A capture probe, kit and method for detection of full-length genome of dianba bandavirus

Abstract

The application provides a capture probe, a kit and a method for detecting full-length genome of Dabie bandavirus, and relates to the technical field of virus detection.The application provides a capture probe group capable of efficiently, specifically and uniformly capturing the full-length genome of SFTSV, and the capture probe group is composed of probes with nucleotide sequences shown in SEQ ID NO: 1-679.The application also provides a kit containing the probe group and a method for detecting the full-length genome of SFTSV based on the probe group and a second-generation sequencing technology.The technical scheme of the application combines innovative probe design with a standardized target enrichment process, and systematically solves the main technical obstacles in obtaining the full-length genome of SFTSV in the prior art, and significantly improves multiple key indicators such as coverage, sensitivity, universality, throughput, cost-effectiveness and data quality, thereby providing a powerful and practical technical tool for molecular typing, variation monitoring, transmission chain tracing and vaccine research of SFTSV.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to a capture probe, kit, and method for detecting the full-length genome of Dabie Bandar virus. Background Technology

[0002] Dabie Bandar virus (SFTSV) belongs to the genus *Leptaviridae* of the family Bunyaviridae. It is a single-stranded RNA virus whose genome consists of three segments: a large segment (L), a medium segment (M), and a small segment (S). This virus is the pathogen of fever with thrombocytopenia syndrome (SFTS), which is prevalent in multiple regions of my country, including Henan, Hubei, and Shandong provinces, and has a high mortality rate. Whole-genome sequencing of SFTSV is of great significance for viral tracing, genotyping, mutation monitoring, and vaccine design.

[0003] Currently, the main techniques for obtaining the whole genome sequence of SFTSV include Sanger sequencing, multiplex PCR amplicon sequencing, and metagenomic next-generation sequencing (mNGS). Sanger sequencing typically requires designing multiple primer pairs for segmental amplification, which is cumbersome, has low throughput, and is inefficient for samples with low viral load, making it difficult to obtain a complete genome sequence. Multiplex PCR amplicon sequencing involves designing multiple pairs of dozens or even hundreds of specific primers in the same reaction system to simultaneously amplify target regions at different locations on the viral genome, and then splicing and analyzing the obtained sequences. This method effectively avoids the low throughput problem of Sanger sequencing, but it still suffers from low primer amplification efficiency and the inability of primers to bind due to viral mutations, resulting in missing amplification of some segments of the viral genome, fragmented sequencing coverage, and a high risk of genomic gaps, incomplete sequence splicing, and distorted genotyping and variant analysis results. While metagenomic sequencing does not require pre-set primers, the sequencing data contains a very high proportion of host background nucleic acids, resulting in a low proportion of effective viral data, high sequencing costs, and difficulty in effectively covering complex regions such as the ends of the genome.

[0004] Liquid-phase hybridization capture combined with targeted next-generation sequencing (SNG) is a targeted enrichment technology developed in recent years. Its principle involves designing specific probes for the target pathogen's genome, enriching the target fragment through hybridization, and then performing high-throughput sequencing. This technology can significantly improve the sequencing depth of the target sequence and reduce background interference. However, current technologies lack high-coverage, homogeneous capture probe combinations specifically designed for the entire SFTSV genome (covering the L, M, and S fragments). Some existing general-purpose virus capture sets have limited coverage of SFTSV and cannot effectively address sequence differences between different SFTSV genotypes, leading to coverage gaps or uneven capture efficiency in low-load samples. These shortcomings collectively result in difficulties in obtaining complete or inaccurate SFTSV whole-genome data, hindering precise molecular epidemiological research and real-time monitoring and early warning based on high-resolution genomic data.

[0005] Therefore, there is an urgent need in this field to develop a capture probe system that can comprehensively cover the entire SFTSV genome and has high sensitivity and specificity, in order to solve the technical bottlenecks faced by existing technologies in molecular tracing and mutation monitoring of SFTSV. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] Existing technologies for obtaining the complete genome of Dabie Bandar virus (SFTSV) suffer from several technical challenges, including low throughput, insufficient sensitivity to low-load samples, uneven genome coverage, strong primer dependence, poor error tolerance, and low cost-effectiveness. These issues collectively lead to difficulties in obtaining complete or inaccurate SFTSV genome data, hindering precise molecular epidemiological research and real-time monitoring and early warning based on high-resolution genomic data.

[0008] The primary objective of this invention is to provide a set of capture probes capable of efficiently, specifically, and uniformly capturing the full-length genome segments L, M, and S.

[0009] Another objective of this invention is to provide a kit containing the probe set and a method for detecting the full-length genome of SFTSV based on the probe set and next-generation sequencing technology, so as to achieve high-sensitivity detection of SFTSV in clinical samples with low viral load and stably obtain full-length genome sequences with high coverage and high accuracy, thereby providing reliable technical support for molecular typing, mutation monitoring, transmission chain tracing and vaccine development of SFTSV.

[0010] (II) Technical Solution

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] In a first aspect, the present invention provides a capture probe set for detecting the full-length genome of Dabie Bandar virus (SFTSV), the capture probe set consisting of probes with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 679.

[0013] Furthermore, each probe in the probe set is a single-stranded DNA oligonucleotide, preferably 100 nucleotides (nt) in length.

[0014] Furthermore, the 5' or 3' end of each probe in the probe group is preferably labeled with biotin.

[0015] Secondly, the present invention provides a detection kit for the full-length genome of Dabie Bandar virus, the detection kit comprising a capture probe with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 679, streptavidin-labeled magnetic beads, and a library hybridization reaction reagent.

[0016] Furthermore, the streptavidin-labeled magnetic beads are used to bind to biotin on the capture probe, thereby separating the probe-target DNA complex from the reaction system using magnetic separation technology.

[0017] Furthermore, the library reaction reagent includes:

[0018] (1) TargetSeqOne® Hyb Buffer v2: Provides a stable hybridization buffer system;

[0019] (2) Hyb Human Block: Human genome blocking sequence used to reduce non-specific binding of human background nucleic acids;

[0020] (3) TargetSeq® Blocking Oligo: a universal blocking oligonucleotide used to block probe sequences and prevent the adapter from binding nonspecifically to the probe;

[0021] (4) RNase inhibitors: protect RNA samples and RNA components in subsequent steps.

[0022] Furthermore, the detection kit may also include a complete or partial set of reagents for constructing an SFTSV genomic cDNA library and subsequent amplification to form a complete "library construction-capture-sequencing" solution, wherein the reagents comprise at least one of the following:

[0023] ①RNA fragmentation reagents: such as buffer solutions containing divalent cations, used to randomly fragment RNA;

[0024] ② Reverse transcription reaction reagents: including reverse transcriptase, random primers / oligo dT primers, dNTPs, buffer, etc., used to synthesize first-strand cDNA;

[0025] ③ Reagents for cDNA second-strand synthesis reaction: including DNA polymerase I, RNase H, dUTP / dNTPs, buffer, etc., used to synthesize second-strand cDNA and introduce dUTP labeling;

[0026] ④ Adapter ligation reagents: including Y-shaped adapters compatible with sequencing platforms, DNA ligase, buffers, etc., used to ligate sequencing adapters with indexes to both ends of cDNA fragments;

[0027] ⑤ Purification reagents: such as magnetic bead purification reagents, used for product purification between each step;

[0028] ⑥ PCR pre-reaction reagents: including PCR premix (PCR MasterMix) containing uracil-DNA glycosylase (UDG enzyme) and / or unique paired-end index primers (UDI primers), used to pre-amplify libraries with ligation adapters and introduce complete sequencing indexes;

[0029] ⑦ Post-capture PCR amplification reaction reagents: including Post PCR primers and Post PCR Master Mix, used to perform PCR amplification of the target DNA fragments after hybridization capture and elution in a limited number of cycles to obtain a sufficient final library for sequencing.

[0030] Thirdly, the present invention provides the application of the capture probe or the kit in whole-genome sequencing of Dabie Bandar virus.

[0031] Fourthly, this invention provides a method for detecting the full-length genome of the Dabie Bandar disease area. The method is based on capture probes with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 679 and next-generation sequencing technology. The method is a standardized and reproducible procedure. The method involves hybridizing the SFTSV genomic cDNA library of the sample to be tested with the capture probe set to obtain hybridization products. The hybridization products are separated by magnetic beads to obtain DNA fragments hybridized with the probes. The DNA fragments are then captured onto a template and amplified by PCR. The obtained PCR amplification products are subjected to next-generation sequencing. The sequencing results are analyzed by bioinformatics to obtain the full-length SFTSV genome.

[0032] Specifically, the method includes the following steps:

[0033] Step 1: Construction of the SFTSV genomic cDNA library

[0034] ① Sample processing and RNA extraction: Total RNA is extracted from clinical samples (such as patient serum, tick homogenate) or virus cultures and quantified;

[0035] ②RNA fragmentation: Take an appropriate amount of total RNA and treat it with RNA fragmentation reagent at high temperature (e.g., 94℃) for several minutes to randomly break it into fragments of appropriate length (e.g., ~200 nt).

[0036] ③ First-strand cDNA synthesis: Using fragmented RNA as a template, random primers and reverse transcriptase are added to synthesize first-strand cDNA.

[0037] ④ Second-strand cDNA synthesis: Add the second-strand synthesis reaction reagent to the first-strand synthesis product, and incorporate dUTP into the reaction system to label the second strand, thereby synthesizing double-stranded cDNA.

[0038] ⑤ End repair, A-tail addition and adapter ligation: The double-stranded cDNA is repaired by adding an A-tail to the 3' end, and then Y-shaped sequencing adapters with specific index sequences are ligated to both ends of the cDNA fragment.

[0039] ⑥ Pre-PCR Amplification and Purification: Using a PCR premix containing UDG enzyme and UDI primers, perform PCR amplification on the adapter-ligated library for a limited number of cycles (e.g., 8-12 cycles). After amplification, purify the library using magnetic beads to remove primer dimers and other impurities, and screen for libraries with the target fragment size. This yields a "pre-capture library" containing the SFTSV sequence (if available) and all background nucleic acids.

[0040] Step 2: Targeted Hybridization and Capture

[0041] ① Preparation of hybridization reaction system: Prepare the hybridization solution in a sterile, nuclease-free PCR tube according to the following system:

[0042] TargetSeqOne® Hyb Buffer v2: 13 µL

[0043] Hyb Human Block: 5 µL

[0044] TargetSeq® Blocking Oligo: 2 µL

[0045] RNase Block: 5 µL

[0046] Nuclease-Free Water: 3 µL

[0047] TargetSeq® Target Probes (i.e., the probe mixture of this invention): 2 µL

[0048] Pre-capture library DNA: After quantification, add an appropriate amount to make the total system 30 µL.

[0049] ② Hybridization reaction: Mix the above mixture thoroughly, centrifuge briefly, and then place it in a PCR instrument to run the hybridization program: First, incubate at 80℃ for 5 minutes to denature the DNA into single strands; then immediately cool down to 50℃ and incubate at this temperature for 12-18 hours to allow the probe to fully hybridize with the complementary SFTSV cDNA sequence in the library.

[0050] Step 3: Magnetic Bead Capture and Washing

[0051] ① Magnetic bead preparation: Take an appropriate amount of streptavidin magnetic beads, wash them with binding buffer and resuspend them.

[0052] ②Capture and Binding: Mix the hybridization reaction solution from step two with the prepared streptavidin magnetic beads and incubate by rotation at room temperature for 30-45 minutes. During this period, the streptavidin on the magnetic beads will bind specifically and with high affinity to the biotin labeled at the 5' end of the hybridization probe, thereby forming a "magnetic bead-streptavidin-biotin-probe-target cDNA" complex.

[0053] ③ Magnetic separation and washing: Place the reaction tube on a magnetic rack and carefully aspirate the supernatant after the solution has clarified. Retain the magnetic bead-complex. Wash the magnetic beads multiple times using a series of precisely formulated washing buffers (such as low-salt, high-salt, and SDS-containing buffers) to thoroughly remove unhybridized library DNA, non-specifically bound nucleic acids, and other impurities.

[0054] Step 4: Target elution and capture followed by PCR amplification

[0055] ① Target elution: Add low-salt elution buffer or sterile water to the cleaned magnetic beads, and incubate under heating conditions (such as 95℃) for several minutes to dissociate the captured SFTSV target cDNA from the probe-magnetic bead complex, and obtain DNA elution buffer enriched with SFTSV sequence.

[0056] ②PCR Amplification: Using the elution buffer as a template, prepare the post-capture PCR amplification system (e.g., 24 µL elution buffer, 1 µL PostPCR primers, 25 µL PostPCR Master Mix, total volume 50 µL). Run the amplification program on the PCR instrument (e.g., 95℃ for 1 minute; then 98℃ for 20 seconds, 60℃ for 30 seconds, 72℃ for 30 seconds, for 8-12 cycles; final extension at 72℃ for 5 minutes). This step aims to perform limited amplification of the trace-enriched target DNA to obtain a "final capture library" that meets the concentration and total amount requirements for sequencing.

[0057] Step 5: Library quality control and high-throughput sequencing

[0058] ① Library purification and quality control: Magnetic beads are used to purify the captured PCR products. The library is precisely quantified using a fluorometer or Qubit, and the fragment size distribution is detected using a bioanalyzer or fragment analyzer. The concentration and fragment size of a qualified library should meet the requirements of the selected sequencing platform (e.g., Illumina MiSeq, MGI DNBSEQ-T7, etc.).

[0059] ② Sequencing: The libraries of multiple samples are mixed in a certain proportion and paired-end sequencing is performed on a second-generation sequencing platform (such as PE150). The sequencing data is produced in FASTQ format.

[0060] Step Six: Bioinformatics Analysis and Genome Acquisition

[0061] Bioinformatics analysis was performed on the sequencing data to assemble the full-length SFTSV genome sequence. The workflow is as follows:

[0062] ① Raw data quality control: Use software such as Fastp to filter the raw sequencing data, remove low-quality bases, adapter sequences and excessively short reads, and obtain high-quality data (Clean reads).

[0063] ② Remove host background: Use alignment software such as Bowtie2 to align clean reads to the host (human, mouse, etc.) reference genome and collect reads that fail to align. This part is the suspected pathogen data after removing the host background.

[0064] ③ Sequence alignment and preliminary assembly: Using software such as BWA, the host-de-hosted reads were aligned to the SFTSV reference genome database. Simultaneously, assembly software such as MEGAHIT was used to perform de novo assembly of these reads to obtain contiguous sequences.

[0065] ④ Virus typing and reference genome determination: The assembled contiguous sequences are aligned again to the SFTSV typing reference sequence set. The alignment score determines the most likely genotype of the virus in the sample, and the representative sequence of this type is selected as the reference genome for subsequent analysis.

[0066] ⑤ Variation Detection and Genome Correction: Clean reads were precisely aligned to the reference genome identified in step 4 using BWA. Variation detection was performed using tools such as SAMtools and BCFtools to identify single nucleotide polymorphisms (SNPs) and insertions / deletions. Then, software such as iVar was used to filter and correct the detected variant sites based on sequencing depth and quality values, generating a high-quality variant list.

[0067] ⑥ Full-length genome generation: Based on the corrected variation information, the reference genome sequence is patched to generate an accurate full-length L, M, and S fragment genome sequence representing the SFTSV strain of the sample. This sequence can be used for subsequent evolutionary analysis, molecular typing, source tracing studies, etc.

[0068] (III) Beneficial Effects

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] 1. This invention achieves high coverage and high uniformity in genome capture, completely solving the coverage gap problem.

[0071] The probe set provided by this invention can achieve nearly 100% theoretical coverage of the full-length genome of the three SFTSV L, M, and S fragments. After capture, the sequencing depth (coverage) of each region is highly uniform, effectively avoiding the genome coverage gaps caused by amplification failures in existing Sanger sequencing and multiplex PCR methods.

[0072] Technical reasons:

[0073] ① Folded probe design: The 679 probes in this invention are arranged in a high-density folded pattern, achieving multi-layer coverage of the target region. Even if a mutation occurs in a certain part of the genome, causing a decrease in the binding efficiency of individual probes, adjacent redundant probes can still effectively capture the sequence in that region, greatly enhancing the fault tolerance of the technical solution.

[0074] ② Advantages of probe capture principle: Unlike primer extension, which relies on precise matching at the 3' end, long probe (100 nt) hybridization allows for a certain degree of mismatch within the probe without completely losing binding ability. This characteristic makes this invention more adaptable and versatile than primer-based methods when dealing with sequence variations among different SFTSV genotypes.

[0075] ③ Optimization of coverage uniformity: The probe design comprehensively evaluated factors such as GC content and thermodynamic stability to avoid probe "missing" or "accumulating" in complex regions such as high GC or high AT, ensuring that the entire genome is captured evenly, thereby obtaining sequencing data with uniform depth, laying the foundation for accurate variant frequency analysis.

[0076] 2. This invention significantly improves detection sensitivity and is suitable for samples with low viral load.

[0077] The method of this invention has extremely high detection sensitivity for low abundance SFTSV RNA and can successfully obtain the full-length genome sequence from clinical samples with extremely low viral copy numbers (such as serum from early patients and some tick samples), thus overcoming the bottleneck of low detection rate of traditional methods for low viral load samples.

[0078] Technical reasons:

[0079] ① Highly efficient targeted enrichment: This invention uses probe hybridization to capture and specifically enrich SFTSV sequences, which account for a very small percentage, from samples containing massive amounts of host background nucleic acids. This "background removal and target enrichment" process allows limited sequencing throughput to be concentrated on the viral genome, effectively amplifying the viral signal.

[0080] ② Complete process optimization: From RNA fragmentation and library construction to hybridization capture and washing, this invention provides a standardized kit and operating procedure, which minimizes nucleic acid loss between steps and optimizes the hybridization efficiency of low-concentration targets, thereby ensuring reliable sequencing data can be obtained from trace amounts of starting materials.

[0081] 3. This invention overcomes primer dependence limitations and possesses excellent new variant detection capabilities.

[0082] This invention does not rely on specific primer amplification, and therefore is completely unaffected by sequence mutations in the primer binding region. It can effectively detect and capture SFTSV strains carrying novel variants, providing a reliable tool for real-time viral evolution monitoring and epidemic early warning.

[0083] Technical reasons:

[0084] ① Broad spectrum of probe design: The probe design is based on the sequences of all known strains, covering most known variants. The shingled design itself also has the ability to buffer against unknown point mutations.

[0085] ② The inclusiveness of the capture principle: Even if a new variant is located in the binding region of a probe, the fragment can still be captured as long as it does not affect the overall hybridization. More importantly, the variant region can almost always be covered by other stacked probes, thus ensuring that the new variant site itself can be stably sequenced and detected, unlike PCR where primer failure can lead to the complete loss of information in that region.

[0086] 4. This invention significantly improves detection throughput and efficiency while reducing the overall cost per sample.

[0087] This invention supports high-throughput operations, enabling simultaneous parallel library construction and capture of dozens or even hundreds of samples. The experimental workflow is standardized and highly compatible with automation. Compared to Sanger sequencing, it offers efficiency improvements of tens of times; compared to mNGS, it reduces the required sequencing data volume by 1-2 orders of magnitude while achieving the same or higher viral genome coverage depth, resulting in a significant reduction in overall cost.

[0088] Technical reasons:

[0089] ① Integrated workflow: The kit integrates all the core reagents required for library construction and capture, simplifying experimental preparation.

[0090] ② High-throughput characteristics of hybridization capture: Libraries of multiple samples can be captured in the same batch of hybridization reactions, with standardized operating procedures.

[0091] ③ The economics of targeted sequencing: Since background sequences are effectively removed, the target sequence accounts for a very high proportion in the sequencing data. Therefore, high-depth viral genome data can be obtained without investing a large amount of sequencing throughput, saving valuable sequencing resources and costs.

[0092] 5. This invention yields high-quality data, directly supporting in-depth molecular epidemiological research.

[0093] The full-length SFTSV genome sequence obtained using the sequencing method of this invention is complete and accurate, and can be directly used for in-depth research such as high-resolution molecular typing, detailed phylogenetic analysis, precise tracing of transmission chains, and identification of virulence or antigenicity-related variant sites, providing key data support for precise prevention and control.

[0094] Technical reasons:

[0095] ① High depth and high uniformity ensure accurate detection of variants: High and uniform sequencing depth enables even low-frequency variants in the sample to be effectively identified, resulting in low false negative and false positive rates in variant detection.

[0096] ② Obtain the full-length sequence: This covers all coding and non-coding regions of the genome. The latter often contains important regulatory information, which is crucial for understanding the biological characteristics of viruses. This is a part that traditional targeted sequencing often ignores.

[0097] In summary, the technical solution of this invention, through the organic combination of innovative probe design and standardized targeted enrichment process, systematically solves the main technical obstacles faced by existing technologies in obtaining the full-length genome of SFTSV. It has achieved significant improvements in multiple key indicators such as coverage, sensitivity, universality, throughput, cost-effectiveness, and data quality, providing a powerful and practical technical tool for molecular typing, variant monitoring, transmission chain tracing, and vaccine development of SFTSV. Attached Figure Description

[0098] Figure 1 This is a schematic diagram of the SFTSV probe design scheme.

[0099] Figure 2 This is a schematic diagram of the SFTSV whole genome capture sequencing method.

[0100] Figure 3 This is a schematic diagram of the SFTSV whole genome capture sequencing data analysis workflow.

[0101] Figure 4A bar chart showing the statistical results of SFTSV genome capture alignment rate and coverage.

[0102] Figure 5 This is a map showing the genomic coverage depth of the three SFTSV L, M, and S segments. Detailed Implementation

[0103] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0104] Example 1

[0105] Design and acquisition of capture probes targeting the full-length genome of SFTSV (L, M, and S segments)

[0106] The capture probe set consists of probes with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 679. Each probe is a single-stranded DNA oligonucleotide, preferably 100 nucleotides (nt) in length. To facilitate the subsequent separation and enrichment of the hybridization complex, the 5' or 3' end of the capture probe is preferably labeled with biotin.

[0107] Probe Design Fundamentals:

[0108] The probe set of this invention is designed based on the complete L, M, and S fragment genome sequences of all publicly available SFTSV strains (covering known genotypes such as AF) collected from public databases such as NCBI GenBank and GISAID, and is performed after comparative analysis. The probe design strategy is as follows: Figure 1 As shown.

[0109] Probe design strategy:

[0110] ① Sequence collection: Collect all available reference sequences and representative variant sequences of SFTSV to build a local reference sequence database.

[0111] ② Shingled Probe Design: A shingled probe placement strategy is employed. Specifically, candidate probe sequences are generated by sliding across all the aforementioned reference sequences in 10-nucleotide increments, ensuring that each segment of the target genome region is covered by multiple probes (i.e., multiple layers). This design greatly enhances the tolerance to point mutations, insertions, deletions, and other variations that may exist in the genome, ensuring that even if a single or multiple base variations occur in the probe binding region, they can still be effectively captured by adjacent probes.

[0112] ③ Thermodynamic Optimization: The thermodynamic stability (e.g., melting temperature Tm) of each candidate probe was calculated to avoid using probe sequences that easily form strong secondary structures (e.g., hairpins, dimers), ensuring that the probes have optimal binding specificity and efficiency under hybridization conditions. Using the appropriate probe hybridization buffer system as the calculation benchmark, the target melting temperature Tm of the probe was set at 68±5 ℃, with hairpin structure free energy ΔG > −9 kcal / mol and probe dimer ΔG > −10 kcal / mol as screening thresholds. A large number of original candidate probes were generated using a sliding step of 10 nt. After batch calculation of thermodynamic parameters, strong secondary structures, Tm deviations from the threshold, and highly interacting sequences were eliminated. For genomic segments with probe gaps after screening, the truncation sites were slightly shifted, and the sequence base composition was fine-tuned to recalculate the thermodynamic parameters, ensuring continuous shingled coverage. After sequence shift optimization, the Tm of the finished probes was uniformly narrowed to the range of 68.2–72.8 ℃, with no strong hairpins or strong probe dimers.

[0113] ④ Specificity assessment: The designed probe sequence is compared with the host genome database (such as human, mouse, tick and other potential hosts of SFTSV) to ensure that there are no probes with high homology with the host genome, so as to minimize the background of non-specific hybridization.

[0114] ⑤ Redundancy Removal and Merging: All candidate probes were clustered and redundancy removed, identical sequences were eliminated, and highly similar probes were merged and optimized to generate a final combination of 679 highly specific and high-coverage probes. Evaluation and theoretical calculations show that this probe set theoretically covers nearly 100% of all SFTSV sequences in the database.

[0115] ⑥ Synthesis and Modification: The above 796 probe sequences were synthesized by a professional oligonucleotide synthesis company using a high-throughput electrochemical synthesis method, and biotin was introduced to modify the probe ends during the synthesis process. After passing quality inspection, the synthesized probes were mixed in equimolar amounts to prepare a ready-to-use TargetSeq® Target Probes mixture.

[0116] Example 2

[0117] A detection kit for the full-length genome of Dabie Bandar virus, the kit comprising the following components:

[0118] ①Capture probe: A mixed probe solution containing the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 796 (TargetSeq® Target Probes).

[0119] ②Streptavidin-labeled magnetic beads: used to bind to biotin on the capture probe, thereby separating the probe-target DNA complex from the reaction system by magnetic separation technology.

[0120] ③ Library hybridization reaction reagents: used to create an optimal probe-target hybridization environment, ensuring high specificity and efficiency of hybridization. The reagents are:

[0121] (1) TargetSeqOne® Hyb Buffer v2: Provides a stable hybridization buffer system.

[0122] (2) Hyb Human Block: Human genome blocking sequence used to reduce non-specific binding of human background nucleic acids.

[0123] (3) TargetSeq® Blocking Oligo: a universal blocking oligonucleotide used to block probe sequences and prevent the adapter from binding nonspecifically to the probe.

[0124] (4) RNase inhibitors: protect RNA samples and RNA components in subsequent steps.

[0125] Furthermore, the kit includes a complete or partial set of reagents required for constructing an SFTSV genomic cDNA library and subsequent amplification, forming a complete "library construction-capture-sequencing" solution. These reagents include:

[0126] ①RNA fragmentation reagents: such as buffer solutions containing divalent cations, used to randomly fragment RNA.

[0127] ② Reverse transcription reaction reagents: including reverse transcriptase, random primers / oligo dT primers, dNTPs, buffer, etc., used to synthesize first-strand cDNA.

[0128] ③ cDNA second-strand synthesis reaction reagents: including DNA polymerase I, RNase H, dUTP / dNTPs, buffer, etc., used to synthesize second-strand cDNA and introduce dUTP labeling.

[0129] ④ Adapter ligation reagents: including Y-shaped adapters compatible with sequencing platforms, DNA ligase, buffers, etc., used to ligate sequencing adapters with indexes to both ends of cDNA fragments.

[0130] ⑤ Purification reagents: such as magnetic bead purification reagents, used for product purification between each step.

[0131] ⑥ PCR pre-reaction reagents: including PCR MasterMix containing uracil-DNA glycosylase (UDG enzyme) and / or unique paired-end index primers (UDI primers), used to pre-amplify the library containing the ligator and introduce the complete sequencing index.

[0132] ⑦ Post-capture PCR amplification reaction reagents: including Post PCR primers and Post PCR Master Mix, used to perform PCR amplification of the target DNA fragments after hybridization capture and elution in a limited number of cycles to obtain a sufficient final library for sequencing.

[0133] Example 3

[0134] A method for detecting the full-length genome of Dabie Bandar virus using the capture probe described in Example 1 or the kit described in Example 2 combined with next-generation sequencing, the flowchart of which is shown below. Figure 2 As shown.

[0135] 1 Method

[0136] Step 1: Construction of the SFTSV genomic cDNA library

[0137] ① Sample processing and RNA extraction: Total RNA was extracted from clinical samples (such as patient serum, tick homogenate) or virus cultures and quantified.

[0138] ②RNA fragmentation: Take an appropriate amount of total RNA and treat it with RNA fragmentation reagent at high temperature (e.g., 94℃) for several minutes to randomly break it into fragments of appropriate length (e.g., ~200 nt).

[0139] ③ First-strand cDNA synthesis: Using fragmented RNA as a template, random primers and reverse transcriptase are added to synthesize first-strand cDNA.

[0140] ④ Second-strand cDNA synthesis: Add the second-strand synthesis reaction reagent to the first-strand synthesis product, and incorporate dUTP into the reaction system to label the second strand, thereby synthesizing double-stranded cDNA.

[0141] ⑤ End repair, A-tail addition and adapter ligation: The double-stranded cDNA is repaired by adding an A-tail to the 3' end, and then Y-shaped sequencing adapters with specific index sequences are ligated to both ends of the cDNA fragment.

[0142] ⑥ Pre-PCR Amplification and Purification: Using a PCR premix containing UDG enzyme and UDI primers, perform PCR amplification on the adapter-ligated library for a limited number of cycles (e.g., 8-12 cycles). After amplification, purify the library using magnetic beads to remove primer dimers and other impurities, and screen for libraries with the target fragment size. This yields a "pre-capture library" containing the SFTSV sequence (if available) and all background nucleic acids.

[0143] Step 2: Targeted Hybridization and Capture

[0144] ① Preparation of hybridization reaction system: Prepare the hybridization solution in a sterile, nuclease-free PCR tube according to the following system:

[0145] TargetSeqOne® Hyb Buffer v2: 13 µL

[0146] Hyb Human Block: 5 µL

[0147] TargetSeq® Blocking Oligo: 2 µL

[0148] RNase Block: 5 µL

[0149] Nuclease-Free Water: 3 µL

[0150] TargetSeq® Target Probes (i.e., the probe mixture of this invention): 2 µL

[0151] Pre-capture library DNA: After quantification, add an appropriate amount to make the total system 30 µL.

[0152] ② Hybridization reaction: Mix the above mixture thoroughly, centrifuge briefly, and then place it in a PCR instrument to run the hybridization program: First, incubate at 80℃ for 5 minutes to denature the DNA into single strands; then immediately cool down to 50℃ and incubate at this temperature for 12-18 hours to allow the probe to fully hybridize with the complementary SFTSV cDNA sequence in the library.

[0153] Step 3: Magnetic Bead Capture and Washing

[0154] ① Magnetic bead preparation: Take an appropriate amount of streptavidin magnetic beads, wash them with binding buffer and resuspend them.

[0155] ②Capture and Binding: Mix the hybridization reaction solution from step two with the prepared streptavidin magnetic beads and incubate by rotation at room temperature for 30-45 minutes. During this period, the streptavidin on the magnetic beads will bind specifically and with high affinity to the biotin labeled at the 5' end of the hybridization probe, thereby forming a "magnetic bead-streptavidin-biotin-probe-target cDNA" complex.

[0156] ③ Magnetic separation and washing: Place the reaction tube on a magnetic rack and carefully aspirate the supernatant after the solution has clarified. Retain the magnetic bead-complex. Wash the magnetic beads multiple times using a series of precisely formulated washing buffers (such as low-salt, high-salt, and SDS-containing buffers) to thoroughly remove unhybridized library DNA, non-specifically bound nucleic acids, and other impurities.

[0157] Step 4: Target elution and capture followed by PCR amplification

[0158] ① Target elution: Add low-salt elution buffer or sterile water to the cleaned magnetic beads, and incubate at 95°C for several minutes under heating conditions to dissociate the captured SFTSV target cDNA from the probe-magnetic bead complex, obtaining a DNA elution buffer enriched with the SFTSV sequence.

[0159] ②PCR Amplification: Using the elution buffer as a template, prepare the post-capture PCR amplification system (PCR amplification system: 24 µL elution buffer, 1 µL Post PCR primers, 25 µL Post PCR Master Mix, total volume 50 µL). Run the amplification program on the PCR instrument (amplification program: 95℃ for 1 minute; then 98℃ for 20 seconds, 60℃ for 30 seconds, 72℃ for 30 seconds, cycle 8-12; final extension at 72℃ for 5 minutes). This step aims to perform limited amplification of the trace-enriched target DNA to obtain the "final capture library" that meets the concentration and total amount requirements for sequencing.

[0160] Step 5: Library quality control and high-throughput sequencing

[0161] ① Library purification and quality control: Magnetic beads are used to purify the captured PCR products. The library is precisely quantified using a fluorometer or Qubit, and the fragment size distribution is detected using a bioanalyzer or fragment analyzer. The concentration and fragment size of a qualified library should meet the requirements of the selected sequencing platform (e.g., Illumina MiSeq, MGI DNBSEQ-T7, etc.).

[0162] ② Sequencing: Libraries from multiple samples are mixed in a certain proportion and paired-end sequencing is performed on a next-generation sequencing platform (such as PE150). Sequencing data are generated in FASTQ format.

[0163] Step Six: Bioinformatics Analysis and Genome Acquisition

[0164] Bioinformatics analysis was performed on the sequencing data to assemble the full-length SFTSV genome sequence. The workflow is as follows:

[0165] ① Raw data quality control: Use software such as Fastp to filter the raw sequencing data, remove low-quality bases, adapter sequences and excessively short reads, and obtain high-quality data (Clean reads).

[0166] ② Remove host background: Use alignment software such as Bowtie2 to align clean reads to the host (human, mouse, etc.) reference genome and collect reads that fail to align. This part is the suspected pathogen data after removing the host background.

[0167] ③ Sequence alignment and preliminary assembly: Using software such as BWA, the host-de-hosted reads were aligned to the SFTSV reference genome database. Simultaneously, assembly software such as MEGAHIT was used to perform de novo assembly of these reads to obtain contiguous sequences.

[0168] ④ Virus typing and reference genome determination: The assembled contiguous sequences are aligned again to the SFTSV typing reference sequence set. The alignment score determines the most likely genotype of the virus in the sample, and the representative sequence of this type is selected as the reference genome for subsequent analysis.

[0169] ⑤ Variation Detection and Genome Correction: Clean reads were precisely aligned to the reference genome identified in step 4 using BWA. Variation detection was performed using tools such as SAMtools and BCFtools to identify single nucleotide polymorphisms (SNPs) and insertions / deletions. Then, software such as iVar was used to filter and correct the detected variant sites based on sequencing depth and quality values, generating a high-quality variant list.

[0170] ⑥ Full-length genome generation: Based on the corrected variation information, the reference genome sequence is patched to generate an accurate full-length L, M, and S fragment genome sequence representing the SFTSV strain of the sample; this sequence can be used for subsequent evolutionary analysis, molecular typing, source tracing studies, etc.

[0171] Example 4

[0172] This embodiment uses the method described in Example 3 to perform whole-genome sequencing on multiple SFTSV-positive real clinical samples to evaluate the total read mapping rate, coverage rate, and coverage uniformity (30X Coverage rate) of the capture sequencing protocol for actual clinical samples, thus verifying the clinical applicability and stability of the present invention.

[0173] 1. Sample Source

[0174] This experiment selected 5 SFTSV-positive clinical samples with different viral loads. All samples were identified as positive by quantitative real-time PCR.

[0175] 2 methods

[0176] (I) Construction of whole genome library:

[0177] (1) RNA was extracted from the sample using the QIAamp Viral RNA Mini kit (catalog number 52906, Qiagen GmbH, Germany). The RNA concentration was quantified using the Qubit RNA HS assay kit and the Qubit Fluorometer (LifeTechnologies, USA).

[0178] (2) RNA fragmentation:

[0179] Beforehand, remove the RNA sample from the -80℃ freezer. Beforehand, remove the Fast First Strand Buffer and Fast Frag Buffer from the IGT® Fast Stranded RNA Library Prep Kit v2.0 (purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd., catalog number: C10032) from the -20℃ freezer, place them on an ice box to thaw, briefly vortex to mix and then centrifuge briefly. Place them on an ice box for later use. The fragmentation reaction system is: 13 μL of RNA sample and 4 μL of Fast Frag Buffer. After adding the sample, use a pipette to mix or vortex to mix, and then centrifuge briefly. Set the PCR instrument parameters as follows: 105℃ hot cap temperature, 94℃ for 7 min, and 4℃ incubation. Place the reaction solution on the PCR instrument and run the program. When the PCR instrument temperature drops to 4℃, remove the PCR tube and centrifuge briefly to obtain fragmented RNA. Proceed to step (3) immediately.

[0180] (3) Reverse transcription:

[0181] Beforehand, remove the Fast First Strand Buffer from the IGT® Fast Stranded RNA Library Prep Kit v2.0 (purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd., catalog number: C10032) from the -20℃ freezer, place it on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly. Place it on an ice box for later use. Remove the Fast First Strand Enzyme from the kit from the -20℃ freezer, invert to mix, and then centrifuge briefly. Place it on an ice box for later use. Prepare the reaction mixture on the ice box: 17 μL fragmented RNA, 6 μL Fast First Strand Buffer, and 2 μL Fast First Strand Enzyme. After preparation, pipette and mix (avoid vigorous shaking), then centrifuge briefly. Set the PCR instrument parameters as follows: 105℃ hot lid temperature, 25℃ for 10 min, 42℃ for 15 min, and 70℃... Keep warm at 4℃ for 15 min, place the reaction solution on the PCR instrument, run the program, and when the temperature of the PCR instrument drops to 4℃, take out the PCR tube, centrifuge briefly to obtain cDNA, and immediately proceed to step (4).

[0182] (4) cDNA double strand synthesis, addition of "A" to the 3' end:

[0183] Beforehand, remove the Fast Second Strand Buffer with dUTP from the IGT® Fast Stranded RNA Library Prep Kit v2.0 (purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd., catalog number: C10032) from the -20℃ freezer, place it on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly. Place it on an ice box for later use. Remove the Fast Second Strand Enzyme from the -20℃ freezer, invert to mix, and then centrifuge briefly. Place it on an ice box for later use. Prepare the reaction mixture on the ice box: 25 μL cDNA, 30 μL Fast Second Strand Buffer with dUTP, and 5 μL Fast Second Strand Enzyme. After preparation, use a pipette to mix (avoid vigorous shaking), briefly centrifuge, and set the PCR instrument parameters as follows: 105℃ hot lid temperature, 16℃ for 30 min, and 72℃... Keep warm at 4℃ for 15 min, place the reaction solution on the PCR instrument, run the program, and when the temperature of the PCR instrument drops to 4℃, take out the PCR tube, centrifuge briefly, and immediately proceed to step (5).

[0184] (5) Connector connection:

[0185] Take the Adapter (purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd., catalog number: C10042) out of the -20℃ freezer in advance, place it on an ice box to thaw, briefly vortex to mix and then centrifuge briefly, and place it on an ice box for later use; according to the amount of RNA to be added for library construction, dilute the Adapter (15μM) to 1.5μM in advance, take the Fast Ligation Buffer from the IGT® FastStranded RNA Library Prep Kit v2.0 (purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd., catalog number: C10032) out of the -20℃ freezer in advance, place it on an ice box to thaw, briefly vortex to mix and then centrifuge briefly, and place it on an ice box for later use; take the Fast Ligase Mix out of the -20℃ freezer, invert to mix and then centrifuge briefly, and place it on an ice box for later use; prepare the reaction system on the ice box: Step (4) reaction end 60μL sample, 30μL Fast Ligation Buffer, Fast Ligase Mix 5 μL of Adapter and 5 μL of PCR tube are prepared and mixed by pipetting (avoid vigorous shaking). Centrifuge briefly and set the PCR instrument parameters (with the hot cap closed) to 20℃ for 15 min and 4℃ for incubation. Place the PCR tube on the PCR instrument and run the program. When the temperature of the PCR instrument drops to 4℃, remove the PCR tube, centrifuge briefly, and immediately proceed to step (6).

[0186] (6) Purification after ligation:

[0187] Prepare an 80% ethanol-water solution in advance using anhydrous ethanol and nuclease-free water, and keep it at room temperature for later use (use freshly prepared 80% ethanol-water solution for magnetic bead purification if possible). The magnetic beads used for purification are IGT® Pure Beads (purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd., catalog number: C80662). Take the purified magnetic beads out of the 4℃ refrigerator in advance, mix them well and equilibrate at room temperature for 30 min. Vortex the purified magnetic beads that have been equilibrated to room temperature for later use. Add 0.45 times the volume (45 μL) of purified magnetic beads to 100 μL of reaction solution after step (5), vortex to mix, and let stand at room temperature for 5 min. Centrifuge briefly, place the PCR tube on a magnetic rack for 3 min, and wait for the solution to become clear. Keep the PCR tube on the magnetic rack, carefully remove the supernatant, and add 200 μL of ethanol to the PCR tube. Add 80% ethanol aqueous solution and let stand for 30 seconds; keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL of 80% ethanol aqueous solution to the PCR tube again, let stand for 30 seconds, discard the supernatant; cap the tube, centrifuge briefly to remove the residual ethanol to the bottom of the tube, place it on the magnetic rack, and carefully use a 10 μL pipette to remove the residual ethanol at the bottom, being careful not to pick up the magnetic beads; keep the PCR tube on the magnetic rack and let stand at room temperature for 4 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely; add 22 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, vortex to mix, and let stand at room temperature for 2 minutes; centrifuge briefly, place the PCR tube on the magnetic rack for 2 minutes, and wait for the solution to become clear; use a pipette to draw 20 μL of supernatant, transfer it to a new PCR tube, label it, and proceed to step (7).

[0188] (7) Pre-PCR reaction:

[0189] Beforehand, remove the PCR Master Mix with UDG from the IGT® Fast Stranded RNA Library Prep Kit v2.0 (purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd., catalog number: C10032) from the -20℃ freezer, place it on an ice box to thaw, mix by inversion and centrifuge briefly, and place it on an ice box for later use; beforehand, remove the UDI Primer from the kit from the -20℃ freezer, place it on an ice box to thaw, mix briefly by vortex and centrifuge briefly, and place it on an ice box for later use; prepare the PCR reaction solution on the ice box: 20μL of the sample from step (6) after the reaction, 25μL of PCR Master Mix with UDG, and 5μL of UDI Primer. Record the index number used. After preparation, use a pipette to mix (avoid vigorous shaking), centrifuge briefly, and set the PCR program as follows: 105℃ hot lid temperature; 98℃ 1min; 98℃ 10s, 60℃ 30s, 72℃ 30s, 11 cycles; 72℃ for 5min; keep warm at 4℃, place the PCR tube on the PCR instrument, run the program, when the temperature of the PCR instrument drops to 4℃, take out the PCR tube, centrifuge briefly, and immediately proceed to step (8).

[0190] (8) Purification after PCR amplification:

[0191] Prepare an 80% ethanol-water solution in advance using anhydrous ethanol and nuclease-free water, and keep it at room temperature for later use (use freshly prepared 80% ethanol-water solution for magnetic bead purification if possible). The magnetic beads used for purification are IGT® Pure Beads (purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd., catalog number: C80662). Take the purified magnetic beads out of the 4℃ refrigerator in advance, mix them well and keep them at room temperature for 30 min. Vortex the purified magnetic beads that have been equilibrated to room temperature and keep them for later use. Add 0.9 times the volume of purified magnetic beads (45 μL) to the 50 μL reaction solution after step (7), vortex and mix well, and let stand at room temperature for 5 min. Centrifuge briefly, place the PCR tube on a magnetic rack for 3 min, and wait for the solution to become clear. Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 200 μL of 80% ethanol-water solution to the PCR tube, and let stand for 30 s. Keep the PCR tube on the magnetic rack, remove the supernatant, and add another 200 μL of ethanol-water solution to the PCR tube. Prepare an 80% ethanol aqueous solution, let it stand for 30 seconds, and discard the supernatant. Cap the tube and centrifuge briefly to remove residual ethanol to the bottom. Place the PCR tube on a magnetic rack and carefully use a 10 μL pipette to remove any remaining ethanol from the bottom, being careful not to aspirate the magnetic beads. Keep the PCR tube on the magnetic rack and let it stand at room temperature for 5 minutes to allow the magnetic beads to dry completely and the residual ethanol to evaporate. Add 30 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, vortex to mix, and let it stand at room temperature for 2 minutes. Centrifuge briefly, and place the PCR tube on the magnetic rack for 2 minutes until the solution becomes clear. Use a pipette to transfer 28 μL of the supernatant to a new PCR tube and label it. Take 1 μL of the library and use the Qubit dsDNA HS Assay Kit reagent to determine the library concentration on a Qubit 4.0 Fluorometer, recording the concentration. Take 1 μL of the sample and use a fragment analyzer to determine the fragment length.

[0192] (ii) Liquid-phase hybridization capture

[0193] All reagents used in this step were purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd.

[0194] (1) Preparations for the hybridization capture experiment:

[0195] Remove the Hyb Human Block and RNase Block from the -20°C freezer, place them on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly before storing on an ice box. Remove the matching TargetSeq® Blocking Oligo from the -20°C freezer, place it on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly before storing on an ice box. Remove the capture probe designed in Example 1 from the -80°C freezer, place it on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly before storing on an ice box. Remove the whole genome library to be used for hybridization capture from the -20°C freezer, place it on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly before storing on an ice box. Remove the TargetSeq One® Hyb Buffer v2, thaw it at room temperature, briefly vortex to mix, and then centrifuge briefly (if there is precipitation, the TargetSeq One® Hyb Buffer v2 needs to be heated in a 37°C water bath until the reagent is completely dissolved before use).

[0196] (2) Hybridization of whole genome library with capture probe:

[0197] When hybridizing a single library, add 750 ng of the library to a PCR tube and label it. Place the PCR tube in a vacuum centrifuge, open the PCR tube cap, and concentrate until dry. After the library is concentrated, prepare the hybridization reaction solution: TargetSeq One® Hyb Buffer v2 13 μL, Hyb Human Block 5 μL, TargetSeq® Blocking Oligo 2 μL, Nuclease-Free Water 8 μL, and TargetSeq® DNA Probes HT (i.e., the probe mixture of this invention) 2 μL. After preparation, add the hybridization reaction solution to the dry library, vortex for 30 seconds to ensure that the DNA at the bottom of the tube is dissolved, and then briefly centrifuge. Set the PCR instrument parameters as follows: 85℃ hot cap temperature, 80℃ for 5 min, and 50℃ for 12 h of incubation. Place the hybridization reaction solution on the PCR instrument and run the program. The hybridization time is 12 h. Perform step (3) 30 min before the program ends.

[0198] (3) Preparations before the capture experiment:

[0199] Remove TargetSeq® Cap Beads from the 4°C freezer beforehand, mix thoroughly, and equilibrate at room temperature for 30 min. Take out Wash Buffer 1 (if precipitation occurs, heat Wash Buffer 1 in a 37°C water bath until the precipitate is completely dissolved before use). Take out TargetSeq One® Wash Buffer 2 v2 and preheat it in a 50°C water bath. Add 50 µL of TargetSeq® Cap Beads to a new PCR tube, place it on a magnetic rack for 1 min, and discard the supernatant after the solution becomes clear. Remove the PCR tube from the magnetic rack, add 180 µL of Binding Buffer, and mix by pipetting or vortexing to resuspend the magnetic beads. After brief centrifugation, place the PCR tube on a magnetic rack for 1 min, and discard the supernatant after the solution becomes clear. Repeat the Binding Buffer resuspension, centrifugation, and supernatant discarding process twice, washing the magnetic beads three times in total with Binding Buffer. Remove the PCR tube from the magnetic rack, add 180 µL of Binding Buffer... Mix the buffer by suction or vortexing, and immediately proceed to step (4).

[0200] (4) Target region DNA capture:

[0201] Keep the hybridization product from step (2) on the PCR instrument. Add 180 μL of TargetSeq® CapBeads prepared in step (3) to the hybridization product and mix well by aspiration. Cap the tube and remove it from the PCR instrument. Place it on a vertical spin mixer at 8 rpm and incubate at room temperature for 30 min. Remove the PCR tube, centrifuge briefly, and place it on a magnetic rack for 2 min. After the solution becomes clear, discard the supernatant. Remove the PCR tube from the magnetic rack, add 150 µL of Wash Buffer 1 to the PCR tube, and gently aspirate to resuspend the magnetic beads. Replace the cap and place the tube on a vertical spin mixer at room temperature for 15 min at 8 rpm. Remove the PCR tube, centrifuge briefly, and place it on a magnetic rack for 2 min. After the solution becomes clear, discard the supernatant. Remove the PCR tube from the magnetic rack and add 150 µL of TargetSeq One® Wash Buffer 2 preheated to 50°C. v2, gently pipette and mix, briefly centrifuge, place on a metal bath, incubate at 50℃ for 10 min; remove the PCR tube, briefly centrifuge, place on a magnetic rack for 2 min, and discard the supernatant after the solution becomes clear; repeat the steps of adding TargetSeq One® Wash Buffer 2 v2 for incubation, centrifugation, and discarding the supernatant twice, for a total of three washes of the magnetic beads with TargetSeq One® Wash Buffer 2 v2 at 50℃; keep the PCR tube on the magnetic rack, add 200µL of 80% ethanol aqueous solution to the PCR tube, let stand for 30 s, then completely discard the ethanol solution, and air dry the magnetic beads at room temperature to allow the residual ethanol to evaporate completely; add 24µL of Nuclease-Free Water to the PCR tube, remove the PCR tube from the magnetic rack, briefly vortex to mix and resuspend the magnetic beads, and perform the capture-after-PCR amplification reaction.

[0202] (III) Post-capture PCR amplification:

[0203] All reagents used in this step were purchased from Aijitaikang Biotechnology (Beijing) Co., Ltd.

[0204] (1) Take the Post PCR Master Mix and Post PCR Primer out of the -20℃ freezer in advance, place them on an ice box to thaw, and then temporarily store them on an ice box. Briefly vortex the Post PCR Master Mix and Post PCR Primer, and then centrifuge them briefly. Prepare the PCR reaction solution: 24 μL of magnetic bead suspension, 1 μL of Post PCR Primer, and 25 μL of Post PCR Master Mix. After preparation, use a pipette to mix them thoroughly. After mixing, quickly transfer them to the PCR instrument and set the PCR instrument program as follows: 105℃ hot lid temperature; 95℃ 1 min; 98℃ 20s, 60℃ 30s, 72℃ 30s, 10 cycles; 72℃ 5 min; 4℃ incubation. Place the PCR reaction solution on the PCR instrument and run the program. After the program is completed, proceed to the next step of magnetic bead purification.

[0205] (2) Purification after amplification: Take out the purified magnetic beads, mix well and equilibrate at room temperature for 30 min. Add 1.1 times the volume of magnetic beads (55 μL) to the PCR product in step (1), vortex to mix well, and let stand at room temperature for 5 min; centrifuge briefly, and place the PCR tube on a magnetic rack for 3 min until the solution is clear; keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL of 80% ethanol aqueous solution to the PCR tube, and let stand for 30 s; keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL of 80% ethanol aqueous solution to the PCR tube again, let stand for 30 s and then completely discard the supernatant; keep the PCR tube on the magnetic rack, let stand at room temperature for 3 min, and air dry the magnetic beads to allow the residual ethanol to evaporate completely; add 25 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, vortex to mix, and let stand at room temperature for 2 min; briefly centrifuge, place the PCR tube on the magnetic rack for 2 min, and wait for the solution to clarify; use a pipette to draw 23 μL of supernatant and transfer it to a new PCR tube to obtain the capture library. Store the capture library in a -20℃ freezer. The capture library can be stored in a -20℃ freezer for one month; take 1 μL of the capture library and use the Qubit dsDNA HS Assay Kit reagent to determine the library concentration on a Qubit 4.0 Fluorometer, and record the library concentration; take 1 μL of the library and use a fragment analyzer for fragment quality control.

[0206] (iv) Sequencing on the instrument:

[0207] One μL of the captured library was taken and quantified using the Qubit ds DNA HS Assay Kit, and the library concentration was recorded. Another 1 μL of the sample was taken and fragment length was determined using the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit). The library length was between 250 bp and 400 bp. The probe hybridization captured library was sequenced using the NovaSeq 6000 (Illumina) sequencing platform to obtain the raw sequencing data.

[0208] (V) Analysis of raw data from SFTSV whole-genome capture sequencing

[0209] The raw sequencing data may contain a small number of reads containing adapter information, low-quality bases, etc. To ensure the quality of information analysis, the raw reads need to be initially filtered to obtain clean reads. Subsequent analyses are based on clean reads. The data filtering mainly involves the following: using an 8bp sliding window to cut off sequences with an average base quality value of less than 20; removing the adapter sequence at the end of the sequence; if the first or last base of the sequence is less than 20, the base will be cut off directly; generally, if the remaining sequence length is less than 40 (paired ends) after the removal is completed, the sequence pair is discarded.

[0210] Removing host sequences: Since the sequencing data may contain residual host genome sequences, we first use Bowtie2 sequencing data to align with the host reference genome to obtain the sequences that could not be aligned (unmapR1 & unmapR2). These unmapped sequences are the viral sequence information that we may actually need.

[0211] Viral genome alignment: The unmapped sequences were aligned to the viral genome using bwa software (with the designed reference species sequence information as the alignment template). The reads aligned to the genome were assembled into contig sequences using MEGAHIT software. The contig sequences were then aligned to the host genome using blastn software. The host sequence was removed again to obtain the non-human contig file. The non-human contig sequence was then aligned to the viral genome sequence to obtain the possible viral typing and sorting results-virus.txt.

[0212] To obtain the full-length SFTSV virus genome sequence: The result-virus-top1 genotyping sequence, which ranks first in the possible virus genotyping ranking, was used as the reference genome. Clean reads were aligned to this reference genome using bwa software to obtain the aligned bam file. Mutation analysis was performed using samtools software to obtain the variant sites. The variant sites were then corrected using iVar software to obtain the final full-length SFTSV virus genome sequence.

[0213] The SFTSV whole genome capture sequencing raw data analysis workflow is as follows: Figure 3 As shown.

[0214] 3 Results

[0215] To intuitively evaluate the sequencing capture effect of this scheme, this experiment verified the effect through multi-dimensional indicators, including sequence alignment rate, overall genome coverage of the sample, 30× effective coverage ratio, and sequencing depth distribution of each region of the SFTSV whole genome. The corresponding detection results are presented through bar charts of alignment rate and coverage, genome coverage depth distribution map and sample sequencing data statistics table.

[0216] 3.1 Statistical Analysis of High-Depth Coverage Effect

[0217] Figure 4 A bar chart shows the effective coverage of the SFTSV whole genome at 30× for each clinical sample. Sequencing depths of 30× and above are the core threshold for ensuring the accuracy of viral genome mutation detection and sequence assembly, directly determining the reliability and validity of the genome sequence. Statistical results show that the proportion of SFTSV whole genome coverage at 30× in all tested clinical samples was at a high level (reaching 99.96%). The high-depth coverage effect was stable for samples with different viral loads, and even low viral load clinical samples could achieve efficient capture and high-depth coverage without significant sample differences. This demonstrates that the probe set of this invention has strong compatibility, stable capture efficiency, and excellent adaptability to complex clinical samples, effectively solving the technical problems of incomplete viral genome coverage and insufficient depth in low viral load samples.

[0218] Figure 5This is a map showing the sequencing coverage depth distribution of the entire SFTSV genome in a typical clinical sample. As can be seen from the figure, after capture sequencing using the capture probes with the optimized shingled design of this invention, the entire L, M, and S fragments of the SFTSV genome are effectively and continuously covered without any breaks, with no significant gaps or extreme differences in depth. Compared to the problems of localized coverage loss, insufficient coverage of hypervariable regions, and uneven depth redundancy in conserved regions that exist in traditional amplicon sequencing, this approach achieves balanced capture of both hypervariable and conserved regions of the viral genome. The sequencing depth fluctuates smoothly across the entire genome, with no significant enrichment or deletion sites, demonstrating excellent sequencing coverage uniformity. Furthermore, the average sequencing depth of each fragment is above 5000x, enabling complete coverage of the variable regions of the entire SFTSV genome and accurate capture of various viral mutation sites, providing comprehensive genomic data support for viral typing and mutation tracing.

[0219] 3.2 Summary and Analysis of Core Sequencing Data from Samples

[0220] Table 1 presents the core sequencing data of multiple real clinical SFTSV-positive samples, including key parameters such as viral load (CT value), Clean Data, viral sequence alignment rate, whole genome coverage, average sequencing depth, and 30X coverage. The data results show that after capture and sequencing using this protocol, all clinical samples exhibited high effective viral sequence alignment rates, thorough removal of host background interference, and SFTSV whole genome coverage exceeding 99.5%, with some samples achieving 100% whole genome coverage. Furthermore, the sequencing depth was uniform among samples, data reproducibility was good, and there were no significant differences in core sequencing indicators. This fully demonstrates that the capture probe set and sequencing system designed in this invention can stably achieve efficient enrichment and accurate sequencing of the entire SFTSV genome in real clinical samples, with significantly better coverage integrity, depth uniformity, and data stability than traditional sequencing protocols.

[0221] Table 1. Statistical table of core sequencing data for clinical SFTSV-positive samples.

[0222]

[0223] In summary, the SFTSV whole-genome capture sequencing scheme based on shingled optimized design can be adapted to real clinical samples with different viral loads. It has the advantages of complete whole-genome coverage, uniform sequencing depth, high coverage ratio of high depth, and stable and reliable data. It can effectively meet the scientific research and clinical testing needs of SFTSV clinical tracing, mutation monitoring, and genome evolution analysis, and has extremely high practicality and promotion value.

[0224] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A capture probe set for detecting the full-length genome of Dabie Bandar Virus (SFTSV), characterized in that, The capture probe set consists of probes with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO:

679.

2. A detection kit for the full-length genome of Dabie Bandar virus, characterized in that, The detection kit consists of a capture probe with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 679, streptavidin-labeled magnetic beads, and a library hybridization reaction reagent.

3. The application of the capture probe as described in claim 1 or the kit as described in claim 2 in the whole genome sequencing of Dabie Bandar virus.

4. A method for detecting the full-length genome of the Dabie Bandar disease area, characterized in that, The method is based on capture probes with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 679 and next-generation sequencing technology. The method involves hybridizing the SFTSV genomic cDNA library of the sample to be tested with the capture probe set to obtain hybridization products. The hybridization products are separated by magnetic beads to obtain DNA fragments hybridized with the probes. The DNA fragments are then captured onto the template and amplified by PCR. The obtained PCR amplification products are subjected to next-generation sequencing. The sequencing results are analyzed by bioinformatics to obtain the full-length SFTSV genome.

5. The method for detecting the full-length genome of the Dabie Bandar disease area according to claim 1, characterized in that, The method specifically includes the following steps: Step 1: Construction of the SFTSV genomic cDNA library ① Sample processing and RNA extraction: Total RNA is extracted from clinical samples or viral cultures and quantified; ②RNA fragmentation: Take an appropriate amount of total RNA and treat it with RNA fragmentation reagent at high temperature (e.g., 94℃) for several minutes to randomly break it into fragments of appropriate length (e.g., ~200 nt); ③ First-strand cDNA synthesis: Using fragmented RNA as a template, random primers and reverse transcriptase and other reagents are added to synthesize first-strand cDNA; ④ Second-strand cDNA synthesis: Add the second-strand synthesis reaction reagent to the first-strand synthesis product, and incorporate dUTP into the reaction system to label the second strand, thereby synthesizing double-stranded cDNA; ⑤ End repair, A-tail addition and adapter ligation: The double-stranded cDNA is repaired by adding an A-tail to the 3' end, and then Y-shaped sequencing adapters with specific index sequences are ligated to both ends of the cDNA fragment. ⑥ Pre-PCR amplification and purification: Using PCR premix containing UDG enzyme and UDI primers, the library with ligated adapters is amplified by PCR for a limited number of cycles; after amplification, the library is purified using magnetic beads to remove impurities such as primer dimers and to screen out libraries of the target fragment size; at this point, a "pre-capture library" containing the SFTSV sequence (if present) and all background nucleic acids is obtained. Step 2: Targeted Hybridization and Capture ① Preparation of hybridization reaction system: Prepare the hybridization solution in a sterile, nuclease-free PCR tube according to the following system: TargetSeqOne® Hyb Buffer v2: 13 µL Hyb Human Block: 5 µL TargetSeq® Blocking Oligo: 2 µL RNase Block: 5 µL Nuclease-Free Water: 3 µL TargetSeq® Target Probes (i.e., the probe mixture of this invention): 2 µL Pre-capture library DNA: After quantification, add an appropriate amount to make the total volume 30 µL; ② Hybridization reaction: Mix the above mixture thoroughly, centrifuge briefly, and then run the hybridization program in a PCR instrument: First, incubate at 80℃ for 5 minutes to denature the DNA into single strands; then immediately cool to 50℃ and incubate at this temperature for 12-18 hours to allow the probe to fully hybridize with the complementary SFTSV cDNA sequence in the library. Step 3: Magnetic Bead Capture and Washing ① Magnetic bead preparation: Take an appropriate amount of streptavidin magnetic beads, wash and resuspend them with binding buffer; ②Capture and Binding: Mix the hybridization reaction solution from step two with the prepared streptavidin magnetic beads and incubate by rotation at room temperature for 30-45 minutes. During this period, the streptavidin on the magnetic beads will bind specifically and with high affinity to the biotin labeled at the 5' end of the hybridization probe, thereby forming a "magnetic bead-streptavidin-biotin-probe-target cDNA" complex; ③ Magnetic separation and washing: Place the reaction tube on a magnetic rack and carefully discard the supernatant after the solution has clarified. Retain the magnetic bead-complex. Wash the magnetic beads multiple times using a series of precisely formulated washing buffers to thoroughly remove unhybridized library DNA, non-specifically bound nucleic acids, and other impurities; Step 4: Target elution and capture followed by PCR amplification ① Target elution: Add low-salt elution buffer or sterile water to the cleaned magnetic beads, incubate under heating conditions for several minutes, and dissociate the captured SFTSV target cDNA from the probe-magnetic bead complex to obtain DNA elution buffer enriched with SFTSV sequence. ②PCR amplification: Using the elution buffer as a template, prepare the capture PCR amplification system and run the amplification program on the PCR instrument. This step aims to perform limited amplification of the trace enriched target DNA to obtain the "final capture library" that meets the concentration and total amount requirements for sequencing. Step 5: Library quality control and high-throughput sequencing ①Library purification and quality control: Magnetic beads are used to purify the captured PCR products, a fluorometer or Qubit is used to accurately quantify the library, and a bioanalyzer or fragment analyzer is used to detect the fragment size distribution of the library. The concentration and fragment size of the qualified library should meet the requirements of the selected sequencing platform (such as Illumina MiSeq, MGI DNBSEQ-T7, etc.). ② Sequencing: Libraries from multiple samples are mixed in a certain proportion and paired-end sequencing is performed on a next-generation sequencing platform (such as PE150). The sequencing data is output in FASTQ format. Step Six: Bioinformatics Analysis and Genome Acquisition Bioinformatics analysis was performed on the sequencing data to assemble the full-length SFTSV genome sequence. The workflow is as follows: ① Raw data quality control: Use software such as Fastp to filter the raw sequencing data, remove low-quality bases, adapter sequences and too short reads, and obtain high-quality clean reads; ② Remove host background: Use alignment software such as Bowtie2 to align clean reads to the host reference genome and collect reads that fail to align. This part is the suspected pathogen data after removing the host background. ③ Sequence alignment and preliminary assembly: Using software such as BWA, the host-de-host reads are aligned to the SFTSV reference genome database. At the same time, using assembly software such as MEGAHIT, these reads are assembled de novo to obtain contig sequences. ④ Virus typing and reference genome determination: The assembled contiguous sequences are aligned again to the SFTSV typing reference sequence set. The alignment score determines the most likely genotype of the virus in the sample, and the representative sequence of this type is selected as the reference genome for subsequent analysis. ⑤ Variant Detection and Genome Correction: Clean reads were precisely aligned to the reference genome identified in step 4 using BWA. Variant detection was performed using tools such as SAMtools and BCFtools to identify single nucleotide polymorphism sites and insertions / deletions. Then, software such as iVar was used to filter and correct the detected variant sites based on sequencing depth and quality values, generating a high-quality variant list. ⑥ Full-length genome generation: Based on the corrected variation information, the reference genome sequence is "patched" to generate an accurate full-length L, M, and S fragment genome sequence representing the SFTSV strain of the sample. This sequence can be used for subsequent evolutionary analysis, molecular typing, source tracing studies, etc.

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