Targeted sequencing method for pathogenic microorganisms

Abstract

The invention discloses a targeted sequencing method for pathogenic microorganisms, and belongs to the technical field of gene detection.The targeted sequencing method comprises the following steps that free nucleic acid in a to-be-detected sample is extracted and subjected to fragmentation treatment, a targeted sequence of the pathogenic microorganisms is specifically captured after a library is constructed, then the captured sequence is enriched with magnetic beads, and a target sequence is obtained; eluting the enriched product, and removing a sequence which is not specifically bound; amplifying the obtained specific capture sequence by using a universal primer to obtain an amplification product; and sequencing the amplification product on a machine, and removing a host sequence by a bioinformatics analysis method to obtain gene information of the pathogenic microorganisms. According to the method, the pathogen detection sensitivity of a sample in which pathogenic microorganisms mainly exist in the form of free nucleic acid is improved, the sequencing data volume is effectively reduced, and the method has relatively high application value on pathogen detection of samples in which microorganisms such as blood and cerebrospinal fluid exist in the form of free nucleic acid.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene detection, and particularly relates to a pathogenic microorganism targeted sequencing method. BACKGROUND

[0002] High-throughput sequencing (NGS) technology provides a high-throughput detection technical means for clinical infectious diseases, mainly including metagenomic high-throughput sequencing (mNGS) and targeted high-throughput sequencing (tNGS). Among them, mNGS is shotgun sequencing, which directly sequences all nucleic acids in the sample in a non-biased manner, and realizes the detection of viruses, bacteria, fungi, parasites and non-classical microorganisms by combining a pathogenic microorganism database and a specific algorithm. This method has gradually become an important means for clinical difficult and unknown pathogenic microorganism testing. However, the sensitivity of mNGS DNA sequencing in detecting microorganisms in clinical samples is related to the content of human nucleic acid in the sample, and is greatly affected by human genome. When the concentration of pathogenic microorganism in the clinical sample is low, false negatives are likely to occur. Secondly, the sequencing cost of mNGS is high. The shotgun sequencing of metagenomic sequencing requires a high-quality sequence of more than 20 million (20 M). However, only 1% of the reads in these sequencing data are non-human, and only a small part of them belong to pathogenic microorganisms. In most clinical samples, the nucleic acid abundance of pathogenic microorganisms is relatively low. In order to improve the sensitivity of microorganisms, two common methods are as follows: 1) in the wet experiment link, remove human DNA; 2) in the dry experiment link, increase the sequencing throughput.

[0003] At present, the kits sold on the market are mostly based on the difference in cell membrane structure to perform selective lysis, preferentially release human genome, and perform enzyme digestion removal. However, this method is not suitable for samples such as blood and cerebrospinal fluid in which microbial genomes exist in the form of free nucleic acids. Secondly, the method based on methylation CpG binding domain protein for human host removal has low removal efficiency and will lose part of the nucleic acids of other non-hosts such as fungi or parasites, and is not suitable for eukaryotic microorganisms such as fungi and parasites which have similar methylation patterns with hosts. The application of this method has obvious limitations.

[0004] Therefore, at present, in the detection of bloodstream infections and central nervous system infections, there are problems of low pathogen load, high host background in part of the samples and no effective removal of host nucleic acids. Based on the multiple PCR technology, the amplification effect of cfDNA is poor, and the technical difficulties faced by sterile body fluids cannot be effectively solved, that is, the precise detection of such samples cannot be realized, and false negatives are likely to occur. SUMMARY

[0005] In view of the problems of low pathogen load, high host background and no effective dehosting method, the present application provides a targeted sequencing method of pathogenic microorganisms, which enriches the proportion of pathogens in the library through capture probe technology, improves the sensitivity of pathogen detection in sterile body fluids such as blood, cerebrospinal fluid and urine, and effectively reduces the amount of sequencing data.

[0006] A targeted sequencing method of pathogenic microorganisms, comprising the following steps:

[0007] Nucleic acid extraction: extracting free nucleic acid in the sample to be tested;

[0008] Library construction: fragmenting the free nucleic acid obtained above to construct a library;

[0009] Probe capture: adding the library and specific probes to a capture buffer solution system for incubation, specifically capturing the target sequence of pathogenic microorganisms, then enriching the captured sequence with magnetic beads, and then eluting the enrichment product to remove non-specifically bound sequences;

[0010] Sequence amplification: amplifying the specific capture sequence obtained above using universal primers to obtain an amplification product;

[0011] Sequencing and analysis: sequencing the amplification product above and removing host sequences using bioinformatics analysis methods to obtain genetic information of pathogenic microorganisms.

[0012] The conventional probe liquid-phase capture method is developed from the targeted capture detection technology in the fields of genetics and tumor, and there are mature probe capture kits on the market. Using pathogen-specific probes in combination with kits can achieve specific enrichment of pathogenic microorganism genomes.

[0013] Generally, the capture time of commercially available liquid-phase capture kits is overnight hybridization, or in order to adapt to the application scenario of rapid detection of pathogenic microorganisms, the hybridization time is simply modified to 2-4 hours. However, when commercial capture kits are combined with pathogen probes for application to sterile body fluid samples such as blood, cerebrospinal fluid and urine, the problem of insufficient sensitivity and too long liquid-phase capture time still exists.

[0014] The inventors conducted systematic research and analysis, discovering that commercially available kits are designed and optimized for applications with short probe lengths (100-120 nt) and long targets (300-500 bp insert fragments). However, the cfDNA fragment length distribution in sterile body fluid samples is 80-180 bp, often resulting in long probes and short targets, leading to performance that fails to achieve the expected positive enrichment capacity. Secondly, the commercially available kits are designed for stable conditions where most probes have targets and the target concentrations are similar. However, when applied to suspected infection scenarios, most probes do not have actual targets; only about one in a thousand probes may contain the corresponding target. This reduces the probability of probes specifically binding to the target, thus failing to achieve good hybridization capture.

[0015] Based on the above research and analysis, this invention does not use differential lysis method. Instead, it first extracts free nucleic acids and uses the free nucleic acids to construct a DNA library, RNA library, or DNA and RNA co-construction library according to the conventional mNGS process to obtain the capture library. The capture library is then hybridized with pathogen-specific probes to positively enrich the abundance of pathogenic microorganisms in the sample. This improves the sensitivity of pathogen detection, reduces the amount of sample sequencing data, and thus reduces costs.

[0016] In some embodiments, the capture buffer solution system comprises the following components:

[0017] NaCl 0.5-1.2M;

[0018] Phosphate buffer system 20±0.5mM;

[0019] EDTA 5±0.2 mM;

[0020] Hybridization blocking agent 5±0.1×;

[0021] SDS 0.1±0.02wt%;

[0022] Human repetitive sequence blocking agent 12.5±2.5μg;

[0023] 100±5 pmol of joint sealant;

[0024] Understandably, the amounts of each component in the above capture buffer solution system are the concentrations in the hybridization capture system (also known as the working concentrations). The specific solution preparation can be finely adjusted according to the library, probe feeding volume, etc. It should be noted that the working concentration of the hybridization blocking agent is higher than usual. This is because the number of probes and the total amount of nucleic acids are both high in the above capture buffer solution system. In a smaller hybridization system, it is necessary to reduce non-specific hybridization and increase the concentration of the hybridization blocking agent to achieve a better hybridization capture effect.

[0025] In some embodiments, the concentration of the phosphate buffer system is 20 mM, the concentration of the EDTA is 5 mM, the concentration of the hybridization blocking agent is 5×, the concentration of the SDS is 0.1 wt%, the concentration of the human repetitive sequence blocking agent is 10-15 μg, and the concentration of the linker blocking agent is 100 pmmol.

[0026] In some embodiments, the concentration of NaCl is 0.6-1.2M, preferably 0.7-1.2M or 0.8-1.2M, more preferably 0.9-1.1M, for example 1.0M. The concentration of NaCl in the capture buffer solution system has a significant impact on the capture results; using NaCl within the above-mentioned concentration range results in superior hybridization capture efficiency.

[0027] In some of these embodiments, the pH of the phosphate buffer system is 7.2.

[0028] In some of these embodiments, the pH of the EDTA (ethylenediaminetetraacetic acid) is 8.

[0029] In some embodiments, the hybridization blocking agent is Denhardt's. This Denhardt's hybridization blocking agent can be a commercially available reagent, and its function is to reduce the non-specific binding of the probe to non-target nucleic acids or membranes, thereby reducing background signal and improving the signal-to-noise ratio.

[0030] In some embodiments, the human repetitive sequence blocking agent is Human Cot-1. This human repetitive sequence blocking agent is a library of genomic DNA fragments extracted from human placenta and cut to a length concentrated between 50-300 bp. The sequences are mainly composed of highly repetitive elements such as Alu, Kpn, LINE, and SINE, and are used to block human repetitive sequences.

[0031] In some embodiments, the joint sealant is Universal Blocker.

[0032] In some embodiments, the Universal Blocker comprises a 5' extension, an index recognition segment, and a 3' extension from the 5' end to the 3' end. The 5' extension is complementary to the upstream sequence of the library adapter index. The index recognition segment is paired with the index sequence and all bases are replaced with xanthine to achieve universal blocking. The 3' extension is complementary to the downstream sequence of the library adapter index, and the end of the 3' extension is modified with MGB and NFO.

[0033] In some of the above embodiments, the 5' extension starts from the index sequence and extends towards the 5' end, with one LNA modification added every 6 bases, for a total of 3; and / or, the 3' extension starts from the index sequence and extends towards the 3' end, first adding one LNA modification every 3 bases, then adding one LNA modification every 6 bases, and finally adding one LNA modification every 3 bases.

[0034] Through the above adjustments, the connector sealant forms a closed structure with locking at both ends (LNA), a universal base in the middle (I base), and a sealed tail end (MGB-NFO). This can improve the TM value after the Universal Blocker binds to the connector sequence and prevent sequence elongation.

[0035] In some embodiments of the probe capture step, the library is used in an amount of 500 ng-6 μg, preferably 4 ± 1 μg, and specific probes are added at a concentration of 0.08-0.4 fmol per probe.

[0036] In some embodiments, the specific probe is a double-stranded DNA with a length of 110±10 bp. Based on the characteristics of probe capture, the binding between the probe and the target, under suitable conditions, mainly depends on sequence complementarity, and the binding strength is primarily affected by the length of perfectly complementary sequences. Therefore, in the scenario of free microbial nucleic acid capture in this invention, the binding force is mainly affected by the length of the free nucleic acid. Thus, considering experimental convenience and reagent availability, this invention preferably maintains a conventional probe length and improves the positive enrichment of the target sequence by optimizing liquid-phase capture and non-specific elution conditions.

[0037] In some embodiments, the similarity between the specific probe and the host sequence is less than 50% within any 50 bp of the probe sequence.

[0038] In some embodiments, the incubation conditions are: denaturation at 95°C for 2 minutes, followed by reaction at 57±0.5°C for 25±10 minutes, preferably: denaturation at 95°C for 2 minutes, followed by reaction at 57±0.5°C for 25±10 minutes.

[0039] In some embodiments, the magnetic beads are avidin-labeled magnetic beads, and the specific probe is a biotin-labeled probe.

[0040] In some embodiments, the elution method in the probe capture step is: incubation and elution using sodium citrate, sodium chloride, and SDS (sodium dodecyl sulfate).

[0041] In some of these embodiments, the mixture is incubated at 63±1°C for 5±3 min using 2× to 4× sodium citrate chloride (SSC) and 0.1±0.02wt% SDS, followed by elution and discarding of the supernatant. This process is repeated 1 to 3 times, for example, twice.

[0042] In some embodiments, the elution method is as follows:

[0043] 1) Initial wash: Incubate with 4× sodium citrate and sodium chloride (SSC) and 0.1wt% SDS at 63±0.5℃ for 3±0.5 min, discard the supernatant; repeat the elution twice.

[0044] 2) Wash again: Incubate with 2× sodium citrate and sodium chloride (SSC) and 0.1 wt% SDS at 63±0.5℃ for 3±0.5 min, and discard the supernatant.

[0045] In some embodiments, the sample to be tested is a sterile body fluid sample, preferably one of blood, cerebrospinal fluid or urine, more preferably peripheral blood or cerebrospinal fluid.

[0046] In some of these embodiments, the pathogenic microorganism includes at least one of bacteria, fungi, DNA viruses, RNA viruses, chlamydia, mycoplasma, and parasites.

[0047] In some implementations, the nucleic acid extraction step involves the following method: taking the sample to be tested, centrifuging to separate human cells, aspirating the supernatant, adding protein lysis buffer and proteinase K, and incubating to allow the nucleic acid to be free in the lysis system, thereby obtaining free nucleic acid.

[0048] In some of the above embodiments, the centrifugation conditions are 4±1℃, 3000±200×g for 10±0.5 min, for example, 4℃, 3000×g for 10 min; the protease lysis buffer is cetyltrimethylammonium bromide (CATB) buffer; more preferably, the amount of the protease lysis buffer is 1±0.1×, and / or the amount of proteinase K is 1.5-3 U / mL.

[0049] In some implementations, the free nucleic acid is further purified after extraction. Understandably, the purification method can follow conventional practices in the art.

[0050] In some implementations, during the library construction step, the fragment length of the library is 180-500 bp, preferably 180-400 bp, more preferably, the peak value of the fragment is 200-300 bp, and most preferably 220-250 bp, for example, 220 bp. Because the peak fragment length of cell-free nucleic acids from microorganisms in samples such as blood, cerebrospinal fluid, or urine is approximately 50-60 bp shorter than that of human cell-free nucleic acids, around 90 bp, a peak value of 200 bp corresponds to the 90 bp length of the microbial cell-free nucleic acid plus the 130 bp of the library's general structure. A peak value of 220 bp indicates a high abundance of microbial cell-free nucleic acids in the library.

[0051] In some implementations, during the sequencing and analysis steps, the sequencing data is <20M, preferably <10M, <5M, <3M, <2M, and more preferably 1M.

[0052] The aforementioned "specific probes" refer to probes that obtain the genomic sequences of the target pathogen and its closely related species, and then use bioinformatics software for multiple sequence alignment to find sequence regions present in all target pathogen strains but absent in other species. They can be designed using conventional methods, such as the design described in the reference "PMID:35474894-Cell RepMethods-2021-Probe design for simultaneous, targeted capture of diversemetagenomic targets".

[0053] In the above reagent concentrations, "×" indicates "times" and is a relative concentration. For example, 5× for hybridization blocking agent means that the concentration of hybridization blocking agent in the system is 5 times the working concentration recommended in the instructions. Similarly, 4× for sodium citrate and sodium chloride means that the concentration of sodium citrate and sodium chloride in the system is 4 times the conventional working concentration.

[0054] The term "host" as used above refers to an organism that provides a place for pathogenic microorganisms to survive and reproduce, and may therefore be infected or otherwise affected by them. A host can be a human, an animal, or even certain plants or microorganisms, depending on the host range of the pathogen. In this invention, it primarily refers to humans; therefore, "host sequence" mainly refers to a human-derived sequence.

[0055] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0056] The reagents and raw materials used in this invention are all commercially available.

[0057] The positive and progressive effects of this invention are as follows:

[0058] The targeted sequencing method for pathogenic microorganisms of the present invention includes the extraction of free nucleic acids from pathogenic microorganisms, obtaining a target library by constructing a DNA library, RNA library, or DNA and RNA co-construction library based on conventional mNGS procedures, rapidly hybridizing the target library with pathogen-specific probes, positively enriching the abundance of pathogenic microorganisms in the sample, improving pathogen detection sensitivity, reducing the amount of sample sequencing data, and reducing costs.

[0059] Furthermore, the targeted sequencing method for pathogenic microorganisms of the present invention can complete the liquid phase capture process within 1.5 hours, which greatly reduces the capture time and shortens the turnaround period. Detailed Implementation

[0060] The present invention is further illustrated below by way of examples, but these examples do not limit the invention to the scope of the embodiments described. Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions, or as selected according to the product instructions. Reagents in the following examples, unless otherwise specified, are commercially available.

[0061] Example 1

[0062] A targeted sequencing method for pathogenic microorganisms includes the following steps:

[0063] I. Sample

[0064] Sixteen peripheral blood samples that tested positive for known pathogens (collected from the First Affiliated Hospital of Guangzhou Medical University).

[0065] II. Methods

[0066] 1. Nucleic acid extraction

[0067] 1.1 Extraction of free nucleic acid: Take the above blood sample, centrifuge at 3000×g at 4℃ for 10 min to separate the human cells contained in the sample, and take out the upper serum layer for extraction (note that the cell layer should not be aspirated). Incubate 600 μL of centrifuged supernatant with 360 μL of protein lysis buffer (Vazyme, RM201) and 20 μL of proteinase K (Vazyme, RM201) at room temperature for 10 min to allow the nucleic acid to be free in the lysis system, and the test sample containing free nucleic acid can be obtained.

[0068] 1.2 Nucleic acid purification: Add 50 μL of magnetic bead suspension to each tube of sample to be tested, then add 1.55 mL of phosphate buffer (pH 5.0), shake thoroughly to mix, and let stand at room temperature for 5 min to allow free nucleic acid molecules to be adsorbed onto the magnetic beads. After brief centrifugation, place on a magnetic rack for 2 min, discard the supernatant, then add 500 μL of buffer B (Tris-EDTA, pH 8.0) to wash the magnetic beads and remove proteins bound to the nucleic acids. Add 500 μL of buffer C (10 mM Tris-HCl and 1 mM EDTA, pH 9.0), let stand for 30 s, place on a magnetic rack, discard the supernatant, and repeat this step 1-3 times.

[0069] 1.3 Nucleic acid drying: Thoroughly remove any remaining buffer C, and place the magnetic beads bound to nucleic acid at room temperature for 3-5 minutes until the magnetic beads are dry.

[0070] 1.4 Nucleic acid lysis: Add 60 μL of EB buffer (10 mM Tris-Cl, pH 8.5) to the purified magnetic beads, let stand for 5 min, adsorb on a magnetic rack for 2 min, and collect the supernatant.

[0071] 2. Library Construction

[0072] 2.1 One-tube construction: DNase I enzyme was used to incubate the supernatant containing total nucleic acid at 37±0.5℃ for 5±0.1 min.

[0073] 2.2 Blocking: The human ribosomal rRNA sequence was blocked by a modified probe (Vazyme, N460-01) on the nucleic acid after incubation to prevent the human rRNA sequence from entering subsequent steps.

[0074] 2.3 One-strand synthesis: The cDNA one-strand was synthesized in the reverse transcription system using random primers according to conventional methods.

[0075] 2.4: Second-strand synthesis: cDNA second-strand synthesis was performed using conventional methods.

[0076] 2.5 Fragmentation: The double-stranded cDNA and DNA in the above-obtained products were fragmented by using non-restriction endonucleases (Fargmentase and / or Endonuclease V), and the DNA fragments were repaired by end-repair enzymes.

[0077] 2.6 Connecting adapters: Add adapters and ligase to the solution obtained in the above steps, and perform adapter ligation in a buffer system.

[0078] 2.7 Purification: The ligation product is purified using magnetic beads to remove incorrectly ligated, short adapter fragments, and residual adapters.

[0079] 2.8 Amplification: The purified product is amplified.

[0080] 2.9 Purification: The amplification products were purified using magnetic beads to remove small fragments, resulting in a single and concentrated peak shape in the library. The library fragments were distributed in the range of 180-400bp, with a peak value of 250bp.

[0081] 2.10 Library Pooling: Calculate the input quality of each library based on the amount of data required for each library, mix the pre-defined libraries, and then purify the mixed libraries using magnetic beads.

[0082] 3. Probe capture

[0083] The prepared library is subjected to microbial-specific probe hybridization, magnetic bead enrichment, non-specific elution, PCR enrichment, and purification to obtain a sequencing library obtained by forward enrichment probe capture, as detailed below.

[0084] 3.1 Capture: Take the purified library from the pooling process described above (4000 ng in this example), add 0.08-0.4 fmol of specific probes per probe, and incubate in a capture buffer solution. The capture buffer solution consists of: 1 M NaCl, 20 mM phosphate buffer (pH 7.2), 5 mM EDTA (pH 8), 5× Denhardt's blocking agent (IGene Tech), 0.1 wt% SDS, 10-15 μg Human Cot-1 (IGene Tech), and 100 pmmol Universal Blocker (IGene Tech) adapter blocking agent, and incubate together.

[0085] The incubation conditions were: denaturation at 95℃ for 2 minutes, reaction at 57±0.5℃ for 25 minutes, followed by enrichment using avidin-labeled magnetic beads.

[0086] 3.2 Elution: The non-specifically bound sequences of the above enriched products were removed using an elution buffer, while the specifically bound sequences were retained.

[0087] The specific method is as follows:

[0088] 1) Initial wash: Incubate with 4× sodium citrate and sodium chloride (SSC) and 0.1wt% SDS at 63±0.5℃ for 3±0.5 min, discard the supernatant; repeat the elution twice.

[0089] 2) Wash again: Incubate with 2× sodium citrate and sodium chloride (SSC) and 0.1 wt% SDS at 63±0.5℃ for 3±0.5 min, and discard the supernatant.

[0090] 4. Sequence amplification

[0091] 4.1 Amplification: The specifically bound sequence is amplified using universal primers to obtain the amplification product.

[0092] 4.2 Purification: The amplification products were purified using magnetic beads.

[0093] 4.3 Circulation: Following conventional methods, the purified product was subjected to single-strand circulation and nanosphere preparation. Specifically, the purified library and short adapters were thermally denatured and then placed on ice for later use. The denatured library was added to the single-strand circulation reaction system for circulation. The circulated product was digested with the uncirculated library and purified using RNA magnetic beads. The purified product was subjected to primer hybridization and rolling circle amplification to obtain nanospheres, which are the sequencing library.

[0094] 5. Sequencing.

[0095] The sequencing libraries constructed in step 3.4 above were sequenced as the experimental group. Simultaneously, the uncaptured libraries from 16 samples (the libraries obtained in step 2.10 above) were sequenced as the control group for the conventional mNGS methodology of this invention (hereinafter referred to as the mNGS group). Furthermore, the 16 samples were sequenced using a commercially available kit (IGeneTech). TM The TargetSeq One® Hyb & Wash Kit v2.0 was used for capture and sequencing as a positive control group of this invention.

[0096] The positive control sequencing method is consistent with this invention in terms of nucleic acid extraction and library construction, followed by library mixing and concentration, library-probe hybridization, streptavidin bead capture, post-capture PCR amplification, library quality control, and sequencing. Specific testing methods and conditions are based on IGeneTech. TM Follow the TargetSeq One® Hyb& Wash Kit v2.0 instruction manual, and determine the fragment size according to the operating procedure of the Bioptic Qsep1 fragment analyzer.

[0097] 6. Bioinformatics analysis.

[0098] Using conventional methods, host sequences are removed through bioinformatics analysis to obtain pathogen gene information.

[0099] III. Results

[0100] The results of the above 16 samples are shown in the table below. The number of pathogenic microorganisms detected in the mNGS group is the number of microbial sequences obtained under the normalized 20M data volume, the number of microbial sequences in the experimental group of this invention is the number of microbial sequences normalized to 1M data volume, and the number of microbial sequences in the positive control group is the number of microbial sequences normalized to 1M data volume.

[0101] Table 1. Sequencing results of 16 clinical samples obtained using different methods

[0102]

[0103]

[0104] Note: RPM (Reads Per Million) is the percentage of every million sequencing reads.

[0105] Experimental results show that even with 1M data volume, the number of sequences detected by the targeted sequencing method of this invention is much higher than that of mNGS with 20M sequences, and correspondingly higher than that of the positive control group based on commercial kits for capture and sequencing.

[0106] Furthermore, the detection methods for the above-mentioned commercially available positive control group and the experimental time for the experimental group of this invention are summarized in the following table.

[0107] Table 2. Comparison of Experimental Times

[0108]

[0109] The above comparison shows that the targeted sequencing method of the present invention takes significantly less time than conventional methods.

[0110] IV. Conclusion

[0111] The above results indicate that the targeted sequencing method of the present invention can effectively increase the number of microbial sequences in the detection results when detecting clinical samples. Compared with the traditional mNGS method, the data volume can be reduced to 0.1-1M, the sequencing cost can be reduced to 1 / 20, the number of sequences can be increased by 1-2 orders of magnitude compared with the detection results based on commercial capture kits, and the operation time can be controlled within 1.5h, shortening the turnaround time.

[0112] Example 2

[0113] In this embodiment, a reference sample was used as the test sample for experimental verification. The remaining experimental methods were carried out in accordance with the targeted sequencing method for pathogenic microorganisms in Example 1.

[0114] The experimental samples used in this embodiment are enterprise reference materials (MPs), which contain 12 microorganisms purchased from the American Type Culture Collection (ATCC), Beijing Beina Biotechnology Co., Ltd. (BNCC), and Bondsheng Biotechnology Co., Ltd. Specifically, these are: Klebsiella pneumoniae (ATCC11296), Haemophilus influenzae (ATCC10211), Enterococcus faecalis (ATCC19433), Streptococcus pneumoniae (ATCCBAA-255), Mycobacterium tuberculosis complex (BNCC 263834), Mycobacterium abscessus (BNCC 186002), Aspergillus fumigatus (BNCC338385), Cryptococcus guillier (ATCC34877), Human herpesvirus type 4 (EBV) (Bondsheng), Human herpesvirus type 1 (hSV1) (Bondsheng), Enterovirus (EV71) (Bondsheng), and Influenza B virus (IVB) (Bondsheng).

[0115] The reference standard MP was configured as a detection limit reference standard (MP1, MP2, MP3, MP4) and a negative reference standard (MP0) at different concentrations.

[0116] The reference standard was prepared using physiological saline as the matrix and human mesenchymal stem cells as the human source cells. The MP reference standard was prepared according to the concentrations shown in Table 3.

[0117] The preparation method of free nucleic acid reference (Mcf) is as follows: nucleic acid is extracted by grinding 600 μL of MP1-MP4 samples with glass beads to break the cell wall and then lysing them. The extracted nucleic acid is then broken into nucleic acid fragments of 40-180 bp by an ultrasonic instrument (Covaris). The free nucleic acid reference is prepared into a volume of about 600 μL using enzyme-free physiological saline, which corresponds to the preparation of reference MP1-4 to obtain free nucleic acid reference Mcf 1-4, for later use.

[0118] Table 3. Composition of MP Reference Material

[0119]

[0120] Nucleic acid extraction, library construction, probe capture, and sequencing were performed according to the method in Example 1; simultaneously, sequencing of the uncaptured library (mNGS group) was performed as a control for the conventional mNGS method; the positive control was performed according to a commercially available kit (IGeneTech). TM The targetSeq One® Hyb & Wash Kit v2.0 method was used for capture and sequencing; the number of pathogenic microorganisms detected in the mNGS group was the number of microbial sequences obtained under the normalized 20M data volume, the experimental group of this invention was the number of microbial sequences normalized to 1M data volume, and the positive control group was the number of microbial sequences normalized to 1M data volume. The detection results are shown in the table below.

[0121] Table 4. Detection results of different free nucleic acid reference materials

[0122]

[0123] The above results indicate that when using standardized Mcf1-Mcf4 detection limit references as samples, even with 1M data volume, the number of detected sequences is significantly higher than that of the mNGS group with 20M sequences. Correspondingly, it is also higher than the positive control group based on capture and sequencing using commercially available kits.

[0124] Example 3

[0125] In this embodiment, reference material Mcf3 was used as the test sample to investigate the effect of different NaCl concentrations in the buffer solution system on hybridization capture. The remaining experimental methods were performed in accordance with the targeted sequencing method for pathogenic microorganisms in Example 1.

[0126] Different experimental groups were designed with NaCl concentrations of 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.1M, and 1.2M in the capture buffer solution system; the free nucleic acid reference sample Mcf3 from Example 2 was used as the detection object. The detection results are shown in the table below.

[0127] Table 5. Detection results of experimental groups with different NaCl concentrations

[0128]

[0129] The above results indicate that the NaCl concentration in the capture buffer solution system has a significant impact on the hybridization capture system. The highest number of captured microbial sequences was obtained when the NaCl concentration was 1.0 M, representing the optimal Na ion concentration for the hybridization reaction system.

[0130] Example 4

[0131] This embodiment uses free nucleic acid reference material as the test sample for experimentation. By digesting the reference material with enzymes to obtain fragmented nucleic acid, the free nucleic acid form in clinical samples is simulated, or the cellular form of the reference material is retained, and the detection effect of the targeted sequencing method of the present invention in different sample types is compared.

[0132] The test samples included free nucleic acid reference material Mcf3 (prepared according to the method in Example 2) and reference material MP3 in cellular form (see Table 3). The cellular reference material was prepared according to the composition of the free nucleic acid reference material, without the nucleic acid extraction and ultrasonic fragmentation processes required for the preparation of the free nucleic acid reference material. The corresponding cellular reference material sample was numbered MP3.

[0133] MP3 and Mcf3 samples were sequenced according to the targeted sequencing method in Example 1, with the uncaptured library being sequenced (mNGS group) as a control for the conventional mNGS method; the positive control was sequenced using a commercially available kit (IGeneTech). TM The targetSeq One® Hyb & Wash Kit v2.0 method was used for capture and sequencing; the number of pathogenic microorganisms detected in the mNGS group was the number of microbial sequences obtained under the normalized 20M data volume, the experimental group of this invention was the number of microbial sequences normalized to 1M data volume, and the positive control group was the number of microbial sequences normalized to 1M data volume. The detection results are shown in the table below.

[0134] Table 6. Comparison of detection results between simulated free nucleic acid samples and cellular samples

[0135]

[0136] Experimental results show that the targeted sequencing method of this invention outperforms commercial kits in detecting samples in the form of free nucleic acids, and is comparable to the detection performance of commercial kits in detecting samples in the form of cells.

[0137] Example 5

[0138] In this embodiment, free nucleic acid reference material was used as the test sample to investigate the effect of different elution conditions in the capture buffer solution system on hybridization capture. The remaining experimental methods were carried out in accordance with the targeted sequencing method for pathogenic microorganisms in Example 1. At the same time, a conventional mNGS detection method group as in Example 1 was set up as a control (hereinafter referred to as the mNGS group).

[0139] The groups are set up as follows:

[0140] 1) Experimental group 1 (3 cycles of moderate to low stringent elution):

[0141] Initial wash: Incubate with 4× sodium citrate and sodium chloride (SSC) and 0.1 wt% SDS at 63±0.5℃ for 3±0.5 min, discard the supernatant; repeat the elution twice.

[0142] Wash again: Incubate with 2× sodium citrate and sodium chloride (SSC) and 0.1 wt% SDS at 63±0.5℃ for 3±0.5 min, and discard the supernatant.

[0143] 2) Experimental Group 2 (6 stringent elutions): Following the commercially available kit (IGeneTech) TMThe elution was performed according to the TargetSeq One® Hyb & Wash Kit v2.0 instructions, specifically using 6× sodium citrate and sodium chloride (SSC) and 0.1 wt% SDS at 63±0.5℃ for 6 elutions. The first 3 elutions were performed by blowing and aspirating 20 times and then discarding the supernatant. The last 3 elutions were performed by incubating at 63±0.5℃ for 3 min and then discarding the supernatant.

[0144] 3) Experimental group 3 (3 times of high-strength elution): Incubate with 6× sodium citrate and sodium chloride (SSC) and 0.1wt% SDS at 63±0.5℃ for 3±0.5 min, discard the supernatant; repeat the elution twice.

[0145] 4) Experimental group 4 (3 rounds of medium-to-high stringent elution):

[0146] Initial wash: Incubate with 6× sodium citrate and sodium chloride (SSC) and 0.1 wt% SDS at 63±0.5℃ for 3±0.5 min, discard the supernatant; repeat the elution twice.

[0147] Wash again: Use 4×SSC + 0.1% SDS, incubate at 63±0.5℃ for 3±0.5 min, then discard the supernatant.

[0148] 5) Experimental group 5 (strict elution in 3 times): Incubate with 4× sodium citrate and sodium chloride (SSC) and 0.1wt% SDS at 63±0.5℃ for 3±0.5 min, discard the supernatant; repeat elution 2 times.

[0149] 6) Experimental group 6 (3 times of low-tight elution): Incubate with 2× sodium citrate and sodium chloride (SSC) and 0.1wt% SDS at 63±0.5℃ for 3±0.5 min, discard the supernatant; repeat the elution twice.

[0150] The experimental results are shown in the table below.

[0151] Table 7. Effects of different elution conditions on hybridization capture. Experimental results.

[0152]

[0153] The above results of the Mcf3 sample under different elution conditions show that for samples mainly composed of free nucleic acids, highly stringent and repeated non-specific elutions are unnecessary. Medium-stringent elution conditions can effectively elute the non-specific binding of the probe to non-target regions without causing target region loss due to excessively short target fragments or short probe-target binding areas. Two subsequent low-stringent elutions can then wash away residual nucleic acids on the tube wall, ultimately achieving a good positive enrichment effect, simplifying the operation process, and shortening the time required.

[0154] Example 6

[0155] This embodiment investigates the effect of different types of universal blockers in the capture buffer solution system on hybridization capture. The test samples were 8 peripheral blood samples and 5 cerebrospinal fluid clinical samples that were positive for known pathogens. The remaining experimental methods were the same as the targeted sequencing method for pathogens in Example 1.

[0156] Based on the different types of Universal Blockers, the experiments were divided into the following groups, and screening tests were conducted according to the usage data provided by the suppliers:

[0157] Experimental Group 1: The Universal Blocker consists of a 5' extension, an index recognition segment, and a 3' extension, sequentially from the 5' to the 3' end. The 5' extension is complementary to the upstream sequence of the library adapter index. Starting from the index sequence, the 5' extension extends towards the 5' end, adding one LNA modification every 6 bases, for a total of 3 modifications. The index recognition segment pairs with the index sequence, and all bases are replaced with xanthine, serving as a universal blocking agent. The 3' extension is complementary to the downstream sequence of the library adapter index. Starting from the index sequence, the 3' extension extends towards the 3' end, first adding one LNA modification every 3 bases, then every 6 bases, and finally every 3 bases. The 3' extension is modified with MGB and NFO at the end, while the rest follows the conventional blocking agent design. This modification increases the TM value of the Universal Blocker after binding to the adapter sequence, preventing sequence elongation. It was synthesized and provided by Sangon Biotech.

[0158] Experimental Group 2: Universal Blocker is the IGene Tech Universal Blocker used in Example 1.

[0159] The test results using different sealing agents are shown in the table below.

[0160] Table 8. Effects of different blocking agents on hybridization capture

[0161]

[0162]

[0163] The above results indicate that both blocking agents can effectively detect the target microbial sequence. Experimental group 1 is slightly better than the other experimental groups. Therefore, the Universal Blocker blocking agent of experimental group 1 is recommended.

[0164] Example 7

[0165] This embodiment examines the performance of the targeted sequencing method of the present invention in different types of biological samples.

[0166] Using seven known positive peripheral blood samples and seven bronchoalveolar lavage fluid clinical samples as test subjects, three groups of experiments were set up respectively, referring to the method in Example 1.

[0167] mNGS group: Sequencing of uncaptured libraries, RPM20 is the number of microbial sequences obtained under normalized 20M data volume.

[0168] Positive control group: according to the commercially available kit (IGeneTech) TM The TargetSeq One® Hyb & WashKit v2.0 method was used for capture and sequencing, with RPM being the number of microbial sequences obtained under normalized 1M data volume.

[0169] Experimental group: The number of microbial sequences under the targeted sequencing method of Example 1, normalized to 1M data volume using RPM.

[0170] The results are shown in the table below.

[0171] Table 9. Comparative Experiment Results of Different Sample Types

[0172]

[0173]

[0174] The results show that the targeted sequencing method of the present invention has a significant advantage in increasing the number of sequences in blood samples, i.e., samples in which microbial nucleic acids mainly exist in free form, with an 8-fold increase in sequence number; however, in samples in which microorganisms exist in cellular form, there is no advantage in increasing the number of sequences, and the overall number of sequences decreases to 0.8 times that of the conventional capture kit method.

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A targeted sequencing method for pathogenic microorganisms, characterized in that, Includes the following steps: Nucleic acid extraction: Extracting free nucleic acids from the sample to be tested; Library construction: The obtained free nucleic acids are fragmented to construct a library; Probe capture: The above library and specific probes are added to the capture buffer solution system for incubation to specifically capture the target sequence of pathogenic microorganisms. The captured sequences are then enriched with magnetic beads, and the enriched products are eluted to remove non-specifically bound sequences. Sequence amplification: The specific capture sequence obtained above was amplified using universal primers to obtain the amplification product; Sequencing and analysis: The amplified products were sequenced and the host sequence was removed using bioinformatics analysis methods to obtain the genetic information of the pathogenic microorganism.

2. The targeted sequencing method for pathogenic microorganisms as described in claim 1, characterized in that, The capture buffer solution system comprises the following components: NaCl 0.5-1.2M; Phosphate buffer system 20±0.5mM; EDTA 5±0.2 mM; Hybridization blocking agent 5±0.1×; SDS 0.1±0.02wt%; Human repetitive sequence blocking agent 12.5±2.5μg; 100±5 pmmol of sealant for the joint; Preferably, the concentration of the phosphate buffer system is 20 mM, the concentration of the EDTA is 5 mM, the concentration of the hybridization blocking agent is 5×, the concentration of the SDS is 0.1 wt%, the concentration of the human repetitive sequence blocking agent is 10-15 μg, and the concentration of the linker blocking agent is 100 pmmol.

3. The targeted sequencing method for pathogenic microorganisms as described in claim 2, characterized in that, The concentration of NaCl is 0.6-1.2M, preferably 0.7-1.2M or 0.8-1.2M, more preferably 0.9-1.1M, for example 1.0M.

4. The targeted sequencing method for pathogenic microorganisms as described in claim 2, characterized in that, Meets at least one of the following conditions: (1) The pH of the phosphate buffer system is 7.2; (2) The pH of the EDTA is 8; (3) The hybridization blocking agent is Denhardt's; (4) The human repetitive sequence blocking agent is Human Cot-1; (5) The linker sealing agent is Universal Blocker; preferably, the Universal Blocker includes a 5' extension, an index recognition segment and a 3' extension segment from the 5' end to the 3' end, wherein the 5' extension segment is complementary to the upstream sequence of the library linker index, the index recognition segment is paired with the index sequence and all bases are replaced with xanthine to play a universal blocking role, the 3' extension segment is complementary to the downstream sequence of the library linker index, and the end of the 3' extension segment is modified with MGB and NFO; More preferably, the 5' extension starts from the index sequence and extends towards the 5' end, with one LNA modification added every 6 bases, for a total of 3; and / or, the 3' extension starts from the index sequence and extends towards the 3' end, first adding one LNA modification every 3 bases, then adding one LNA modification every 6 bases, and finally adding one LNA modification every 3 bases.

5. The targeted sequencing method for pathogenic microorganisms as described in claim 1, characterized in that, The probe captures at least one of the following conditions: (1) The amount of the library used is 500 ng-6 μg, preferably 4 ± 1 μg, and the specific probe is added at a concentration of 0.08-0.4 fmol per probe. (2) The specific probe is a double-stranded DNA with a length of 110±10 bp; (3) The similarity between the specific probe and the host sequence is less than 50% within any 50 bp of the probe sequence; (4) The incubation conditions are: denaturation at 95°C for 2±1 minutes and reaction at 57±0.5°C for 25±20 minutes, preferably: denaturation at 95°C for 2 minutes and reaction at 57±0.5°C for 25±10 minutes; (5) The magnetic beads are avidin-labeled magnetic beads, and the specific probe is a biotin-labeled probe.

6. The targeted sequencing method for pathogenic microorganisms as described in any one of claims 1-5, characterized in that, In the probe capture step, the elution method is as follows: incubation and elution using sodium citrate, sodium chloride, and SDS; Preferably, the mixture is incubated at 63±1℃ for 5±3 min using 2× to 4× sodium citrate and sodium chloride and 0.1±0.02wt% SDS. After elution, the supernatant is discarded, and the process is repeated 1 to 3 times, for example, 2 times. More preferably, the elution method is as follows: 1) Initial wash: Incubate with 4× sodium citrate and sodium chloride and 0.1wt% SDS at 63±0.5℃ for 3±0.5 min, then discard the supernatant; Repeat the washing process twice; 2) Wash again: Incubate with 2× sodium citrate and sodium chloride and 0.1 wt% SDS at 63±0.5℃ for 3±0.5 min, and discard the supernatant.

7. The targeted sequencing method for pathogenic microorganisms as described in any one of claims 1-5, characterized in that, The sample to be tested is a sterile body fluid sample, preferably one of blood, cerebrospinal fluid or urine, more preferably peripheral blood or cerebrospinal fluid.

8. The targeted sequencing method for pathogenic microorganisms as described in any one of claims 1-5, characterized in that, The pathogenic microorganisms include at least one of bacteria, fungi, DNA viruses, RNA viruses, chlamydia, mycoplasma, and parasites.

9. The targeted sequencing method for pathogenic microorganisms as described in any one of claims 1-5, characterized in that, Meets one or both of the following conditions: (1) In the nucleic acid extraction step, the nucleic acid extraction method is as follows: take the sample to be tested, centrifuge to separate human cells, aspirate the supernatant, add protein lysis buffer and proteinase K, and incubate to allow the nucleic acid to be free in the lysis system, thereby obtaining free nucleic acid; preferably, the centrifugation conditions are 4±1℃, 3000±200×g centrifugation for 10±0.5min, for example, 4℃, 3000×g centrifugation for 10min; the proteinase lysis buffer is hexadecyltrimethylammonium bromide buffer; more preferably, the amount of proteinase lysis buffer is 1±0.1×, and / or the amount of proteinase K is 1.5-3U / mL; (2) In the library construction step, the fragment length of the library is 150-500bp, preferably 180-400bp, more preferably, the peak value of the fragment of the library is 200-300bp, and most preferably 220-250bp, for example 220bp.

10. The targeted sequencing method for pathogenic microorganisms as described in any one of claims 1-4, characterized in that, In the sequencing and analysis steps, the sequencing data is <20M, preferably <10M, <5M, <3M, <2M, and more preferably 1M.

Images