Capture probe group for detecting pathogenic microorganisms and application of capture probe group

Abstract

The invention discloses a capture probe group for detecting pathogenic microorganisms and application of the capture probe group. The nucleic acid sequence of the capture probe group comprises a sequence as shown in SEQ ID NO. 1 to SEQ ID NO. 101. The capture probe group can cover and detect 119 types of bacteria, 32 types of fungi, 41 types of DNA viruses and 71 types of RNA viruses and covers common infectious disease pathogens, the kit containing the capture probe can cover and detect 88 types of drug-resistant and virulence genes, the detection performance of pathogenic microorganisms is greatly improved, and the sensitivity and specificity are improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a capture probe group for detecting pathogenic microorganisms and an application thereof. Background Art

[0002] Infectious diseases have always been one of the main causes of human morbidity and mortality. In the early 20th century, the discovery of antimicrobial drugs changed modern medicine and effectively treated infectious diseases, while also providing great help for surgery, transplantation, and cancer treatment. However, there are many types of pathogens that cause infections. In order to improve the effectiveness of infection treatment, effective and rapid detection of pathogenic microorganisms has become an important issue.

[0003] Currently, commonly used detection methods include traditional culture, immunological testing, serological testing, and genetic testing. Microbial culture is still the gold standard for pathogen detection, but the long time and low positive rate are still the disadvantages of traditional methods. Immunological testing and serological testing are fast, but they can detect fewer pathogens and have limitations. In recent years, genetic testing technology has developed rapidly, such as PCR technology and gene sequencing technology, which have improved speed, throughput, and sensitivity compared to traditional methods, and can better detect pathogens.

[0004] Metagenome is a new term proposed by Handelsam et al. in 1998, and is defined as the sum of all the genetic materials of microorganisms in the biological environment. It takes the entire microbial community (pathogenic microorganisms) in a specific habitat (lesion site) as the research object, directly extracts the DNA of all microorganisms from environmental samples without cultivation, constructs a metagenomic library, and uses genomic research strategies to study the genetic composition of all microorganisms contained in environmental samples and their community functions (species identification of pathogens).

[0005] However, there are also many problems in the clinical application of metagenomics. Since it detects all microorganisms in the sample, the content of human genes in the sample is high and the content of pathogenic genes is low, the amount of data required for metagenomics is large, which increases the sequencing cost; and most of the pathogenic genes are masked by human genes, resulting in low sensitivity, easy to cause false negatives, and reduced clinical compliance rate.

[0006] At present, the effective method to reduce human hosts and increase pathogenic microbial genes is mainly to remove the host. For example, after DNA extraction, methylation treatment is performed, and magnetic beads selectively bind to CpG methylated host DNA for removal, but this solution is costly and requires relatively complete DNA; or before nucleic acid extraction, host cells are lysed with mild detergents (such as saponin) to release DNA, and then naked host DNA is digested with DNaseⅠ. This method is relatively effective and can usually increase the microbial enrichment efficiency by 1000 times, but this method will also affect mycoplasma and free pathogen DNA, and may miss some pathogens, resulting in false negatives.

[0007] It can be seen that all methods of removing host genes positively have some defects, which are prone to false negatives and reduce clinical compliance. Another way to reduce the host is positive enrichment, which reduces the host impact by increasing pathogenic genes, such as 16s amplicon sequencing. This method adds a large number of primers to amplify the conservative and specific segments on the microorganisms in the clinical samples. Combined with the second-generation sequencing, the pathogenic microorganisms in the clinical samples can be detected. This method can effectively reduce the host level and improve specificity and sensitivity, but the number of targets it detects is limited, the primer panel design is more difficult, and the final result is only the 16s rRNA gene of the pathogen.

[0008] Hybridization capture technology uses modified probes and streptavidin magnetic beads to specifically bind to pathogens in clinical samples, and then uses streptavidin magnetic beads to capture them. It can effectively enrich pathogenic microorganisms, and it still has the ability to detect homologous or unknown pathogens, retaining the advantages of metagenomic detection. This method can greatly reduce human sources, reduce the use of sequencing data, and greatly improve the specificity and sensitivity of pathogen detection. At present, this product uses capture technology to enrich pathogens, but there are also many problems and difficulties, such as it cannot be applied to a variety of different samples such as FFPE and cfDNA samples, the number of probes is small and cannot achieve the improvement of a large number of pathogens, and the hybridization capture time is too long and requires overnight hybridization.

[0009] Therefore, there is an urgent need to provide a highly sensitive and specific enrichment method to improve metagenomic detection, and to be applicable to more clinical samples and target more comprehensive pathogenic genes. Summary of the invention

[0010] In view of the deficiencies in the prior art and actual needs, the present invention provides a capture probe group for detecting pathogenic microorganisms and its application. The present invention also provides a capture probe kit, which utilizes a liquid phase hybridization method to capture a conventional mNGS library, thereby improving the sensitivity and specificity while retaining the advantages of metagenomic detection. The capture probe kit uses fast hybridization technology and a large panel probe group to greatly reduce the hybridization capture time and increase the pathogen enrichment range, and is suitable for a variety of different types of samples.

[0011] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a capture probe group for detecting pathogenic microorganisms, wherein the nucleic acid sequence of the capture probe group includes sequences shown as SEQ ID NO.1-SEQ ID NO.101.

[0013] The capture probe group of the present invention is designed as single-stranded DNA, and the probe sequence is designed by performing bioinformatics analysis based on the pathogen information library. The probes in the capture probe group are all segments that specifically bind to the corresponding targets. The probes are combined with the denatured single-stranded target library in the hybridization buffer. The hybridization time under this technology is only 30 minutes.

[0014] The capture probes of the present invention are all modified with biotin, and can be specifically captured by streptavidin magnetic beads, and the genes not captured are cleaned with a washing solution at high temperature, and finally the captured products are PCR amplified and purified, and then sequenced on a machine to detect pathogenic microorganisms in the sample.

[0015] The nucleic acid sequence of the capture probe group in the present invention includes a pathogenic microorganism probe sequence and a drug-resistant gene probe sequence. The pathogenic microorganism probe sequence is specifically shown in Table 1, and the drug-resistant gene probe sequence is specifically shown in Table 2.

[0016] Table 1

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] Table 2

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] Preferably, the capture probe group specifically captures any one or a combination of at least two of the following pathogenic microorganism genome coding region sequences: Aspergillus flavus complex, Aspergillus fumigatus, Aspergillus niger complex, Aspergillus terreus complex, Candida glabrata, Candida albicans, Candida pseudoglabrata, Candida parapsilosis, Candida tropicalis, Pichia kudriavzevii, Candida guillainensis, Cryptococcus gattii, Cryptococcus neoformans, Fusarium, Histoplasma capsulatum, Mucor spp., Mucor agglomerata, Mucor branched, Mucor irregular, Mucor racemosus, Pneumocystis jirovecii, Rhizomucor spp., Rhizomucor microsporus, Rhizopus spp., Rhizopus delbrueckii, Rhizopus microsporus, Rhizopus oryzae, Scedosporium spp., Scedosporium apiosporum, Scedosporium bodensis, Talaromyces marneffei, Trichosporon assasii, and Streptococcus constellus. cocci, Prevotella intermedia, sweet Nocardia, Actinobacillus urinariae, Nocardia dermatophytes, Corynebacterium diphtheriae, Mycobacterium asiaticum, Mycobacterium avium, Mycobacterium avium complex, Mycobacterium cryptic, Mycobacterium gordonii, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoe, nontuberculous mycobacteria, Mycobacterium scrofulae, Mycobacterium schrenckii, Mycobacterium simianum, Mycobacterium surga, Mycobacterium tuberculosis complex, Mycobacterium toad, Mycobacterium abscessus, Mycobacterium chelonae-abscessus complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium smegmatis, Nocardia, Nocardia abscessus, Nocardia asteroides, Nocardia brasiliensis, Nocardia concave, Nocardia st. georgia, Nocardia glanders, Nocardia otitis media, Nocardia terpenoides, Micromonas microstomiasis, Horse red cocci, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus anginosus group, Streptococcus intermedius, Streptococcus pneumoniae, Streptococcus pyogenes, Trophicola whipplei, Acinetobacter baumannii, Acinetobacter johnsii, Acinetobacter ursinus, Bacteroides fragilis, Bordetella homblynsis, Bordetella parapertussis, Bordetella pertussis, Brucella, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia pseudomallei, Burkholderia contamination, Burkholderia polyphaga, Anopheles elizabethkinensis, Elizabethkinensis meningoseptica, Enterobacter cloacae complex, Escherichia coli, Fusobacterium necroticum, Fusobacterium nucleatum, Haemophilus influenzae, Klebsiella aerogenes, Klebsiella oxytoca, pneumonia Klebsiella, Klebsiella mutans, Legionella spp., Legionella Bozeman, Legionella pneumophila, Legionella Long Beach, Legionella Mikdade, Moraxella catarrhalis, Neisseria meningitidis, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia, Nocardia africana, Nocardia neoformans, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Coxiella burnetii, Mycoplasma pneumoniae, Ureaplasma parvum, Ureaplasma urealyticum, Enterococcus faecium, Enterococcus faecalis, Proteus vulnerariae, Enterobacter cloacae, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium marinum, Haemophilus parainfluenzae, Vibrio parahaemolyticus, Vibrio vulnificus, Rickettsia typhi, Streptococcus dysgalactiae, Mycoplasma hominis, Rickettsia prowazekii, Orientia tsutsugamushi,Clostridium perfringens, Morganella morganii, Salmonella enterica, Aspergillus nidulans, Clostridium tetani, Neisseria gonorrhoeae, Treponema pallidum, Yersinia pestis, Bacillus anthracis, Mycobacterium leprae, Leptospira renalis, Vibrio cholerae, Monkeypox virus, BK polyomavirus, Epstein-Barr virus, Herpes simplex virus type 2, Cytomegalovirus, Human polyomavirus type 2, Human polyomavirus type 3, Human polyomavirus type 4, Human herpesvirus 6A, Human herpesvirus 6B, Human herpesvirus type 7, Human herpesvirus type 8, Varicella-zoster virus, Human adenovirus, Hepatitis B virus, Human parvovirus B19, Human adenovirus type 11, Human adenovirus type 14, Human adenovirus type 1, Human adenovirus type 21, Human adenovirus type 2, Human adenovirus type 34 type, human adenovirus type 35, human adenovirus type 3, human adenovirus type 4, human adenovirus type 55, human adenovirus type 57, human adenovirus type 5, human adenovirus type 6, human adenovirus type 7, human adenovirus group B, human adenovirus group C, human adenovirus group D, swine herpesvirus, human epileptovirus type 1, human epileptovirus type 2, human epileptovirus type 3, human bocavirus type 1, human bocavirus type 2, human bocavirus type 3, human bocavirus type 4, human coronavirus 229E, human coronavirus HKU1, human coronavirus NL63, human coronavirus OC43, human metapneumovirus, mumps virus type 2, mumps virus type 4, human respiratory virus type 1, human respiratory virus type 3, measles virus, mumps virus, rubella virus , dengue virus type 1, dengue virus type 2, dengue virus type 3, dengue virus type 4, Middle East respiratory syndrome coronavirus, influenza B virus, influenza C virus, new coronavirus, Japanese encephalitis virus, influenza D virus, Hantavirus, human immunodeficiency virus type 1, human immunodeficiency virus type 2, rabies virus, human poliovirus type 1, Zika virus, Astrovirus, Hepatitis C virus, human rotavirus type A, human rotavirus type B, human rotavirus type C, rhinovirus, rhinovirus type A, rhinovirus type B, rhinovirus type C, new bunyavirus, influenza A virus H10N1, influenza A virus H2N2, influenza A virus H7N1, influenza A virus H7N2, influenza A virus H7N7, influenza A virus H9N2, human hepatitis A virus, hepatitis GB virus C, coxsackievirus A10, coxsackievirus A16, coxsackievirus A2, coxsackievirus A5, coxsackievirus A6, coxsackievirus B3, echovirus E18, echovirus E30, enterovirus, enterovirus A, enterovirus A71, enterovirus B, enterovirus C, enterovirus D, enterovirus D68, influenza A virus, influenza B virus Victoria strain, influenza B virus Yamagata strain, human respiratory syncytial virus A, human respiratory syncytial virus B, influenza A virus, influenza A virus H1N1, influenza A virus H3N2,Influenza virus H5N1, Influenza virus H7N9, Acanthamoeba cabodzii, Acanthamoeba heli, Acanthamoeba pisiformis, Acanthamoeba parviflora, Babesia divergentis, Babesia ovata, Balamuthia baboon, Clonorchis sinensis, Cryptosporidium meleagridis, Cryptosporidium parvum, Cyclospora kayannensis, Leishmania ethiopiae, Leishmania amazonica, Leishmania braziliensis, Leishmania donovani, Leishmania gerbilis, Leishmania guyanaensis, Leishmania infantum, Leishmania mexicanum, Leishmania panama, Leishmania tropicalis, Naegleria fowleri, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Intermediate Plasmodium vivax, Schistosoma japonicum, Strongyloides stercoralis, Taenia solium, Toxoplasma gondii, Trichuris trichuris, Trypanosoma cruzi, Wuchereria bancrofti, Acanthamoeba dijonnais, Acanthamoeba lugdunensis, Acanthamoeba, Acanthamoeba quina, Acanthamoeba ridge, Acanthamoeba triangularis, Angiostrongylus cantonensis, Cryptosporidium belli, Cryptosporidium hominis, Cryptosporidium ratis, Cryptosporidium theridii, Cryptosporidium spp., Cryptosporidium traveler, Echinococcus canadensis, Echinococcus granulosus, Entamoeba histolytica, Leishmania chaga, Leishmania, Paragonimus westermani, Brassica oleracea, Trichinella spiralis, Trichomonas oralis, Trichomonas vaginalis, Schistosoma mansoni or Echinococcus multilocularis.

[0032] Preferably, the capture probe group further includes sequences as shown in SEQ ID NO.102-SEQ ID NO.147.

[0033] Preferably, the capture probe group also specifically captures any one or a combination of at least two of the following drug-resistant genome sequences: CMV_UL97, CMV_UL54, MTB_ahpC, MTB_CycA, MTB_alr, MTB_ddlA, MTB_embA, MTB_embC, MTB_ethR, MTB_fbiA, MTB_fbiB, MTB_fbiC, MTB_fgd1, MTB_kasA, MTB_pepQ, MTB_rpoC, MTB_ddn, MTB_ald, MTB_fbiD, MTB_Mmpl5, MTB_atpE, MTB_eis, MTB_gidB, MTB_rplC, iroB, iroC, iroD, iroE, iroN, irp1, irp2, peg-344, peg-589, tox, pt xA, ptxB, ptxC, iutA, rmpA, rmpA2, iucA, iucB, iucC, iucD, rmtF, rmtE, LEN-1, GIM-2, GIM-1, S PM-1, vgaA, NmcA, IMI-1, SME-1, aac6-Ii, aac6, blaCTX-M-151, blaDHA-4, blaIMP-7, DIM, erm A. ermB, fosA3, mcr1, mecA, mecB, nimA, nimB, OXA-23, OXA-51, qnrA9, qnrB12, qnrS11, SIM, su l1, tetA, vanA, vanE, vanG, QnrA1, QnrA2, QnrC, QnrS2, QnrS1, SHV-1, KPC-3, CTX-M-2, or qnrB8.

[0034] Preferably, the capture probe set is modified with biotin.

[0035] Preferably, the length of the probes in the capture probe set is 85-95 bp (eg, 85 bp, 88 bp, 90 bp, 94 bp, 95 bp).

[0036] Preferably, the GC content of the probes in the capture probe set is 40%-60% (eg, 40%, 45%, 50%, 55%, 60%).

[0037] In a second aspect, the present invention provides a kit for detecting pathogenic microorganisms, wherein the kit comprises the capture probe set described in the first aspect.

[0038] Preferably, the kit further comprises a positive quality control product, a negative quality control product and a library construction reagent.

[0039] In a third aspect, the present invention provides a library for detecting pathogenic microorganisms, wherein the library for detecting pathogenic microorganisms is constructed using the kit described in the second aspect.

[0040] In a fourth aspect, the present invention provides a capture detection method for detecting pathogenic microorganisms, the capture detection method comprising: extracting nucleic acid from a sample to be tested and constructing a library, mixing the constructed library with the capture probe group described in the first aspect for hybridization capture, performing PCR amplification and purification on the hybridization capture product to obtain a capture library, and performing high-throughput sequencing on the above-mentioned capture library.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) The capture probe group of the present invention covers common pathogens and can be used to detect pathogens in different clinical samples. It also has high detection performance for homologous species. At the same time, the probe group is also designed for drug resistance and pathogen virulence gene segments, and can cover the detection of 119 bacteria, 32 fungi, 41 DNA viruses, and 71 RNA viruses, covering common infectious disease pathogens. The kit containing the capture probe can cover the detection of 88 drug resistance and virulence genes, assisting doctors in providing medication guidance;

[0043] (2) The present invention covers most of the segments of the detected pathogens by designing probes, which can specifically bind to and capture the pathogen segments to be detected in the library, clean up the host genes, and enrich the pathogen genes, thereby greatly improving the detection performance of the pathogens and improving the sensitivity and specificity;

[0044] (3) The human host in the clinical samples of the present invention is as high as 50%-90%. Most of the sequencing data is of human origin, which is useless in pathogen detection. After hybridization capture, the human proportion can be greatly reduced, thereby reducing the data volume requirement for each sample. The data volume is only 0.5-1M, which is 1 / 20 of conventional mNGS, effectively reducing the sequencing cost;

[0045] (4) Common hybridization capture kits on the market require overnight hybridization, but when used in critical illness infection detection, it is often necessary to improve timeliness as much as possible. The method of the present invention uses rapid hybridization technology to significantly reduce the hybridization time while ensuring detection performance. The hybridization capture kit of the present invention uses magnetic bead concentration to reduce experimental time compared to vacuum concentration, thereby ensuring clinical timeliness requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a comparison of the detection limit levels of the conventional library and the capture library;

[0047] Figure 2 This is a graph showing the enrichment multiples of different pathogenic microorganism types. DETAILED DESCRIPTION

[0048] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0049] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0050] Example 1

[0051] Design and synthesis of capture probe sets.

[0052] (1) The probe design takes into account the gene region or intergenic region of the pathogenic microorganism, sequence complexity, GC content, conservation and specificity. Multi-copy gene regions or housekeeping genes and regions with a GC content of 40%-60% are preferred. Conservative specific probes of different species levels are designed according to actual needs. In terms of conservation requirements, the similarity between the probe sequence and all genomes of the species is more than 95%. In terms of specificity requirements, the probe sequence only matches the current target range, and the homology to other targets is less than 80%, and the maximum length of continuous bases matched between the probe and the human genome is strictly controlled, preferably 30, to reduce the non-specificity of the human genome. The probe length is compatible with most of the current lengths, with an average length of about 90bp. The probe group is designed and generated in a tile array manner. Probes with a GC% content of less than 40% are encrypted at a depth of 2× or higher, and probes with biotin nucleolabeled nucleotides are synthesized.

[0053] The probe sequences of pathogenic microorganisms and drug resistance virulence genes are shown in Tables 1 and 2.

[0054] The prepared C1 probe, library blocking solution, adapter blocking solution and lyophilization protectant were mixed thoroughly in the lyophilization eight-tube strip at a ratio of 4:2:5:12, and then Hm (Hybridization Mix, TWIST) preheated at 60°C was added. After centrifugation, the tubes were placed in a lyophilization plate for 24 hours of lyophilization. After lyophilization, the eight rows of tubes were taken out and packaged, which is the C1 enrichment tube in the finished kit. This enrichment tube can better protect the probe and shorten the enrichment time.

[0055] (2) Capture reagent composition

[0056] The capture kit of the present invention includes reagents as shown in Table 3.

[0057] Table 3

[0058]

[0059] The probe, library blocking solution, and adapter blocking solution are stored in C1 enrichment tubes using freeze-drying technology. Kit A is stored at -25 to -15°C, and Kit B is stored at 2 to 8°C.

[0060] The test instruments are shown in Table 4 below, and the test reagents are shown in Table 5 below.

[0061] Table 4

[0062] Serial number name model factory 1 Mini centrifuge LX-400 Kylin-Bell 2 Vortex mixer Vortex2 IKA 3 Nucleic acid quantifier Qubit 4.0 Invitrogen 4 PCR Amplifier VeritiDx96-well Applied Biosystems 5 Constant temperature mixer ThermoMixerC Eppendorf 6 Vacuum Concentrator Concentratorplus Eppendorf

[0063] Table 5

[0064] Serial number name Specifications / Models factory 1 Anhydrous ethanol (analytical grade) >100mL Unlimited 2 Nuclease-free water >50mL Unlimited 3 Qubitds DNA HS assay kit 500rxn ThermoFisherScientific 4 1NNaOH >1mL Unlimited 5 High-throughput sequencing kit (NextSeq500 / 550v2) 1 set Illumina

[0065] Example 2

[0066] Library construction and capture.

[0067] 1. According to the method of macro-gene library construction, the extracted nucleic acid is library-constructed. The source of the nucleic acid can be blood, alveolar lavage fluid, cerebrospinal fluid, wax roll, sputum, etc. The library construction method required for this capture method is not limited by the platform, but it needs to be pre-processed by using the interruption method. The size of the constructed library is 300-600bp.

[0068] 2. Simultaneously, the above nucleic acids are subjected to pathogen quantification using matching qPCR reagents.

[0069] 3. Prepare library mixture by mixing 8-12 libraries with different adapter sequences in equal mass (if the qPCR pathogen quantification is strongly positive, the amount needs to be reduced).

[0070] Preparation before the experiment: Extract the nucleic acid of the sample to be tested and construct a library so that the final library fragment is 300-600bp. After purification, quantitatively detect the library concentration. Take 8-12 libraries with different adapters and take 250ng of each to mix into a library.

[0071] 1. Library enrichment

[0072] 1.1B3 purified magnetic beads were equilibrated at room temperature for 30 min, and then vortexed to mix.

[0073] 1.2 Add 1.8 times the volume of B3 purified magnetic beads to the library to be concentrated (if 1.8 times the volume is less than 50 μL, add 50 μL), vortex to mix, and incubate at room temperature for 5 minutes.

[0074] 1.3 Briefly centrifuge the PCR tube and place it on a magnetic rack. After the solution is clear, carefully remove the supernatant.

[0075] 1.4 Add 6 μL C2 sample diluent and 15 μL C3 enrichment enhancer equilibrated at room temperature to the magnetic beads, resuspend the magnetic beads and shake them thoroughly, and centrifuge the PCR tube for 3 seconds for later use.

[0076] 2. Hybridization

[0077] 2.1 Set up the PCR instrument (heated cover 85°C, volume 100 μL) according to the following program and start the run for preheating. The hybridization PCR program is shown in Table 6.

[0078] Table 6

[0079]

[0080]

[0081] 2.2 Transfer all the concentrated products from step 1.4 (a total of 21 μL, separated into layers, with the lower layer being the magnetic bead suspension) to the C1 enrichment tube, remove all the bubbles from the tube, and place the PCR tube on the PCR instrument from step (1).

[0082] 2. After incubation at 368℃ for 1 minute, take out the PCR tube, oscillate for 20 seconds, and then centrifuge for 3 seconds for later use (at this time, the solution in the tube is layered, and the magnetic bead suspension is at the bottom layer).

[0083] 2.4 Use the SKIP function to end the 68℃∞ program. When the PCR instrument rises to 95℃, put the PCR tube in step 2.3 back into the PCR instrument.

[0084] 2.5 Use the SKIP function to end the 95℃∞ program and enter the 95℃5min program.

[0085] 2. After 65 minutes, the PCR instrument enters the 60°C∞ program to start the hybridization reaction. This step needs to be kept running for at least 30 minutes.

[0086] 3. Preparation of Enrichment Magnetic Beads

[0087] 3.1B2 enrichment magnetic beads were equilibrated at room temperature for 30 min, and then vortexed for 5 s to mix.

[0088] 3.2 For each reaction, pipette 25 μL B2-enriched magnetic beads into a new PCR tube, place it on a magnetic stand for 15 seconds, and discard the supernatant.

[0089] 3.3 Add 100 μL C6 enrichment buffer, pipette and mix the beads, centrifuge for 1 second, place on a magnetic stand for 15 seconds, and discard the supernatant.

[0090] 3.4 Repeat step (3) twice, for a total of three washes.

[0091] 3.5 Add 20 μL C6 enrichment buffer, pipette and mix the magnetic beads, and keep at room temperature for use.

[0092] 4. Capture

[0093] 4.1 After step 2.6 has run for 30 minutes, open the incubation tube on the PCR instrument, add 80 μL of C6 enrichment buffer and oscillate to mix, centrifuge for 1 second and place on the magnetic rack. After the solution is clarified, transfer all the supernatant to the 20 μL of B2 enrichment magnetic bead suspension in step 3.5, oscillate thoroughly to mix and centrifuge for 1 second.

[0094] 4.2 Use the SKIP function to end the 60℃∞ program and enter the 65℃∞ program. Place the PCR tube in step 4.1 on the PCR instrument and incubate for 30 minutes. Pipet and mix every 5 minutes to resuspend the enriched magnetic beads to ensure that there are no magnetic beads clumping at the bottom and no magnetic beads remaining on the tube cap.

[0095] During 4.3, preheat C7 wash solution 1 (320 μL / each reaction) and C8 wash solution 2 (480 μL / each reaction) in a PCR instrument.

[0096] 5. Washing

[0097] 5.1. After the reaction in step 4.2 is completed, remove the samples from the PCR instrument (C7 wash solution 1 and C8 wash solution 2 must remain on the PCR instrument), use the SKIP function to end the 65℃∞ program, and enter the 68℃∞ program.

[0098] 5.2. Centrifuge the sample for 1 second, place it on a magnetic rack, and discard the supernatant after the solution becomes clear;

[0099] 5.3. Add 150 μL of preheated C7 wash solution 1, mix by pipetting, and incubate at 68°C for 5 min. Make sure there are no magnetic beads clumping at the bottom and no magnetic beads remaining on the tube cap.

[0100] 5.4. Place on the magnetic stand for about 5 seconds. After the beads are clarified, the supernatant must be discarded immediately.

[0101] 5.5. Re-add 150 μL of preheated C7 wash solution 1 to resuspend the magnetic beads, then transfer all the magnetic bead suspension to a new PCR tube and incubate at 68°C for 5 minutes to ensure that there are no magnetic bead aggregates at the bottom and no magnetic beads remain on the tube cap;

[0102] 5.6. Place on the magnetic stand for about 5 seconds. After the magnetic beads are clarified, the supernatant must be discarded immediately;

[0103] 5.7. Use the SKIP function to end the 68℃∞ program and enter the 48℃∞ program;

[0104] 5.8. Add 150 μL of preheated C8 wash solution 2, pipette and mix, and incubate at 48°C for 5 min to ensure that there are no magnetic beads clumping at the bottom and no magnetic beads remaining on the tube cap;

[0105] 5.9. Place on the magnetic stand for about 2 seconds. After the magnetic beads are clarified, the supernatant must be discarded immediately;

[0106] 5.10. Repeat steps 5.8-5.9 twice, washing three times in total;

[0107] 5.11. Centrifuge the magnetic beads in step 5.10 for 3 seconds, place them on the magnetic rack for about 5 seconds, and then use a 10μL pipette to carefully remove the remaining liquid at the bottom of the tube;

[0108] 5.12. Add 22 μL NF-Water to resuspend the magnetic beads, pipette and mix, then take 11 μL for downstream amplification steps (the remaining 11 μL should be stored at -20℃ for future use).

[0109] 6. Amplification and Quantification

[0110] 6.1. Prepare the PCR system according to Table 7 below.

[0111] Table 7

[0112]

[0113]

[0114] 6.2. Mix the prepared PCR system and perform the reaction according to the procedure in Table 8 below:

[0115] Table 8

[0116]

[0117] 6.3. PCR product purification

[0118] 6.3.1.B3 After the purified magnetic beads are equilibrated at room temperature for 30 minutes, vortex and oscillate to mix well for later use;

[0119] 6.3.2. Add 38 μL B3 purified magnetic beads to the product of step 6.2, pipette and mix, and incubate at room temperature for 5 min;

[0120] 6.3.3. Briefly centrifuge the PCR tube for 3 seconds and place it on a magnetic rack. After the solution is clear, discard the supernatant;

[0121] 6.3.4. Add 200 μL of freshly prepared 80% ethanol and transfer the PCR tube on the magnetic rack twice to fully wash the magnetic beads. After the solution is clear, aspirate and discard the supernatant;

[0122] 6.3.5. Repeat step 6.3.4 once, washing twice in total;

[0123] 6.3.6. Centrifuge the magnetic beads in step 6.3.5 for 5 seconds, place them on the magnetic rack for about 30 seconds, and then use a 10μL pipette to carefully remove the remaining liquid at the bottom of the tube;

[0124] 6.3.7. After the magnetic beads are dried, add 22 μL NF-Water, pipette to mix and let stand at room temperature for 3 min;

[0125] 6.3.8. Briefly centrifuge the PCR tube for 3 seconds, place it on a magnetic rack, and after the solution is clarified, transfer 20 μL of the supernatant to a new PCR tube and store it at -20°C for later use.

[0126] 6.4. Nucleic acid quantification

[0127] 6.4.1. Use Qubit3.0 / 4.0 to determine the concentration of the product in step 6.3.8) according to the instructions of Qubit dsDNA HS Assay Kit (Invitrogen) and record the concentration. If the concentration is ≥1ng / μL, it is qualified.

[0128] 7. Sequencing

[0129] 7.1. Perform sequencing according to the instructions.

[0130] 8. Bioinformatics Analysis

[0131] 8.1. Analyze and output the test results according to the pathogenic microorganism metagenomic analysis software. Obtain the library quality control results and the number of pathogenic microorganism sequence detections.

[0132] Example 3

[0133] Comparative experiment on detection limits of conventional metagenomic detection and probe hybridization capture method of the present invention.

[0134] 1. Prepare simulation samples

[0135] Jurkat cells were used as simulated human-derived cells, and Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Candida albicans, Aspergillus fumigatus, Cryptococcus gattii, influenza A virus, influenza B virus, and herpes simplex virus type 1 were mixed respectively.

[0136] Each simulated sample was diluted with saline to 4 target concentration gradients (according to 3× gradient dilution), and then added to the corresponding Jurkat cells at a final concentration of 1x105 copies / mL. The microbial concentrations in the simulated samples are shown in Table 9 below:

[0137] Table 9

[0138]

[0139] 2. Library Preparation

[0140] Take 600 μL of pathogen gradient simulation sample, process it with a wall breaking instrument, take 300 μL each, use nucleic acid extraction test or purification reagent to extract and purify DNA or RNA, use co-construction library reagent to construct the library, and pay attention to avoid the linker duplication to obtain a regular library.

[0141] 3. Hybridization capture

[0142] Hybridization capture and purification were performed according to the method of Example 2 to obtain a capture library.

[0143] 4. Sequencing

[0144] The conventional library and the corresponding capture library were run for NGS sequencing on the Illumina Nextseq 550 platform respectively. The conventional library required a data volume of more than 8M, and the capture library required a data volume of more than 1M.

[0145] 5. Data Analysis

[0146] 5.1. Library quality control

[0147] The amount of data generated by sequencing is shown in Table 10.

[0148] Table 10

[0149]

[0150] 5.2. Comparison of target detection results

[0151] The captured library was analyzed by bioinformatics process, and the number of detected sequences and enrichment multiples were counted for every 1M sequencing sequence of the sample. The results of pathogen detection in simulated mixed samples are shown in Table 11 below, and the comparison results of the detection limit level of conventional library and captured library are shown in Figure 1 The capture efficiency results of different pathogenic microorganism types are shown in Figure 2 .

[0152] Table 11

[0153]

[0154] From the above table and Figure 1 and Figure 2 From the above, the pathogen capture probe group of the present invention improves the sensitivity of pathogen detection and significantly increases the virus enrichment multiple without missed detection. At the same time, while reducing the amount of data, it can also increase the detection rate and the number of detection reads, effectively saving costs.

[0155] Example 4

[0156] Comparative performance testing of clinical plasma samples.

[0157] Twelve whole blood samples with high clinical suspicion of infection were collected, and the plasma was collected after centrifugation at 1600g for 10 minutes. The extraction, library construction and hybridization capture were performed in the same process as in Example 2. The captured library was analyzed by the bioinformatics process, and the number of detected sequences and enrichment multiples of the samples were counted. The results of pathogen detection in clinical plasma samples are shown in Table 12 below:

[0158] Table 12

[0159]

[0160]

[0161] It can be seen from the data in Table 12 that the capture method used by the capture probe detected more reads of different types of pathogenic microorganisms in plasma samples, and some pathogens that were negative in mNGs were detected in the capture method. The capture probe has better performance in plasma.

[0162] Example 5

[0163] Comparative testing of pathogens and drug-resistant genes in clinical bronchoalveolar lavage fluid samples.

[0164] 18 bronchoalveolar lavage fluid samples with high clinical suspicion of infection were taken, and the extraction, library construction and hybridization capture were performed according to the same process as Example 2. The captured library was analyzed by the target and drug resistance gene bioinformatics process, and the number of detected sequences and enrichment multiples of the samples were counted. The results of pathogen detection in clinical bronchoalveolar lavage fluid samples are shown in Table 13 below, and the results of drug resistance gene detection in clinical bronchoalveolar lavage fluid samples are shown in Table 14 below.

[0165] Table 13

[0166]

[0167]

[0168] Table 14

[0169]

[0170]

[0171] Example 6

[0172] Comparative performance testing of clinical cerebrospinal fluid samples.

[0173] Twelve cerebrospinal fluid samples with high clinical suspicion of infection were taken, and extraction, library construction and hybridization capture were performed according to the same process as Example 2. The captured library was analyzed by the bioinformatics process, and the number of detected sequences and enrichment multiples of the samples were counted. The results are shown in Table 15 below.

[0174] Table 15

[0175]

[0176] Example 7

[0177] 12 wax roll samples with high clinical suspicion of infection were taken, and extraction, library construction and hybridization capture were performed according to the same process as Example 2. The captured library was analyzed by the bioinformatics process, and the number of detected sequences and enrichment multiples of the samples were counted. The results of pathogen detection in clinical wax roll samples are shown in Table 16 below.

[0178] Table 16

[0179]

[0180] It can be seen from the data in Table 16 that wax roll samples are easily detected as negative in mNGS. After the capture method using this capture probe is applied, the conversion rate to positive is improved, and the number of reads detected in other targets is also greatly improved. Therefore, this capture probe can be better used in the detection of wax roll samples.

[0181] Example 8

[0182] Capture performance test of bronchoalveolar lavage fluid samples suspected of false-negative mNGS.

[0183] Eight bronchoalveolar lavage fluid samples with high clinical suspicion of infection but negative mNGS test results were selected, and extraction, library construction and hybridization capture were performed according to the same process as Example 2. The captured library was analyzed by the bioinformatics process, and the number of detected sequences of the sample was counted. The results are shown in Table 17 below.

[0184] Table 17

[0185]

[0186]

[0187] It can be seen from Table 17 that the capture method used by the capture probe has higher sensitivity. The capture method can increase the positive conversion rate and the detection rate, which is very helpful for the detection of low-load pathogens in clinical practice.

[0188] In summary, the capture probe group of the present invention can cover the detection of 119 types of bacteria, 32 types of fungi, 41 types of DNA viruses, and 71 types of RNA viruses, covering common infectious disease pathogens. The kit containing the capture probe can cover the detection of 88 types of drug resistance and virulence genes, greatly improving the detection performance of pathogenic microorganisms, and improving sensitivity and specificity.

[0189] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A capture probe set for detecting pathogenic microorganisms, characterized in that: The nucleic acid sequences of the capture probe group include sequences shown as SEQ ID NO.1-SEQ ID NO.

101.

2. The capture probe set according to claim 1, characterized in that The capture probe group specifically captures any one or a combination of at least two of the following pathogenic microorganism genome coding region sequences: Aspergillus flavus complex, Aspergillus fumigatus, Aspergillus niger complex, Aspergillus terreus complex, Candida glabrata, Candida albicans, Candida pseudoglabrata, Candida parapsilosis, Candida tropicalis, Pichia kudriavzevii, Candida guillainensis, Cryptococcus gattii, Cryptococcus neoformans, Fusarium, Histoplasma capsulatum, Rhizoctonia solani, Rhizoctonia umbrella-shaped, Rhizoctonia branched, Mucor irregular, Mucor racemosus, Pneumocystis jirovecii, Rhizomucor spp., Rhizomucor microsporus, Rhizopus spp., Rhizopus delbrueckii, Rhizopus microsporus, Rhizopus oryzae, Scedosporium spp., Scedosporium apiosporum, Scedosporium bodensis, Talaromyces marneffei, Trichosporon assasii, Streptococcus constellatus, Intermediate Prevotella, sweet Nocardia, Actinobacillus urinariae, Nocardia dermatophytes, Corynebacterium diphtheriae, Mycobacterium asiaticum, Mycobacterium avium, Mycobacterium avium complex, Mycobacterium cryptic, Mycobacterium gordonii, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoe, nontuberculous mycobacteria, Mycobacterium scrofulae, Mycobacterium schrenckii, Mycobacterium simianum, Mycobacterium surga, Mycobacterium tuberculosis complex, Mycobacterium tomentosa, Mycobacterium abscessus, Mycobacterium chelonae-abscessus complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium smegmatis, Nocardia, Nocardia abscessus, Nocardia asteroides, Nocardia brasiliensis, Nocardia concave, Nocardia st. georgia, Nocardia glanders, Nocardia otitis media, Nocardia terpenoides, Micromonas microsporus, Rhodococcus equi, Aureus aureus Staphylococci, Streptococcus agalactiae, Streptococcus anginosus group, Streptococcus intermedius, Streptococcus pneumoniae, Streptococcus pyogenes, Whipple's Trophic Barrier, Acinetobacter baumannii, Acinetobacter johnsii, Acinetobacter ursinus, Bacteroides fragilis, Homebortella, Bortella parapertussis, Bortella pertussis, Brucella, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia cepacia complex, Burkholderia glanders, Burkholderia pseudomallei, Burkholderia contamination, Burkholderia polyphaga, Anopheles Elizabethkinensis, Elizabethkinensis meningoseptica, Enterobacter cloacae complex, Escherichia coli, Fusobacterium necroticum, Fusobacterium nucleatum, Haemophilus influenzae, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella, Legionella, Bozeman Legionella, Legionella pneumophila, Long Beach Legionella, Mikdade Legionella, Moraxella catarrhalis, Neisseria meningitidis, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia, Nocardia africana, Nocardia neoformans, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Coxiella burnetii, Mycoplasma pneumoniae, Ureaplasma parvum, Ureaplasma urealyticum, Enterococcus faecium, Enterococcus faecalis, Proteus vulnerabilities, Enterobacter cloacae, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium marinum, Haemophilus parainfluenzae, Vibrio parahaemolyticus, Vibrio vulnificus, Rickettsia typhi, Streptococcus dysgalactiae, Mycoplasma hominis, Rickettsia prowazekii, Orientia tsutsugamushi, Clostridium perfringens,Morganella morganii, Salmonella enterica, Aspergillus nidulans, Clostridium tetani, Neisseria gonorrhoeae, Treponema pallidum, Yersinia pestis, Bacillus anthracis, Mycobacterium leprae, Leptospira renalis, Vibrio cholerae, Monkeypox virus, BK polyomavirus, Epstein-Barr virus, Herpes simplex virus type 2, Cytomegalovirus, Human polyomavirus type 2, Human polyomavirus type 3, Human polyomavirus type 4, Human herpesvirus 6A, Human herpesvirus 6B, Human herpesvirus type 7, Human herpesvirus type 8, Varicella-zoster virus, Human adenovirus, Hepatitis B virus, Human parvovirus B19, Human adenovirus type 11, Human adenovirus type 14, Human adenovirus type 1, Human adenovirus type 21, Human adenovirus type 2, Human adenovirus type 34, Human adenovirus type 35 type, human adenovirus type 3, human adenovirus type 4, human adenovirus type 55, human adenovirus type 57, human adenovirus type 5, human adenovirus type 6, human adenovirus type 7, human adenovirus group B, human adenovirus group C, human adenovirus group D, swine herpesvirus, human epileptovirus type 1, human epileptovirus type 2, human epileptovirus type 3, human bocavirus type 1, human bocavirus type 2, human bocavirus type 3, human bocavirus type 4, human coronavirus 229E, human coronavirus HKU1, human coronavirus NL63, human coronavirus OC43, human metapneumovirus, mumps virus type 2, mumps virus type 4, human respiratory virus type 1, human respiratory virus type 3, measles virus, mumps virus, rubella virus, dengue virus type 1, Dengue virus type 2, dengue virus type 3, dengue virus type 4, Middle East respiratory syndrome coronavirus, influenza B virus, influenza C virus, new coronavirus, Japanese encephalitis virus, influenza D virus, Hantavirus, human immunodeficiency virus type 1, human immunodeficiency virus type 2, rabies virus, human poliovirus type 1, Zika virus, Astrovirus, Hepatitis C virus, human rotavirus type A, human rotavirus type B, human rotavirus type C, rhinovirus, rhinovirus type A, rhinovirus type B, rhinovirus type C, new bunyavirus, influenza A virus H10N1, influenza A virus H2N2, influenza A virus H7N1, influenza A virus H7N2, influenza A virus H7N7, influenza A disease Virus H9N2, human hepatitis A virus, hepatitis GB virus C, coxsackievirus A10, coxsackievirus A16, coxsackievirus A2, coxsackievirus A5, coxsackievirus A6, coxsackievirus B3, echovirus E18, echovirus E30, enterovirus, enterovirus A, enterovirus A71, enterovirus B, enterovirus C, enterovirus D, enterovirus D68, influenza A virus, influenza B virus Victoria strain, influenza B virus Yamagata strain, human respiratory syncytial virus A, human respiratory syncytial virus B, influenza A virus, influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1,Influenza A virus H7N9, Acanthamoeba cabodzii, Acanthamoeba heli, Acanthamoeba pisiformis, Acanthamoeba parviflora, Babesia divergentis, Babesia ovale, Balamuthia baboon, Clonorchis sinensis, Cryptosporidium meleagridis, Cryptosporidium parvum, Cyclospora kayannensis, Leishmania ethiopiae, Leishmania amazonica, Leishmania braziliensis, Leishmania donovani, Leishmania gerbilis, Leishmania guianaensis, Leishmania infantum, Leishmania mexicanum, Leishmania panamaensis, Leishmania tropicalis, Naegleria fowleri, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Plasmodium vivax Schistosoma japonicum, Strongyloides stercoralis, Taenia fasciatus, Toxoplasma gondii, Trichuris trichuris, Trypanosoma cruzi, Wuchereria bancrofti, Acanthamoeba dijonnais, Acanthamoeba lugdunensis, Acanthamoeba, Acanthamoeba quina, Acanthamoeba ridge, Acanthamoeba triangularis, Angiostrongylus cantonensis, Cryptosporidium belli, Cryptosporidium hominis, Cryptosporidium ratis, Cryptosporidium theridii, Cryptosporidium spp., Cryptosporidium traveler, Echinococcus canadensis, Echinococcus granulosus, Entamoeba histolytica, Leishmania chaga, Leishmania, Paragonimus westermani, Brassica oleracea, Trichinella spiralis, Trichomonas oralis, Trichomonas vaginalis, Schistosoma mansoni or Echinococcus multilocularis.

3. The capture probe set according to claim 1 or 2, characterized in that: The capture probe group also includes sequences shown as SEQ ID NO.102-SEQ ID NO.

147.

4. The capture probe set according to any one of claims 1 to 3, characterized in that The capture probe group also specifically captures any one or a combination of at least two of the following drug-resistant genome sequences: CMV_UL97, CMV_UL54, MTB_ahpC, MTB_CycA, MTB_alr, MTB_ddlA, MTB_embA, MTB_embC, MTB_ethR, MTB_fbiA, MTB_fbiB, MTB_fbiC, MTB_fgd1, MTB_kasA, MTB_pepQ, MTB_rpoC, MTB_ddn, MTB_ald, MTB_fbiD, MTB_Mmpl5, MTB_atpE, MTB_eis, MTB_gidB, MTB_rplC, iroB, iroC, iroD, iroE, iroN, irp1, irp2, peg-344, peg-589, tox, ptxA , ptxB, ptxC, iutA, rmpA, rmpA2, iucA, iucB, iucC, iucD, rmtF, rmtE, LEN-1, GIM-2, GIM-1, SP M-1, vgaA, NmcA, IMI-1, SME-1, aac6-Ii, aac6, blaCTX-M-151, blaDHA-4, blaIMP-7, DIM, ermA , ermB, fosA3, mcr1, mecA, mecB, nimA, nimB, OXA-23, OXA-51, qnrA9, qnrB12, qnrS11, SIM, su l1, tetA, vanA, vanE, vanG, QnrA1, QnrA2, QnrC, QnrS2, QnrS1, SHV-1, KPC-3, CTX-M-2, or qnrB8.

5. The capture probe set according to any one of claims 1 to 4, characterized in that The capture probe set is modified with biotin.

6. The capture probe set according to any one of claims 1 to 5, characterized in that The length of the probes in the capture probe set is 85-95 bp.

7. The capture probe set according to any one of claims 1 to 6, characterized in that: The GC content of the probes in the capture probe group is 40%-60%.

8. A kit for detecting pathogenic microorganisms, characterized in that: The kit comprises the capture probe set according to any one of claims 1 to 7.

9. A library for detecting pathogenic microorganisms, characterized in that: The library for detecting pathogenic microorganisms is constructed using the kit described in claim 8.

10. A capture detection method for detecting pathogenic microorganisms, characterized in that: The capture detection method comprises: extracting the nucleic acid of the sample to be tested and constructing a library, mixing the constructed library with the capture probe group described in any one of claims 1-7 for hybridization capture, performing PCR amplification and purification on the hybridization capture product to obtain a capture library, and performing high-throughput sequencing on the above capture library.

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