Kit and method for extracting nucleic acid from ophthalmic sample and application of kit and method in metagenome sequencing

Abstract

The invention discloses a kit and a method for extracting nucleic acid from an ophthalmic sample and application of the kit and the method in metagenome sequencing. A method of extracting nucleic acid from an ophthalmic sample includes extracting nucleic acid from the ophthalmic sample using a sample processing system comprising the ophthalmic sample, a carrier RNA, protease K, and a lysis reagent. According to the method, a carrier RNA is added to prevent loss of trace nucleic acid, high-concentration protease K is adopted to enhance digestion of protein, and a chemical reagent is adopted to lyse cells, so that excessive damage and degradation of nucleic acid and low-volume elution caused by severe oscillation of mechanical wall breaking are avoided, and the final concentration of nucleic acid is improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to kits, methods for extracting nucleic acids from ophthalmic samples, and their use in metagenomic sequencing. Background Technology

[0002] Metagenomic sequencing (mNGS) plays a crucial role in the diagnosis of pathogens in ophthalmic samples. Traditional microbiological methods can only identify about 40% of pathogens in ophthalmic infection samples and are ineffective for some difficult-to-culture or uncultured microorganisms. mNGS, employing an unbiased and broad-coverage sequencing approach, can directly detect all nucleic acids in a sample, theoretically enabling the detection of all pathogens present, including bacteria, viruses, fungi, and parasites, significantly improving the detection rate. For example, in the diagnosis of intraocular infections, mNGS achieves a sensitivity of 92.2% and an overall concordance rate of 81.3% using vitreous fluid samples, while achieving a sensitivity of 85.4% and an overall concordance rate of 75.4% using aqueous humor samples—far exceeding the approximately 40% sensitivity of traditional culture methods. By performing metagenomic sequencing on ophthalmic samples, a comprehensive understanding of the composition and function of the normal ocular flora, as well as its changes under different disease states, can be obtained. This contributes to a deeper understanding of the interactions between the ocular microbiota and the host, providing a basis for developing targeted treatments and preventative measures.

[0003] Ophthalmic samples (such as aqueous humor and vitreous fluid) are small in volume, typically only 30-200 μL, and have low nucleic acid content. To meet the needs of subsequent testing, high efficiency in nucleic acid extraction is required. Furthermore, ophthalmic samples contain components such as tear proteins, mucopolysaccharides, and collagen, which easily adsorb nucleic acids and must be removed during extraction, increasing the difficulty of nucleic acid extraction. Nucleic acids in ophthalmic samples may also degrade due to improper handling during sample collection, preservation, or extraction, resulting in compromised nucleic acid integrity and significantly increasing the difficulty of extraction.

[0004] Traditional phenol-chloroform nucleic acid extraction methods are complex to operate, and the reagents are highly toxic, making them unsuitable for small sample volumes. Commercial column extraction methods are relatively simple to operate, but require large sample volumes, repeated centrifugation, and significant nucleic acid loss, making them unsuitable for high-throughput operations.

[0005] In conclusion, how to effectively improve the efficiency of nucleic acid extraction from ophthalmic samples is a problem that urgently needs to be solved. Summary of the Invention

[0006] Based on this, this application provides at least one kit, method for extracting nucleic acids from ophthalmic samples, and its use in metagenomic sequencing.

[0007] In a first aspect of this application, a method for extracting nucleic acids from ophthalmic samples is provided, the method comprising:

[0008] Nucleic acid was extracted from the ophthalmic sample using a sample processing system that included ophthalmic sample, vector RNA, proteinase K, and lysis reagent.

[0009] In a second aspect of this application, a kit for extracting nucleic acids from ophthalmic samples is provided, comprising a lysis reagent, proteinase K, and vector RNA.

[0010] This application provides an efficient and low-cost nucleic acid extraction method to solve the problem of low nucleic acid extraction efficiency in ophthalmic samples. The nucleic acid extraction method involves adding carrier RNA to prevent trace nucleic acid loss, using a high concentration of proteinase K to enhance protein digestion, using chemical reagents to lyse cells to avoid excessive damage and degradation of nucleic acids caused by violent mechanical shaking, and low-volume elution to increase the final nucleic acid concentration. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0012] Figure 1 This is a schematic diagram of the nucleic acid extraction and DNA library construction process in one embodiment of this application. Detailed Implementation

[0013] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0015] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0016] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0017] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0018] In this application, terms such as "especially," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0019] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0020] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0021] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0022] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0023] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0024] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0025] Traditional extraction methods, such as column chromatography, are prone to clogging and low recovery rates; ultrasonic disruption can cause excessive DNA shearing; and conventional magnetic bead methods, which involve mechanical cell disruption, can also cause varying degrees of nucleic acid fragmentation. This application provides an efficient and low-cost nucleic acid extraction method that improves upon the problems of low extraction efficiency and severe nucleic acid fragmentation, thereby increasing the success rate of library construction.

[0026] One aspect of this application provides a method for extracting nucleic acids from ophthalmic samples, the method comprising:

[0027] Nucleic acid was extracted from the ophthalmic sample using a sample processing system that included ophthalmic sample, vector RNA, proteinase K, and lysis reagent.

[0028] This method uses chemical reagents (i.e., lysis reagents) to lyse cells. This chemical cell lysis method is gentler and eliminates the mechanical cell disruption part in the nucleic acid extraction operation, thus avoiding micro-nucleic acid damage and degradation caused by violent oscillations during mechanical cell disruption.

[0029] In some embodiments, the initial concentration of the vector RNA in the sample processing system is 0.5 μg / μL to 1 μg / μL, for example, 0.5 μg / μL, 0.6 μg / μL, 0.7 μg / μL, 0.8 μg / μL, 0.9 μg / μL, 1 μg / μL, or any range or value between two values.

[0030] While not wishing to be limited by any theoretical framework, the inventors believe that carrier RNA plays a role as a nucleic acid binding enhancer in sample processing systems, with its function including at least increasing the extraction efficiency of trace nucleic acids. Furthermore, the inventors discovered that when the concentration of carrier RNA in the sample processing system is too low, it is difficult to achieve the desired efficiency improvement; while when the concentration of carrier RNA in the sample processing system is too high, it may unnecessarily increase costs.

[0031] In some embodiments, the initial concentration of proteinase K in the sample processing system is 20 mg / mL to 40 mg / mL. Exemplary values ​​include, for example, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, or any range or value between two such values.

[0032] While not wanting to be limited by any theory, it was found that increasing the concentration of proteinase K can effectively digest proteins in ophthalmic samples and enhance cell lysis; conversely, when the concentration is too low, it can easily lead to incomplete digestion, excessively high protein background, and affect the efficiency of nucleic acid extraction.

[0033] In this application, the ophthalmic samples include, but are not limited to, conventional ophthalmic sample types in the art, such as aqueous humor, vitreous fluid, and corneal tissue.

[0034] In this application, the lysis reagent may be derived from commercial kits.

[0035] In some embodiments, the cleavage reagent includes one or more of guanidine compounds, detergents, buffer systems, and ethylenediaminetetraacetic acid.

[0036] In some embodiments, the guanidine compound exemplarily includes or is guanidine hydrochloride.

[0037] In some embodiments, the buffer system exemplarily includes or is Tris-HCl.

[0038] In some embodiments, the detergent exemplarily includes or is one or more of SDS and Tween-20.

[0039] In some implementations, the method includes the following steps:

[0040] S100. Mix the ophthalmic sample with lysis reagent, vector RNA and proteinase K, and incubate to obtain the first mixture;

[0041] S200. Add magnetic beads to the first mixture to obtain a second mixture;

[0042] S300. The second mixture is subjected to magnetic adsorption to extract nucleic acids.

[0043] In some implementations, the incubation conditions in step S100 are 65°C for 10 min to 30 min.

[0044] In some embodiments, in step S200, in addition to adding the magnetic beads, anhydrous ethanol is also added.

[0045] In some embodiments, in step S300, after the second mixture is magnetically adsorbed, a first precipitate containing magnetic beads-nucleic acid complex is collected; the first precipitate is washed with washing solution 1, magnetically adsorbed, and a second precipitate containing magnetic beads-nucleic acid complex is collected; the second precipitate is washed with washing solution 2, magnetically adsorbed, and a third precipitate containing magnetic beads-nucleic acid complex is collected; the washing solution 1 and the washing solution 2 contain anhydrous ethanol.

[0046] In some embodiments, the method further includes step S400, adding 500-750 μL of washing solution 2 to the third precipitate, vortexing to mix and fully suspend the magnetic beads; magnetic adsorption is then used to collect the fourth precipitate. The vortexing time can be, for example, 2 minutes.

[0047] In some embodiments, the method further includes step S500, drying the fourth precipitate and adding elution buffer to fully elute the nucleic acids. The volume of the elution buffer is, exemplarily, 30-100 μL.

[0048] We do not want to be limited by any theory, but we have found that reducing the elution volume (e.g., to 30-50 μL) and increasing the nucleic acid concentration can ensure that as much nucleic acid as possible enters the subsequent detection.

[0049] In some embodiments, step S500 includes: adding 30-100 μL LNase-Free ddH2O to the dried fourth precipitate, resuspending the magnetic beads by pipetting, incubating at 56°C for 5 min, and gently shaking every 2 min to ensure complete elution of nucleic acids.

[0050] In some embodiments, the method further includes step S600, placing the centrifuge tube on a magnetic rack and letting it stand until the magnetic beads are completely adsorbed, then transferring the nucleic acid solution to a new centrifuge tube to obtain the extracted nucleic acid.

[0051] Another aspect of this application provides a kit for extracting nucleic acids from ophthalmic samples, which includes a lysis reagent, proteinase K, and vector RNA.

[0052] The lysis reagents are as defined above.

[0053] Another aspect of this application also provides a method for metagenomic sequencing of pathogens in ophthalmic samples, the method comprising:

[0054] Nucleic acid was extracted from ophthalmic samples using the method described above.

[0055] Metagenomic sequencing was performed on the extracted nucleic acids.

[0056] The following are some examples.

[0057] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0058] Example 1

[0059] Table 1

[0060]

[0061] Note: Due to the small sample size, it is not possible to process the same sample differently. Therefore, samples 1 to 11 come from different samples.

[0062] Table 1 shows the results of different processing methods. Based on the extraction concentration and library concentration and quality control, it can be seen that mechanical cell disruption results in low nucleic acid extraction and low library pass rate, while chemical lysis increases the nucleic acid extraction and library success rate.

[0063] Table 2 shows the results of subsequent addition of carrier RNA and PK enhancement. Different ophthalmic samples were tested using the chemical lysis extraction method, and all samples successfully constructed libraries. The sequencing data output was >20M, which met expectations.

[0064] Table 2

[0065]

[0066] Table 3 shows the processing of different ophthalmic samples using only chemical lysis, a relatively gentle extraction method. Library construction was successful in all cases, and according to the sequencing results, the data output was greater than 20M. There was little difference between Clean Reads and Total Reads, indicating high library quality.

[0067] Table 3

[0068]

[0069] For the procedures of nucleic acid extraction and DNA library construction, please refer to [link to documentation]. Figure 1 The details are as follows:

[0070] I. Nucleic Acid Extraction Procedure (Applicable to Samples 1 to 25)

[0071] 1. Add PBS to the sample to 300 μL, add 1 μg Carrier RNA, 150 μL of lysis buffer 1, 150 μL of lysis buffer 2, 30 μL of nucleic acid protectant and 20 μL of proteinase K (20 mg / mL), and vortex to mix for 2 min.

[0072] 2. Proteinase K digestion: Incubate at 65°C for 10 min.

[0073] 3. After protease digestion, centrifuge the sample at 12000 rpm for 1 min and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0074] 4. Magnetic bead binding: Add 350 μL of anhydrous ethanol (or isopropanol) and 15 μL of magnetic bead suspension, vortex to mix, and let stand at room temperature for 10 min, vortexing for 30 sec every 5 min during this period.

[0075] 5. Place the centrifuge tube on the magnetic rack for 2 minutes. Once the magnetic beads are fully adsorbed, carefully remove the liquid with a pipette.

[0076] 6. Add 750 μL of washing solution 1 (please check before use whether 36 mL of anhydrous ethanol has been added), vortex and mix for 2 min to fully suspend the magnetic beads.

[0077] 7. Place the centrifuge tube on the magnetic rack for 1 minute. Once the magnetic beads are fully adsorbed, carefully remove the liquid with a pipette.

[0078] 8. Repeat steps 6 and 7.

[0079] 9. Add 750 μL of washing solution 2 (please check before use whether 80 mL of anhydrous ethanol has been added), vortex and mix for 2 min to fully suspend the magnetic beads.

[0080] 10. Place the centrifuge tube on the magnetic rack for 1 minute. Once the magnetic beads are fully adsorbed, carefully remove the liquid with a pipette.

[0081] 11. Repeat steps 9-10.

[0082] 12. Place the centrifuge tubes on a magnetic rack and let them air dry at room temperature for 5 to 10 minutes.

[0083] Note: Residual ethanol can inhibit subsequent enzymatic reactions, so ensure that all ethanol evaporates completely during drying. However, do not dry for too long, otherwise it may be difficult to wash away nucleic acids.

[0084] 13. Add 50~100 μL of RNase-Free ddH2O, use a pipette to resuspend the magnetic beads, and incubate at 56℃ for 5 min, gently shaking every 2 min to ensure complete elution of nucleic acids.

[0085] 14. Place the centrifuge tube on a magnetic rack and let it stand for 2 minutes until the magnetic beads are completely adsorbed. Carefully transfer the nucleic acid solution to a new centrifuge tube and store it at -20°C.

[0086] II. DNA Library Construction

[0087] 1. DNA fragmentation

[0088] (1) Take out the enzyme breakage mix, thaw and mix well, briefly centrifuge to collect to the bottom of the tube, and place on ice for later use. All the following steps shall be performed on ice.

[0089] (2) Prepare the reaction shown in Table 4 below in the PCR tube.

[0090] Table 4

[0091]

[0092] Note: Add nucleic acid and water first, and add the enzyme-disrupting mix last. Operate on ice.

[0093] (3) Use a pipette to gently blow or shake to mix, and briefly centrifuge to the bottom of the tube to get the reaction solution.

[0094] (4) Place the PCR tube into the sample slot of the amplification instrument and set the reaction program shown in Table 5 below to perform DNA fragmentation, end repair and dA tail addition reaction.

[0095] Table 5

[0096]

[0097] Note: (1) In order to effectively control the fragmentation efficiency, the reaction program can be preset to 4℃. When the module temperature drops to 4℃, the PCR tube can be placed into the PCR instrument.

[0098] (2) If the sample is cfDNA, it is recommended to break down the enzyme for 5 minutes;

[0099] (3) If the sample concentration is too low and the nucleic acid input volume exceeds 30 μL, it is recommended to extend the enzyme digestion time to 8 min;

[0100] (4) It is recommended to set the enzyme digestion interruption time to 6 minutes for 10 ng of nucleic acid;

[0101] 2. Connector connection

[0102] Remove the ligation buffer and ligase from -20℃ in advance, thaw them, mix them well, centrifuge briefly, and place them on ice for later use.

[0103] Dilute the connector (15μM) to 1μM and set aside for use only. Prepare fresh each time you need it.

[0104] Prepare the reaction system shown in Table 6 in the PCR tube.

[0105] Table 6

[0106]

[0107] Place the PCR tube into the sample slot of the amplification instrument, set the reaction program as shown in Table 7, and perform adapter connection.

[0108] Table 7

[0109]

[0110] 3. Purification of ligation products

[0111] Preparation: This kit does not contain anhydrous ethanol; please use commercially available anhydrous ethanol that has been validated for performance. Remove the DNA sorting beads from the refrigerator and allow them to equilibrate at room temperature for at least 30 minutes. Vortex or invert the beads thoroughly to resuspend them, and prepare 80% ethanol.

[0112] (1) Add 80 μL of DNA sorting magnetic beads to the ligation product; vortex mix and incubate at room temperature for 5 min.

[0113] (2) Briefly centrifuge the centrifuge tube and place it in a magnetic rack. After the solution becomes clear (about 2 min), carefully remove the supernatant.

[0114] (3) Keep the centrifuge tube in the magnetic rack at all times, add 200 μL of freshly prepared 80% ethanol, and use the magnetic rack to repeatedly adsorb the DNA sorting magnetic beads back and forth on different sides to fully suspend the DNA sorting magnetic beads for washing. Carefully remove the supernatant.

[0115] (4) Repeat step (3).

[0116] (5) Keep the centrifuge tubes in the magnetic rack at all times and dry them with the lid open (about 2 minutes). Note: The drying time should not be too long, as over-drying of the magnetic beads will affect the purification effect.

[0117] (6) Keep the centrifuge tube in the magnetic rack at all times, add 22.5 μL of Nuclease-Free Water to elute, vortex to mix or gently pipette to mix, and let stand at room temperature for 5 min;

[0118] (7) Briefly centrifuge the PCR tube and place it in a magnetic rack to stand until it becomes clear (about 2 min). Carefully aspirate 20 μL of supernatant into a new PCR tube, being careful not to touch the magnetic beads.

[0119] 4. PCR amplification

[0120] Remove the amplification buffer and Index X from -20°C beforehand, thaw and mix them, briefly centrifuge to collect them to the bottom of the tube, and place them on ice for later use.

[0121] Prepare the reaction system shown in Table 8 in the PCR tube.

[0122] Table 8

[0123]

[0124] Gently pipette or vortex to mix, and briefly centrifuge. Place the PCR tube in the sample slot of the PCR instrument, set the reaction program as shown in Table 9, and perform PCR amplification.

[0125] Table 9

[0126]

[0127] Remark:

[0128] (1) The nucleic acid input is 10 ng, and the recommended number of cycles is 8 cycles;

[0129] (2) The nucleic acid input is 5-10 ng, and the recommended number of cycles is 9 cycles;

[0130] (3) Nucleic acid input is 1-5 ng, and the recommended number of cycles is 10 cycles;

[0131] (3) The nucleic acid concentration is too low, and the recommended number of cycles is 12.

[0132] 5. Library purification

[0133] Preparation: Remove the DNA sorting magnetic beads from the refrigerator and allow them to equilibrate at room temperature for at least 30 minutes. Vortex or invert the beads thoroughly to resuspend them. Prepare 80% ethanol.

[0134] (1) Add 40 μL of DNA sorting magnetic beads to the PCR amplification product; vortex mix and incubate at room temperature for 5 min.

[0135] (2) Briefly centrifuge the centrifuge tube and place it in a magnetic rack. After the solution becomes clear (about 2 min), carefully remove the supernatant.

[0136] (3) Keep the centrifuge tube in the magnetic rack at all times, add 200 μL of freshly prepared 80% ethanol, and use the magnetic rack to repeatedly adsorb the magnetic beads on both sides to fully suspend the magnetic beads and wash them. Carefully remove the supernatant.

[0137] (4) Repeat step (3).

[0138] (5) Keep the centrifuge tubes in the magnetic rack at all times and dry them with the lid open (about 2 minutes). Note: The drying time should not be too long, as over-drying of the magnetic beads will affect the purification effect.

[0139] (6) Remove the centrifuge tube from the magnetic rack, add 25 μL of Nuclease-Free Water, vortex to mix, and incubate at room temperature for 2 min.

[0140] (7) Briefly centrifuge the centrifuge tube and place it in a magnetic rack to separate the DNA sorting beads and liquid. After the solution becomes clear (about 2 min), carefully aspirate the supernatant into a clean tube to complete library construction. Label the tube and store it at -20 ± 5℃ for short-term use or at -70 ± 5℃ for long-term storage.

[0141] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for extracting nucleic acids from ophthalmic samples, characterized in that, The method includes: Nucleic acid was extracted from the ophthalmic sample using a sample processing system that included ophthalmic sample, vector RNA, proteinase K, and lysis reagent.

2. The method as described in claim 1, characterized in that, The initial concentration of the vector RNA in the sample processing system was 0.5 μg / μL to 1 μg / μL.

3. The method as described in claim 1, characterized in that, The initial concentration of proteinase K in the sample processing system was 20 mg / mL to 40 mg / mL.

4. The method as described in claim 1, characterized in that, The ophthalmic samples include one or more of aqueous humor, vitreous humor, and corneal tissue.

5. The method as described in claim 1, characterized in that, The lysis reagent includes one or more of guanidine compounds, detergents, buffer systems, and ethylenediaminetetraacetic acid; wherein: The guanidine compounds include guanidine hydrochloride; The buffer system includes Tris-HCl; The detergent includes one or more of SDS and Tween-20.

6. The method according to any one of claims 1 to 5, characterized in that, The method includes the following steps: The ophthalmic sample was mixed with lysis reagent, vector RNA and proteinase K, and incubated to obtain the first mixture; Magnetic beads are added to the first mixture to obtain a second mixture; The second mixture is subjected to magnetic adsorption to extract nucleic acids.

7. The method as described in claim 6, characterized in that, The method meets one or more of the following conditions 1) to 3): 1) In the step of preparing the first mixture, the incubation conditions are 65℃ for 10 min to 30 min; 2) In the step of preparing the second mixture, in addition to adding the magnetic beads, anhydrous ethanol is also added; 3) In the nucleic acid extraction step, after magnetic adsorption of the second mixture, a first precipitate containing magnetic bead-nucleic acid complex is collected; the first precipitate is washed with washing solution 1, magnetic adsorption is performed, and a second precipitate containing magnetic bead-nucleic acid complex is collected. The second precipitate was washed with washing solution 2, and then magnetically adsorbed to collect a third precipitate containing a magnetic bead-nucleic acid complex; washing solution 1 and washing solution 2 contain anhydrous ethanol.

8. A kit for extracting nucleic acids from ophthalmic samples, characterized in that, It contains lysis reagents, proteinase K, and vector RNA.

9. The kit for extracting nucleic acids from ophthalmic samples as described in claim 8, characterized in that, The kit meets one or more of the following conditions (1) to (3): (1) The lysis reagent as defined in claim 5; (2) The concentration of the vector RNA is 0.5 μg / μL to 1 μg / μL; (3) The concentration of proteinase K is 20 mg / mL to 40 mg / mL.

10. A method for metagenomic sequencing of pathogens in ophthalmic samples, characterized in that, The method includes: Nucleic acid is extracted from ophthalmic samples using the method for extracting nucleic acid from ophthalmic samples as described in any one of claims 1 to 7; Metagenomic sequencing was performed on the extracted nucleic acids.

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