Method for capturing and detecting a target nucleic acid

Abstract

The application relates to a target nucleic acid capturing method and a detection method, which comprises the following steps: concentrating a nucleic acid library to be detected to obtain a nucleic acid library to be detected concentrate; mixing and dissolving the nucleic acid library to be detected concentrate with a probe group freeze-dried product to obtain a hybridization system, and performing a hybridization reaction; and after the hybridization reaction, adding capturing magnetic beads to capture the probe group, and obtaining a target nucleic acid library; wherein the probe group contains a base sequence which is at least partially complementary to the target nucleic acid. The method can improve the performance of hybridization capturing.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a method for capturing and detecting a target nucleic acid. Background Technology

[0002] Microbial detection methods mainly include microbial isolation and culture detection and nucleic acid detection. Nucleic acid detection refers to microbial detection at the nucleic acid level, including methods such as fluorescent PCR and first- and second-generation sequencing. Among nucleic acid detection methodologies related to second-generation sequencing, hybridization capture is a targeted sequencing technology based on molecular hybridization. This technology requires the design of probe sets complementary to the target region. The probe sets capture fragments of the target region, and then unwanted portions are eluted, thereby achieving the goal of enriching the target gene.

[0003] To prepare libraries suitable for probe hybridization capture, nucleic acid extraction and library construction are required for different types of clinical samples. The target library is then mixed with the probe set for hybridization capture in a hybridization buffer, and hybridization is performed for an extended period at a specific temperature. The hybridization efficiency C0t is related to the initial probe and target concentrations C, and the hybridization duration t is related to the buffer coefficient A, i.e.:

[0004] C0t=C×t×A

[0005] As the formula shows, the higher the initial concentration of the probe and target, the higher the hybridization efficiency. However, when multiple sample libraries are added at once, the concentrations of both the sample library and the probe are diluted, significantly reducing the capture efficiency.

[0006] To increase the concentration of the target library and probe set after mixing, existing methods include concentrating the target library and probe set using an evaporation concentrator or enriching the mixture of the target library and probe set using magnetic beads, thereby ensuring that the liquid volume of the hybridization system is as small as possible and the corresponding concentration of the target library and probe set is as high as possible. However, the hybridization and capture efficiency in the liquid phase of the above methods is reduced, which will affect the hybridization and capture performance to varying degrees, ultimately leading to poorer target detection results. Summary of the Invention

[0007] Therefore, one embodiment of this application needs to provide a method for capturing target nucleic acids, which can improve the performance of hybridization capture.

[0008] The technical solutions include the following:

[0009] A method for capturing a target nucleic acid, the method comprising the following steps:

[0010] Concentrate the nucleic acid library to be tested to obtain the concentrated nucleic acid library solution;

[0011] The concentrated nucleic acid library to be tested was mixed and dissolved with the lyophilized probe kit to obtain a hybridization system, and a hybridization reaction was performed; and

[0012] After the hybridization reaction, magnetic beads are added to capture the probe set to obtain the target nucleic acid library;

[0013] The probe set contains a base sequence that is at least partially complementary to the target nucleic acid.

[0014] In one embodiment, nucleic acid is extracted from the sample to be tested, and the nucleic acid library to be tested is constructed.

[0015] Optionally, the sample to be tested may include one or more of the following: serum, plasma, whole blood, sputum, swabs, lavage fluid, tissue, urine, cerebrospinal fluid, food, water, soil, bacterial culture, viral culture, cell line culture, and synthetic plasmids.

[0016] In one embodiment, the nucleic acid library to be tested is concentrated using a drying method or by using concentration magnetic beads.

[0017] In one embodiment, the nucleic acid library to be tested is concentrated using concentrating magnetic beads; after the hybridization reaction and before the capture magnetic beads are added to capture the probe set, the step of removing the concentrating magnetic beads is also included.

[0018] In one embodiment, the step of removing the concentrated magnetic beads includes:

[0019] The hybridization system is diluted with a diluent, and the diluted hybridization system is subjected to solid-liquid separation to obtain supernatant A, from which the concentrated magnetic beads are removed.

[0020] Optionally, the diluent is a solution of the captured magnetic beads used for solid-liquid separation, resulting in a supernatant B.

[0021] Optionally, the volume of the solution containing the captured magnetic beads is 20-200 μL.

[0022] In one embodiment, the supernatant A is mixed with the capturing magnetic beads from which the supernatant B has been separated to capture the probe group.

[0023] A method for detecting nucleic acids, the method comprising the following steps:

[0024] The target nucleic acid library was obtained using the described capture method;

[0025] PCR amplification of the target nucleic acid library; and

[0026] The amplified products were purified and then subjected to high-throughput sequencing to determine whether the sample contained the target nucleic acid.

[0027] In one embodiment, the target nucleic acid includes the nucleic acid of a microorganism in the sample to be tested. Optionally, the microorganism includes one or more of bacteria, fungi, and viruses.

[0028] A kit comprising a probe set for detecting a target nucleic acid, concentrated magnetic beads, and capture magnetic beads; wherein the probe set is a lyophilized probe set; the concentrated magnetic beads are used to adsorb a nucleic acid library to be tested; and the capture magnetic beads are used to capture the probe set; optionally, the kit further comprises a nucleic acid washing solution; optionally, the kit further comprises PCR reagents and high-throughput sequencing reagents for amplifying the target nucleic acid.

[0029] Compared with the prior art, this application has the following beneficial effects:

[0030] In the target nucleic acid capture method of this application, a strategy of separately concentrating the test library and probe set is adopted. First, the probe set is prepared into pre-made lyophilized beads using a lyophilization method, which has no significant impact on the probe set. Lyophilization production is usually carried out before the start of the hybridization capture process, and a maximum of 3456 lyophilized beads can be produced in one run cycle, with the total manual operation time not exceeding 20 minutes. Then, the test library is concentrated using a drying method or a magnetic bead method, with the magnetic bead method taking approximately 8 minutes. The concentrated magnetic bead suspension is mixed with the probe set lyophilized beads and fully dissolved to obtain the hybridization system for hybridization and capture. This application provides a more efficient and rapid hybridization capture operation method, improving the concentration efficiency of multiple test libraries and probe sets, and enhancing capture performance. Attached Figure Description

[0031] Figure 1 The top figure is a flowchart of the traditional vacuum concentration hybridization capture method, the middle figure is a flowchart of the traditional magnetic bead elution hybridization capture method, and the bottom figure is a flowchart of the improved hybridization capture method of this application.

[0032] Figure 2 This is a comparison chart of the RPM of detected species in the hybridization capture test results of Comparative Examples 1-2 and Examples 1-2.

[0033] Figure 3 This is a comparison chart of the RPM of the detected species in the hybridization capture test results of Examples 3 and 4.

[0034] Figure 4 A comparison of the RPM of detected species in hybridization capture tests performed on multiple groups of clinical samples using the methods in Comparative Example 3 and Example 5. Detailed Implementation

[0035] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0036] 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.

[0037] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0038] The inventors discovered some shortcomings in traditional hybrid capture methods:

[0039] After the probe set is mixed with the test library, it is evaporated and concentrated. The evaporation method is not only time-consuming (~1 hour), but the high temperature during evaporation (generally 60°C) can also damage the biotin-modified probe, thus affecting the hybridization capture performance.

[0040] Magnetic bead elution requires eluting magnetic beads enriched with test libraries and probe sets using a small volume of elution buffer. However, due to the inherent losses in the elution operation and the limitation on the elution volume, the elution efficiency is often low (typically 55-65%), meaning that some test libraries and probe sets are wasted.

[0041] Therefore, one embodiment of this application provides a method for capturing target nucleic acids, which has advantages such as saving operation time, reducing operation complexity, and improving hybridization capture efficiency. The method includes steps (1) to (3):

[0042] Step (1): Concentrate the nucleic acid library to be tested to obtain the concentrated solution of the nucleic acid library to be tested.

[0043] In one specific example, the nucleic acid library to be tested is concentrated using a drying method or by using concentration magnetic beads.

[0044] Specifically, the step of concentrating the nucleic acid library to be tested using the drying method includes concentrating at 50-65℃ and 3000-4000×g to obtain a concentrated solution of the library to be tested.

[0045] Specifically, the step of concentrating the nucleic acid library to be tested using concentrating magnetic beads includes: adsorbing the nucleic acid library to be tested with concentrating magnetic beads, removing the supernatant, resuspending it with ddH2O, and obtaining a concentrated solution of the nucleic acid library to be tested. Preferably, the volume of ddH2O is 4-20 μL. With the amount of nucleic acid input remaining constant, the volume of the nucleic acid library to be tested is preferably concentrated to 4-20 μL to increase the nucleic acid concentration.

[0046] In one specific example, concentrated magnetic beads include CB magnetic beads (purified magnetic beads).

[0047] Multiple sample libraries were combined and enriched using 1.8× volume CB magnetic beads. Without washing with 80% ethanol, the supernatant was discarded and the magnetic beads were resuspended with 4-20 μL ddH2O. The entire process took about 8 minutes, which improved the concentration efficiency of multiple libraries.

[0048] In a specific example, nucleic acids are extracted from the sample to be tested, and a nucleic acid library is constructed.

[0049] In one specific example, the sample to be tested includes one or more of the following: serum, plasma, whole blood, sputum, swabs, lavage fluid, tissue, urine, cerebrospinal fluid, food, water, soil, bacterial culture, viral culture, cell line culture, and synthetic plasmids.

[0050] In a specific example, nucleic acids, either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), are extracted from the sample, and a nucleic acid library is constructed using standard library preparation procedures. When the nucleic acid extracted from the sample is deoxyribonucleic acid, the library preparation method is DNA library preparation; when the nucleic acid extracted from the sample is ribonucleic acid, the library preparation method is DNA library preparation, RNA library preparation, or DNA / RNA co-construction library preparation.

[0051] Step (2): Mix and dissolve the concentrated nucleic acid library solution to be tested with the lyophilized probe group to obtain a hybridization system and carry out the hybridization reaction.

[0052] In one specific example, the probe set contains a base sequence that is at least partially complementary to the target nucleic acid.

[0053] In a specific example, the liquid volume of the hybridization system is preferably 4-20 μL.

[0054] In one specific example, the probe assembly is first prepared into freeze-dried pellets free of free water using freeze-drying technology. The freeze-drying operation of the probe assembly can be completed before the hybridization capture process begins. Freeze-drying production is simple and efficient: up to 3456 freeze-dried pellets can be produced in one run cycle, and the total manual operation time does not exceed 20 minutes. This improves the concentration efficiency of the probe assembly.

[0055] In a specific example, the lyophilized probe assembly was freeze-dried using the following procedure:

[0056] Set temperature to -34~-36℃, hold for 3-5 hours; set temperature to -34~-36℃, hold for 3-5 hours, vacuum degree 12-14 Pa; set temperature to -29~-31℃, hold for 1-3 hours, vacuum degree 12-14 Pa; raise temperature to -14~-16℃, raise temperature for 3-5 hours, hold for 1-3 hours, vacuum degree 12-14 Pa; set temperature to -1~1℃, hold for 0.5-1.5 hours, vacuum degree 12-14 Pa; set temperature to 14~16℃, hold for 0.5-1.5 hours; set temperature to 29~31℃, hold for 5-7 hours.

[0057] In one specific example, the hybridization reaction consisted of denaturation at 92-98°C for 1-5 min, followed by standing at 40-80°C for 15 min to overnight.

[0058] Step (3): After the hybridization reaction, capture magnetic beads are added to capture the probe set and obtain the target nucleic acid library.

[0059] In one specific example, the capturing magnetic beads can be connected to the probe set via a connection system comprising a streptavidin-biotin system. Optionally, the capturing magnetic beads are streptavidin magnetic beads, and the probe set is a biotin-modified probe set.

[0060] In a specific example, when using concentrating magnetic beads to concentrate the nucleic acid library to be tested, a step of removing the concentrating magnetic beads is included before capturing the probe set after the hybridization reaction.

[0061] In a specific example, the step of removing concentrated magnetic beads includes: diluting the hybridization system with a diluent, performing solid-liquid separation on the diluted hybridization system to obtain supernatant A, and removing the concentrated magnetic beads. Preferably, the diluent is the solution used to capture the magnetic beads, and the resulting supernatant B (the working solution for capturing the magnetic beads) is used for solid-liquid separation. Using supernatant B to dilute the hybridization system is more beneficial to the subsequent capture performance of the magnetic beads. Optionally, supernatant B is a portion of the liquid phase; leaving a remaining liquid phase in the magnetic beads can prevent them from drying and clumping due to the loss of the working solution, which would affect their capture performance.

[0062] Optionally, the volume of the solution containing the capturing magnetic beads is 20-200 μL, and the concentration of the capturing magnetic beads is generally 5-25 mg / mL. It should be noted that those skilled in the art should select a reasonable concentration of commercially available capturing magnetic beads based on the concentration of the library.

[0063] In a preferred embodiment, the solution containing the capturing magnetic beads undergoes solid-liquid separation, and the resulting supernatant B is used to dilute the hybridization system. The diluted hybridization system is then subjected to solid-liquid separation to obtain supernatant A, after which the concentrated magnetic beads are removed. Further, supernatant A is mixed with the captured magnetic beads from which supernatant B was separated to form a capture probe assembly. Using the supernatant from the capturing magnetic beads to dilute the hybridization system not only makes solid-liquid phase separation on a magnetic rack feasible, preventing the difficulty of directly separating the solid and liquid phases on a magnetic rack when the liquid phase of the hybridization system is small and highly viscous, which could easily lead to the loss of both solid and liquid phases, but also does not affect subsequent capture reactions, thus improving capture performance.

[0064] Specifically, 20-200 μL of streptavidin magnetic bead suspension is subjected to solid-liquid separation using a magnetic rack. 16-160 μL of supernatant (the working solution for the streptavidin magnetic beads) is then used to dilute 4-20 μL of the hybridization system. The diluted hybridization system is then subjected to solid-liquid separation using a magnetic rack. All the supernatant is collected and thoroughly mixed with the remaining 4-40 μL of streptavidin magnetic bead suspension, and the mixture is subjected to capture for 10-100 min. This procedure ensures that only 4-20 μL of the optimal hybridization system is sufficiently diluted, making solid-liquid phase separation using a magnetic rack a feasible step. This eliminates the possibility that purified magnetic beads would reduce the effectiveness of the streptavidin magnetic bead capture probe in the subsequent capture system, thereby improving and stabilizing the hybridization capture performance.

[0065] In one specific example, after capturing the probe set, a step of cleaning the capturing magnetic beads is also included.

[0066] Specifically, the streptavidin magnetic beads were washed 1-5 times with nucleic acid washing buffer at 40-80℃ to remove uncaptured sample nucleic acids and probe groups, and the streptavidin magnetic beads were resuspended in 3-30μL ddH2O.

[0067] One embodiment of this application provides a method for detecting nucleic acids, the method comprising the following steps:

[0068] The target nucleic acid library was obtained using the capture method described above;

[0069] The target nucleic acid library was amplified using PCR; and

[0070] The amplified products were purified and then subjected to high-throughput sequencing to determine whether the sample contained the target nucleic acid, thereby determining whether the sample contained pathogenic microorganisms and the specific species of the pathogenic microorganisms.

[0071] In a specific example, this method can be used for nucleic acid detection for diagnostic purposes.

[0072] In a specific example, this method can also be used for nucleic acid testing for non-diagnostic purposes. For instance, it can be used for detecting pollution sources in the environment (such as sewage) for biosafety control; or for guiding medication use by detecting drug resistance genes; or for investigating and tracing the source of infectious diseases.

[0073] In one specific example, the target nucleic acid includes the nucleic acid of a microorganism in the sample to be tested, said microorganism including one or more of bacteria, fungi, and viruses.

[0074] In a specific example, the virus is a DNA virus and / or an RNA virus.

[0075] In a specific example, the bacteria include, but are not limited to, one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Haemophilus influenzae, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Legionella pneumophila, Mycobacterium tuberculosis, Enterococcus faecalis, Serratia marcescens, Enterobacter cloacae, Streptococcus pneumoniae, Bordetella pertussis, Neisseria meningitidis, Streptococcus agalactiae, and Moraxella catarrhalis.

[0076] In one specific example, the fungi include, but are not limited to, one or more of Candida albicans, Pneumocystis yew, Aspergillus flavus, Aspergillus fumigatus, and Cryptococcus glottii;

[0077] In one specific example, DNA viruses include, but are not limited to, one or more of herpes simplex virus type I, herpes simplex virus type II, and herpes simplex virus type IV.

[0078] In one specific example, RNA viruses include, but are not limited to, one or more of the following: novel coronavirus, influenza A virus, and human metapneumovirus.

[0079] One embodiment of this application also provides a kit comprising the reagents used in the above-described capture method or the reagents used in the above-described method.

[0080] In one specific example, the kit includes a lyophilized probe set for detecting the target nucleic acid, magnetic beads for adsorbing the nucleic acid, and magnetic beads for capturing the probe set.

[0081] In one specific example, the kit also includes a nucleic acid washing solution.

[0082] In one specific example, the kit also includes PCR amplification reagents and high-throughput sequencing.

[0083] In one specific example, the PCR amplification reagents include polymerase and universal adapter primers for amplifying the captured target nucleic acid.

[0084] 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 specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0085] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0086] The following are the main reagents involved in the examples or comparative examples:

[0087] The hybridization capture nucleic acid detection method of this application uses at least the following reagents A) to G):

[0088] A) A DNA library obtained by constructing a library from nucleic acids extracted from the sample to be tested using conventional DNA library construction methods;

[0089] B) A probe kit for nucleic acid detection, containing a base sequence at least partially complementary to the target sequence of the pathogen being detected, and modified with biotin at its 5' end via a C6 structure; the probe kit was designed by the inventors and synthesized by Integrated DNA Technologies (IDT); reagent name: xGen TM Custom Hyb Panels;

[0090] C) Purification magnetic beads for concentrating and purifying nucleic acids (VAHTS™ DNA Clean Beads, Novizan Biosciences Co., Ltd., N411-03);

[0091] D) Streptavidin magnetic beads for capturing biotin-modified probes (Dynabeads Streptavidin Magnetic Beads, ThermoFisher Scientific, 65605D);

[0092] E) Nucleic acid washing solution (10X Wash Buffer I / II from xGen Hybridization and Wash Kit, Integrated DNA Technologies, 1080577);

[0093] F) Polymerase (DreamTaq DNA polymerase, ThermoFisher Scientific, EP1701);

[0094] G) Universal adapter primers for amplifying captured target sequences (GenScript Biotechnology Co., Ltd., SC1568-1).

[0095] In the nucleic acid detection method of this application, by using the reagents A) to G), when the sample contains the target nucleic acid, the following reactions i) to iiiiiii) occur, thereby enabling the detection of the target nucleic acid in the sample using the target-specific sequences detected by the obtained nucleic acid in next-generation sequencing. Simultaneously, the performance and sensitivity of the detection method can be evaluated using the normalized number of target-specific sequences detected by the obtained nucleic acid in next-generation sequencing. The detailed detection procedure of the new method is shown in the appendix. Figure 1 .

[0096] i) Thoroughly mix the purified magnetic beads (C) with multiple A) test libraries, discard the supernatant on a magnetic rack, add 4-20 μL of ddH2O for resuspending, and prepare a library-magnetic bead suspension;

[0097] ii) According to the freeze-drying process shown in Table 1, the B) probe group solution was freeze-dried and concentrated using a freeze dryer to prepare the probe group freeze-dried spheres;

[0098] Table 1. Probe assembly freeze-drying process table

[0099] Target temperature / °C Heating time / h Template vacuum / Pa Isothermal time / h -35 - - 4 -35 - 13 4 -30 0 13 2 -15 4 13 2 0 0 13 1 15 0 0 1 30 0 0 6

[0100] iii) Dissolve the lyophilized beads in the library-magnetic bead suspension and mix thoroughly to prepare a hybridization system. The volume of the hybridization system should be 4-20 μL. Incubate at 95°C for 5 min to allow the double-stranded DNA to unwind, then incubate at 40-80°C for 15 min or overnight to ensure sufficient contact between the target nucleic acid and the probe group. The target single-stranded DNA in the sample nucleic acid and the probe single-stranded DNA will renature to form a probe-target complex.

[0101] iv) Separate 20-200 μL of D) streptavidin magnetic beads on a magnetic rack for solid-liquid phase separation, and add 16-160 μL of the liquid phase to the hybridization system prepared in step iii); then place the diluted hybridization system on a magnetic rack for solid-liquid phase separation, thereby achieving the purpose of separating the C) purified magnetic beads in the hybridization system;

[0102] v) Take all the supernatant from the liquid phase and mix it thoroughly with the remaining 4-40 μL of streptavidin magnetic beads from step iv). D) Ensure that the biotin-modified probe group is in full contact with the streptavidin magnetic beads, thereby achieving the purpose of capturing the probe group with streptavidin magnetic beads. If the probe group binds to the target DNA, then the target DNA will also be captured by the streptavidin magnetic beads.

[0103] vi) Wash the streptavidin beads from step v) 1-5 times with nucleic acid washing buffer E) at 40-80℃ to remove uncaptured sample nucleic acids and probes. Finally, resuspend the streptavidin beads in 3-30 μL ddH2O for PCR amplification.

[0104] vii) According to the PCR system shown in Table 2 and the PCR procedure shown in Table 3, use F) polymerase and G) universal adapter primers to amplify the target DNA captured in the streptavidin magnetic bead suspension obtained in step vi).

[0105] Table 2. PCR amplification system of streptavidin magnetic bead suspension

[0106] reaction system Reagent dosage (μL) Streptavidin magnetic bead suspension 3-30 F) Polymerase 12.5 G) Universal connector primers 1.5

[0107] Table 3. PCR amplification program for streptavidin magnetic bead suspension.

[0108]

[0109] viii) The amplification product obtained in step vii) was purified using magnetic beads in step C). After dilution and denaturation, it was then subjected to next-generation sequencing using a next-generation sequencing platform. The sequencing results were used to determine whether pathogenic microorganisms were present in the sample. The normalized target-specific sequence number (Reads Per Million Reads, RPM) of the detected microorganisms was used to evaluate the hybridization capture performance.

[0110] Detailed explanation of the result interpretation for the nucleic acid detection method in this application:

[0111] i) After the captured target DNA is amplified and sequenced, the sequencing results will be compared and matched with NCBI's NT nucleic acid sequence database. The test sequence that successfully matches a certain pathogen species will be listed as the target-specific sequence of that pathogen species.

[0112] ii) Sum the number of sequences that match the same pathogen species. Considering the potential bias in PCR amplification, all identical target-specific sequences within the same species are counted as one. The sum of the sequence counts for that species is the target-specific sequence count (Reads). Considering the fluctuation in the total sequencing data volume, the target-specific sequence count should be normalized to the target-specific sequence count when the total data volume is 1M, which is the normalized target-specific sequence count (RPM).

[0113] iii) To compare the hybridization capture performance of different operating procedures, after performing hybridization capture on the same test library using different operating procedures, the performance of different operating procedures can be judged by comparing the detection RPM of the same pathogen species in the same library: the higher the RPM of each pathogen species in the same library, the better the hybridization capture performance of the operating procedure.

[0114] The above method can detect more than 3,000 types of target nucleic acid sequences.

[0115] Comparative Example 1

[0116] Comparative Example 1: Hybridization capture: The multi-target test library and probe set were mixed and then concentrated under vacuum for hybridization capture.

[0117] First, at least 10 historical clinical sample libraries were selected. The sample types included at least whole blood samples, sputum samples, nasopharyngeal swab samples, lavage fluid samples, fresh tissue samples, urine and cerebrospinal fluid samples. Each sample included at least one pathogen target species, including at least: Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Haemophilus influenzae, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Legionella pneumophila, Mycobacterium tuberculosis, Enterococcus faecalis, Serratia marcescens, Enterobacter cloacae, Streptococcus pneumoniae, Bordetella pertussis, Neisseria meningitidis, Streptococcus agalactiae, Moraxella catarrhalis; Candida albicans, Pneumocystis jirovecii, Aspergillus flavus, Aspergillus fumigatus, Cryptococcus glottii; Herpes simplex virus type I, Herpes simplex virus type II, Herpes simplex virus type IV; Novel coronavirus, Influenza A virus, Human metapneumovirus, and 28 other pathogenic microorganisms.

[0118] according to Figure 1 The above diagram illustrates the procedure: The library is tested for Qubit concentration. Based on the obtained concentration, equal volumes of nucleic acids are mixed, followed by an equal volume of probe kits. The mixture is then concentrated to complete dryness using a vacuum concentrator (60℃, 3500×g, 50-70 min). 4-20 μL of ddH2O is added to dissolve the evaporated product, which is then incubated at 95℃ for 5 min to allow double-stranded DNA to unwind. Hybridization is then performed at 40-80℃ for 15 min to overnight. Next, 20-200 μL of streptavidin magnetic bead suspension is added for 10-100 min of capture. After washing 1-5 times with nucleic acid washing buffer at 40-80℃, the streptavidin magnetic beads are resuspended in 3-30 μL of ddH2O. The suspension is then subjected to PCR amplification according to Table 2-3. The amplified product is purified with purification magnetic beads, diluted, denatured, and then sequenced using Illumina MiniSeq. The sequencing data will be compared and matched with NCBI's NT nucleic acid sequence database, and the final result will be presented as the number of pathogen species detected plus the number of normalized target-specific sequences (RPM).

[0119] Table 4. List of pathogenic species finally detected in Comparative Example 1 and RPM of each species.

[0120]

[0121]

[0122] Table 4 shows the final detection results of Comparative Example 1. The results show that all 28 pathogenic microorganisms were detected, with detection RPM ranging from 7 to 208,889. During the experiment, the vacuum concentration of the test library and probe set took a total of 55 minutes.

[0123] Comparative Example 2

[0124] Comparative Example 2: Hybridization capture: The multi-target test library and probe set were mixed and then concentrated and eluted with magnetic beads for hybridization capture.

[0125] At least 10 historical clinical samples from Comparison 1 were still selected for library construction. According to Figure 1 The proposed method involves testing the concentration of the constructed test library using Qubit. Based on the obtained concentration, equal volumes of nucleic acids are mixed, followed by an equal volume of probe sets. The total volume of the system is calculated, and 1.0-3.0 times the total volume of purified magnetic beads is added and thoroughly mixed. After 5 minutes, nucleic acid is eluted with 4-20 μL of ddH2O. The eluent is the hybridization system. The eluent is incubated at 95°C for 5 minutes to allow double-stranded DNA to unwind, followed by hybridization at 40-80°C for 15 minutes to overnight. Then, 20-200 μL of streptavidin magnetic bead suspension is added for 10-100 minutes for capture. After washing 1-5 times with nucleic acid washing buffer at 40-80°C, the streptavidin magnetic beads are resuspended with 3-30 μL of ddH2O. The suspension is then subjected to PCR amplification according to Table 2-3. The amplified product is purified with purified magnetic beads, diluted, denatured, and then sequenced using Illumina MiniSeq. The sequencing data will be compared and matched with NCBI's NT nucleic acid sequence database, and the final result will be presented as the number of pathogen species detected plus the number of normalized target-specific sequences (RPM).

[0126] Table 5. List of pathogenic species finally detected in Comparative Example 2 and RPM of each species.

[0127]

[0128]

[0129] Table 5 shows the final detection results of Comparative Example 2. The results show that all 28 pathogenic microorganisms were detected, with detection RPMs ranging from 20 to 217,920. During the experiment, the purification, concentration, and elution of the test library and probe group using magnetic beads took a total of 20 minutes.

[0130] Example 1

[0131] This embodiment of the improved hybridization capture methodology 1: adopts a strategy of separately concentrating the library and the probe set. The probe set is concentrated under vacuum. The multi-target test library is concentrated by purified magnetic beads and resuspended before hybridization capture.

[0132] At least 10 historical clinical samples from Comparative Example 1 were selected for library construction. The constructed test libraries were subjected to Qubit concentration testing. Based on the obtained concentration, equal volumes of nucleic acids were mixed, and 1.0-3.0 times the total volume of purified magnetic beads were added and thoroughly mixed. After 5 minutes, the magnetic beads were resuspended in 4-20 μL of ddH2O to obtain 4-20 μL of concentrated test library magnetic bead suspension. An equal volume of probe kit was concentrated to complete dryness using vacuum concentration. 4-20 μL of the concentrated test library magnetic bead suspension was added to the completely dried probe kit and thoroughly mixed to dissolve. The double-stranded DNA was allowed to unwind at 95°C for 5 minutes, followed by hybridization at 40-80°C for 15 minutes to overnight. Then, 20-200 μL of streptavidin magnetic bead suspension was added for at least 10-100 minutes for capture. After washing 1-5 times with nucleic acid washing buffer at 40-80℃, streptavidin magnetic beads were resuspended in 3-30 μL ddH2O. The suspension was then subjected to PCR amplification according to Table 2-3. The amplified products were purified with purification magnetic beads, diluted, denatured, and then subjected to next-generation sequencing using Illumina MiniSeq. The sequencing data will be compared and matched with the NCBI NT nucleic acid sequence database. The final results will be presented as the number of pathogen species detected plus the number of normalized target-specific sequences (RPM).

[0133] Table 6. List of pathogenic species finally detected in Example 1 and RPM of each species.

[0134]

[0135]

[0136]

[0137] Table 6 shows the final detection results of Example 1. The results show that all 28 pathogenic microorganisms were detected, with detection RPM ranging from 12 to 227,130. Comparing the RPM of each detected target with the RPM of the same target in Comparative Examples 1 and 2, it is evident that the hybridization capture performance of Example 1 is higher than that of Comparative Examples 1 and 2, with median values ​​of 1.17 and 1.46 for each target, respectively. In the experimental procedure, the purification and concentration of the test library using magnetic beads took a total of 8 minutes, and the vacuum concentration of the probe set took a total of 30 minutes, which is superior to the 55 minutes in Comparative Example 1.

[0138] Example 2

[0139] This embodiment of the improved hybridization capture methodology 2 adopts a strategy of separating the concentrated library and probe set. The multi-target test library is still concentrated using the vacuum concentration method, and the probe set is concentrated by freeze drying before hybridization capture.

[0140] At least 10 historical clinical samples from Comparative Example 1 were selected for library construction. The constructed test libraries were subjected to Qubit concentration testing. Based on the obtained concentration, equal nucleic acid masses were mixed, concentrated to complete dryness using a vacuum concentrator, and then thoroughly dissolved in 4-20 μL ddH2O. The probe set was lyophilized beforehand according to the procedure in Table 1. 4-20 μL of the dissolved test library was thoroughly mixed with the lyophilized product and incubated at 40-80℃ for 15 min to overnight. Then, 20-200 μL of streptavidin magnetic bead suspension was added for 10-100 min of capture. After washing 1-5 times with nucleic acid washing buffer at 40-80℃, the streptavidin magnetic beads were resuspended in 3-30 μL ddH2O. The suspension was then subjected to PCR amplification according to Table 2-3. The amplified products were purified with purification magnetic beads, diluted, denatured, and then sequenced using Illumina MiniSeq. The sequencing data will be compared and matched with NCBI's NT nucleic acid sequence database, and the final result will be presented as the number of pathogen species detected plus the number of normalized target-specific sequences (RPM).

[0141] Table 7. List of pathogenic species finally detected in Example 2 and RPM of each species.

[0142]

[0143]

[0144] Table 7 shows the final detection results for Example 2. The results show that all 28 pathogenic microorganisms were detected, with detection RPMs ranging from 36 to 218287. Comparing the RPMs of each detected target with those in Comparative Examples 1 and 2, it is evident that the hybridization capture performance of Example 2 is superior to that of Comparative Examples 1 and 2, with median values ​​of 1.35 and 1.69 for each target, respectively. In the experimental procedure, the vacuum concentration of the test library took 30 minutes, which is better than the 55 minutes in Comparative Example 1.

[0145] Based on the data from Comparative Examples 1-2 and Examples 1-2, it can be seen that the method of purifying the mixture of the test library and probe set with magnetic beads and eluting with ddH2O in Comparative Example 2 had the lowest performance, which is likely related to incomplete elution of the test library or probe set. A comparison of the data from Example 1 and Comparative Example 1 shows that the method of purifying the test library, concentrating it with magnetic beads, and then resuspending it as a magnetic bead suspension before adding it to the hybridization system does not affect the library recovery; its hybridization capture performance is even slightly higher than that of concentrating the test library using vacuum concentration. Compared with Comparative Examples 1-2, Example 2 had the highest target RPM, indicating that the lyophilization method was the best for probe set concentration. This may be because lyophilization does not destroy the modified structure of the probe set, while evaporation (vacuum concentration) may destroy the biotin modification of the probe set due to high temperature.

[0146] Example 3

[0147] This embodiment of the improved hybridization capture methodology 3: adopts a strategy of separately concentrating the library and probe set, using purified magnetic beads to concentrate and resuspend the multi-target test library and probe set, freeze-drying and hybridizing, and then performing the capture operation without removing the purified magnetic beads.

[0148] At least 10 historical clinical samples from Comparative Example 1 were selected for library construction. The constructed test libraries were subjected to Qubit concentration testing. Based on the obtained concentration, equal volumes of nucleic acids were mixed, and 1.0-3.0 times the total volume of purified magnetic beads were added and thoroughly mixed. After 5 minutes, the magnetic beads were resuspended in 4-20 μL of ddH2O to obtain 4-20 μL of concentrated test library magnetic bead suspension. An equal volume of probe kit was concentrated to complete dryness using lyophilization. 4-20 μL of the concentrated test library magnetic bead suspension was added to the completely dried probe kit lyophilized and thoroughly mixed to dissolve. The double-stranded DNA was allowed to unwind at 95°C for 5 minutes, followed by hybridization at 40-80°C for 15 minutes to overnight. Then, 20-200 μL of streptavidin magnetic bead suspension was added for 10-100 minutes for capture. At this point, the system still contained the purified magnetic beads used for concentrating the test library. After washing 1-5 times with nucleic acid washing buffer at 40-80℃, streptavidin magnetic beads were resuspended in 3-30 μL ddH2O. The suspension was then subjected to PCR amplification according to Table 2-3. The amplified products were purified with purification magnetic beads, diluted, denatured, and then subjected to next-generation sequencing using Illumina MiniSeq. The sequencing data will be compared and matched with the NCBI NT nucleic acid sequence database. The final results will be presented as the number of pathogen species detected plus the number of normalized target-specific sequences (RPM).

[0149] Table 8. List of pathogenic species finally detected in Example 3 and RPM of each species.

[0150]

[0151]

[0152] Table 8 shows the final detection results of Example 3. The results show that all 28 pathogenic microorganisms were detected, with detection RPM ranging from 15 to 149,862.

[0153] Example 4

[0154] In this embodiment, the improved hybridization capture methodology 4 is as follows: after concentrating and resuspending the multi-target test library with purified magnetic beads and hybridizing it with the probe group by lyophilization, the purified magnetic beads are removed before the capture operation is performed.

[0155] At least 10 historical clinical samples from Comparison 1 were still selected for library construction. According to Figure 1 The scheme shown in the diagram below, which is the final improved scheme of this invention, involves testing the concentration of the constructed test library using Qubit. Based on the obtained concentration, the nucleic acid masses are mixed in an equal volume, and then 1.0-3.0 times the total volume of purified magnetic beads are added and thoroughly mixed. After 5 minutes, the magnetic beads are resuspended in 4-20 μL of ddH2O to obtain 4-20 μL of concentrated magnetic bead suspension of the test library. An equal volume of probe kit is concentrated to complete dryness using lyophilization. The 4-20 μL of concentrated magnetic bead suspension of the test library is added to the completely dried lyophilized probe kit and thoroughly mixed to dissolve. The mixture is then incubated at 95°C for 5 minutes to allow the double-stranded DNA to unwind, followed by hybridization at 40-80°C for 15 minutes to overnight. At this point, 20-200 μL of streptavidin magnetic bead suspension was subjected to solid-liquid separation using a magnetic rack. 16-160 μL of the supernatant was added to the 4-20 μL hybridization system, and the diluted hybridization system was further separated using a magnetic rack. The entire supernatant of the hybridization system was removed and thoroughly mixed with the remaining 4-40 μL of streptavidin magnetic bead suspension, followed by capture for 10-100 min. At this point, the system no longer contained the purification magnetic beads used for concentrating the target library. After washing 1-5 times with nucleic acid washing buffer at 40-80℃, the streptavidin magnetic beads were resuspended in 3-30 μL of ddH2O. The suspension was then subjected to PCR amplification according to Table 2-3. The amplified product was purified with purification magnetic beads, diluted, denatured, and then sequenced using Illumina MiniSeq. The sequencing data will be compared and matched with NCBI's NT nucleic acid sequence database, and the final result will be presented as the number of pathogen species detected plus the number of normalized target-specific sequences (RPM).

[0156] Table 9. List of pathogenic species finally detected in Example 4 and RPM of each species.

[0157]

[0158]

[0159] Table 9 shows the final detection results of Example 3. The results show that all 28 pathogenic microorganisms were detected, with detection RPMs ranging from 28 to 165,130. Comparing the RPMs of each detected target with those in Example 3, it is evident that the hybridization capture performance of Example 4 is higher than that of Example 3, with a median comparison value of 1.32 for each target. This result indicates that the purified magnetic beads used to concentrate the test library interfere with the efficiency of the streptavidin magnetic bead capture probe set after entering the capture system, affecting capture and amplification performance. Removing the purified magnetic beads further improves the hybridization capture performance.

[0160] Comparative Example 3

[0161] Hybridization capture was performed on prospective clinical samples using the hybridization capture method described in Comparative Example 1: a method involving vacuum concentration after mixing the test library and probe set.

[0162] First, at least 10 prospective clinical samples were selected for library construction. The sample types included at least whole blood samples, sputum samples, lavage fluid samples, and cerebrospinal fluid samples, with unknown targets.

[0163] The constructed test library was subjected to Qubit concentration testing. Based on the obtained concentration, equal volumes of nucleic acids were mixed, and then an equal volume of probe kit was added. The mixture was concentrated to complete dryness using a vacuum concentrator (60℃, 3500×g, 50-70min). After dissolving the evaporated product in 4-20μL ddH2O, the double-stranded DNA was allowed to unwind at 95℃ for 5min, followed by hybridization at 40-80℃ for 15min to overnight. Then, 20-200μL of streptavidin magnetic bead suspension was added for 10-100min capture. After washing 1-5 times with nucleic acid washing buffer at 40-80℃, the streptavidin magnetic beads were resuspended in 3-30μL ddH2O. The suspension was then subjected to PCR amplification according to Table 2-3. The amplified product was purified with purification magnetic beads, diluted, denatured, and then sequenced using Illumina MiniSeq. The sequencing data will be compared and matched with NCBI's NT nucleic acid sequence database, and the final result will be presented as the number of pathogen species detected plus the number of normalized target-specific sequences (RPM).

[0164] Table 10 List of pathogenic species finally detected in Comparative Example 3 and RPM of each species

[0165]

[0166]

[0167] Table 10 shows the final detection results of Comparative Example 3. The results show that a total of 24 pathogenic microorganisms were detected across all targets, with RPM ranging from 6 to 7705. During the experiment, the vacuum concentration of the mixture of the test library and probe set took a total of 50 minutes.

[0168] Example 5

[0169] Hybrid capture was performed on prospective clinical samples using the hybridization capture method described in Example 4: multi-target test library concentration and resuspending with magnetic beads + probe group lyophilization and concentration + removal of purified magnetic beads before capture.

[0170] At least 10 prospective clinical samples from proportion 3 were still selected for library construction. According to Figure 1The scheme shown in the figure below, which is the final improved scheme of this application, involves testing the concentration of the constructed test library using Qubit. Based on the obtained concentration, the nucleic acid masses are mixed in an equal mass, and then 1.0-3.0 times the total volume of purified magnetic beads are added and thoroughly mixed. After 5 minutes, the magnetic beads are resuspended in 4-20 μL of ddH2O to obtain 4-20 μL of concentrated magnetic bead suspension of the test library. An equal volume of probe kit is concentrated to complete dryness using lyophilization. The 4-20 μL of concentrated magnetic bead suspension of the test library is added to the completely dried lyophilized probe kit and thoroughly mixed to dissolve. The mixture is then incubated at 95°C for 5 minutes to allow the double-stranded DNA to unwind, followed by hybridization at 40-80°C for 15 minutes to overnight. At this point, 20-200 μL of streptavidin magnetic bead suspension was subjected to solid-liquid separation using a magnetic rack. 16-160 μL of the supernatant was added to the 4-20 μL hybridization system, and the diluted hybridization system was further separated using a magnetic rack. The entire supernatant of the hybridization system was removed and thoroughly mixed with the remaining 4-40 μL of streptavidin magnetic bead suspension, followed by capture for 10-100 min. At this point, the system no longer contained the purification magnetic beads used for concentrating the target library. After washing 1-5 times with nucleic acid washing buffer at 40-80℃, the streptavidin magnetic beads were resuspended in 3-30 μL of ddH2O. The suspension was then subjected to PCR amplification according to Table 2-3. The amplified product was purified with purification magnetic beads, diluted, denatured, and then sequenced using Illumina MiniSeq. The sequencing data will be compared and matched with NCBI's NT nucleic acid sequence database, and the final result will be presented as the number of pathogen species detected plus the number of normalized target-specific sequences (RPM).

[0171] Table 11 List of pathogenic species finally detected in Example 5 and RPM of each species

[0172]

[0173]

[0174] Table 11 shows the final detection results of Example 5. The results show that a total of 24 pathogenic microorganisms were detected across all targets, with detection RPMs ranging from 11 to 11259. During the experiment, the purification and concentration of the multi-target test library using magnetic beads took a total of 8 minutes. Comparing the RPM of each detected target with the RPM detected in Comparative Example 3, it is evident that the hybridization capture performance of Example 5 is higher than that of Comparative Example 3, with a median comparison value of 1.40 for each target. These results demonstrate that the method of magnetic bead concentration of the test library + lyophilization concentration of the probe group + removal of purified magnetic beads before capture, as described in this invention, has advantages over traditional methods, including higher detection performance and shorter operation time. Specifically, the new method shows an overall improvement in species detection RPM compared to traditional methods without any risk of missed detections; and under the premise of completing the lyophilization production of the probe group in advance, the operation time for test library concentration is shortened by at least 40-50 minutes.

[0175] 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.

[0176] 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 can be used to interpret the content of the claims.

Claims

1. A method for capturing target nucleic acids for non-diagnostic purposes, characterized in that, The method includes the following steps: The nucleic acid library to be tested was concentrated using magnetic beads to obtain a concentrated solution of the nucleic acid library to be tested; The concentrated nucleic acid library to be tested was mixed and dissolved with the lyophilized probe group to obtain a hybridization system, and a hybridization reaction was carried out. as well as After the hybridization reaction, the concentrated magnetic beads are removed, and then the capture magnetic beads are added to capture the probe set to obtain the target nucleic acid library; The probe set contains a base sequence that is at least partially complementary to the target nucleic acid; The steps of concentrating the nucleic acid library to be tested using concentrating magnetic beads include: adsorbing the nucleic acid library to be tested with concentrating magnetic beads, removing the supernatant, resuspending it with 4-20 μL ddH2O to obtain the concentrated nucleic acid library to be tested; The steps for removing the concentrated magnetic beads include: diluting the hybridization system with a diluent, performing solid-liquid separation on the diluted hybridization system to obtain supernatant A, and removing the concentrated magnetic beads.

2. The capture method according to claim 1, characterized in that, Nucleic acid is extracted from the sample to be tested, and the nucleic acid library to be tested is constructed.

3. The capture method according to claim 2, characterized in that, The samples to be tested include one or more of the following: serum, plasma, whole blood, sputum, lavage fluid, tissue, urine, cerebrospinal fluid, bacterial culture, viral culture, cell line culture, and artificially synthesized plasmids.

4. The capture method according to any one of claims 1 to 3, characterized in that, The probe assembly lyophilized product was prepared by the following procedure: Set the temperature to -34~-36℃ and maintain it for 3-5 hours; Set the temperature to -34~-36℃, hold for 3-5 hours, and maintain a vacuum of 12-14 Pa. Set the temperature to -29~-31℃, hold for 1-3 hours, and maintain a vacuum of 12-14 Pa. Heat to -14~-16℃ for 3-5 hours, hold for 1-3 hours, and maintain a vacuum of 12-14 Pa. Set the temperature to -1~1℃, hold for 0.5-1.5h, and maintain a vacuum of 12-14Pa. Set the temperature to 14~16℃ and maintain it for 0.5-1.5 hours; Set the temperature to 29~31℃ and maintain it for 5-7 hours.

5. The capture method according to any one of claims 1 to 3, characterized in that, The hybridization reaction includes 92-98 o After denaturation of C for 1-5 minutes, at 40-80 o Let stand in C for 15 minutes or overnight.

6. The capture method according to claim 1, characterized in that, The diluent is the supernatant B obtained after solid-liquid separation of the solution that captures the magnetic beads.

7. The capture method according to claim 6, characterized in that, The volume of the solution for capturing magnetic beads is 20-200 μL.

8. The capture method according to claim 6, characterized in that, The supernatant A is mixed with the capturing magnetic beads after the supernatant B has been separated to capture the probe group.

9. The capture method according to any one of claims 1 to 3, characterized in that, The capturing magnetic beads can be connected to the probe group via a connection system, which includes a streptavidin-biotin system.

10. The capture method according to claim 9, characterized in that, The capturing magnetic beads are streptavidin magnetic beads, and the probe set is a biotin-modified probe set.

11. The capture method according to any one of claims 1 to 3, characterized in that, After the capture magnetic beads capture the probe group, the process also includes a step of cleaning the capture magnetic beads.

12. A method for detecting nucleic acids for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes the following steps: The target nucleic acid library is obtained by the capture method according to any one of claims 1 to 11; PCR amplification of the target nucleic acid library; and The amplified products were purified and then subjected to high-throughput sequencing to determine whether the sample contained the target nucleic acid.

13. The method according to claim 12, characterized in that, The target nucleic acid includes the nucleic acid of microorganisms in the sample to be tested.

14. The method according to claim 13, characterized in that, The microorganisms include one or more of bacteria, fungi, and viruses.

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