172] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A kit suitable for nucleic acid drug metabolism extraction and detection, characterized in that: The kit comprises: a lysis solution, a binding solution enhancer, a magnetic bead suspension, a deproteinization solution, a rinsing solution and an elution solution; The binding fluid enhancer comprises 60-70% isopropanol, 10-30% ethylene glycol high molecular polymer, and 1.5-2M sodium chloride; the molecular weight of the ethylene glycol high molecular polymer is between 2000-6000; The deproteinization solution comprises: 2-3M guanidine thiocyanate, 50-100mM pH 8.0 Tris-HCl, 5-10% ethylene glycol high molecular polymer, 0.2-1M sodium chloride, and 30-40% anhydrous ethanol; The magnetic beads in the magnetic bead suspension are silicon hydroxyl and carboxyl modified magnetic beads, with a particle size range of 0.1-1 μm and a concentration of 100 mg / ml.

2. The kit for nucleic acid drug metabolism extraction and detection according to claim 1, characterized in that: The lysate comprises: 2-4M guanidine isothiocyanate, 1-4% mass concentration of 3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, 1-3% mass concentration of lithium dodecylsulfonate, 100-200mM Tris-HCl buffer with pH 8.0, 1-2M lithium chloride, and 4-6% reducing agent.

3. The kit for nucleic acid drug metabolism extraction and detection according to claim 1, characterized in that: The washing solution includes: 10-20 mM pH 8.0 Tris-HCl, 50-70% anhydrous ethanol.

4. The kit for nucleic acid drug metabolism extraction and detection according to claim 1, characterized in that: The eluent is nuclease-free water.

5. The kit suitable for nucleic acid drug metabolism extraction and detection according to claim 1, characterized in that: The biological samples involved in the extraction of the kit include but are not limited to: whole blood, serum, plasma or tissue containing nucleic acid drugs; the nucleic acid drugs are DNA drugs or RNA drugs; the DNA is plasmid DNA or linear double-stranded DNA; the RNA is a small molecule RNA of less than 100 nt or a large molecule RNA of several hundred to several thousand nt.

6. A method for extracting nucleic acid drugs using the kit according to any one of claims 1 to 5, characterized in that: The method specifically comprises the following steps: firstly, the sample to be tested is fully mixed with a lysate, then a binding solution enhancer and a magnetic bead suspension are added, then the sample is rinsed with a deproteinizing solution and a rinsing solution in sequence, and finally, the sample is eluted with an elution solution to obtain the nucleic acid drug in the sample to be tested.

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