A pathogen detection composition, kit and use thereof

Abstract

The present application belongs to the field of biotechnology, and in particular relates to a pathogen detection composition, a kit and use thereof, and more particularly relates to a pathogen detection composition, a kit and use thereof based on metagenomic sequencing. Using the composition of the present application, multiple types of targets can be captured and enriched simultaneously in one tube, and multiple pathogens and their derived mutant strains can be quickly identified. At the same time, through enrichment, the amount of data required for subsequent sequencing is significantly reduced, and the possibility of missing detection of low-load pathogens is avoided.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, relates to a pathogen detection composition, kit and use thereof, more particularly, to a pathogen detection composition, kit and use thereof based on metagenomic sequencing. BACKGROUND

[0002] With the acceleration of globalization, imported infectious disease monitoring has become an important part of China's infectious disease monitoring network. The detection of imported infectious disease pathogens mainly relies on technologies centered on fluorescent quantitative PCR and multiplex PCR. Traditional fluorescent quantitative PCR technology amplifies the target sequence of known pathogens through specific primers, which has high sensitivity, but it is limited by the pre-set single target and cannot cope with the detection of pathogen-derived variant strains. Although multiplex PCR can detect multiple pre-set targets (usually 10-30) at the same time, the number of targets is still limited by reaction system interference (such as primer cross-reaction) and the number of fluorescence channels, making it difficult to cover unknown or highly variable pathogens. Accordingly, the field can use metagenomic sequencing (mNGS) to detect the whole pathogen genome without bias through large-scale parallel sequencing. However, traditional mNGS is easily interfered by host nucleic acids, and has limited detection capability for low-load pathogens.

[0003] Therefore, the field needs a detection composition that can more accurately detect pathogens, especially low-load pathogens. SUMMARY

[0004] Therefore, in a first aspect, the present application provides a pathogen detection composition, comprising at least 4 types of targets in the probe set as shown below:

[0005] An Ebola-capturing probe set as shown in SEQ ID NO. 1-3;

[0006] An Oropouche virus-capturing probe set as shown in SEQ ID NO. 4-6;

[0007] A Corynebacterium diphtheriae-capturing probe set as shown in SEQ ID NO. 7-9;

[0008] A Bordetella pertussis-capturing probe set as shown in SEQ ID NO. 10-12;

[0009] An Enterohemorrhagic Escherichia coli O157:H7-capturing probe set as shown in SEQ ID NO. 13-15;

[0010] A Salmonella enterica-capturing probe set as shown in SEQ ID NO. 16-18;

[0011] A probe set for capturing Dengue fever as shown in SEQ ID NO. 19~21;

[0012] A probe set for capturing Klebsiella pneumoniae as shown in SEQ ID NO. 22~24;

[0013] A probe set for capturing Vibrio parahaemolyticus as shown in SEQ ID NO. 25~27;

[0014] A probe set for capturing Yellow fever virus as shown in SEQ ID NO. 28~30;

[0015] A probe set for capturing Hantavirus as shown in SEQ ID NO. 31~33;

[0016] A probe set for capturing Cholera CTX gene / Ol group / Ol 39 group as shown in SEQ ID NO. 34~36;

[0017] A probe set for capturing Chikungunya virus as shown in SEQ ID NO. 37~39;

[0018] A probe set for capturing Poliovirus as shown in SEQ ID NO. 40~42;

[0019] A probe set for capturing Influenza A virus as shown in SEQ ID NO. 43~45;

[0020] A probe set for capturing Mycobacterium tuberculosis as shown in SEQ ID NO. 46~48;

[0021] A probe set for capturing Staphylococcus aureus as shown in SEQ ID NO. 49~51;

[0022] A probe set for capturing Coxsackievirus as shown in SEQ ID NO. 52~54;

[0023] A probe set for capturing Crimean-Congo hemorrhagic fever virus as shown in SEQ ID NO. 55~57;

[0024] A probe set for capturing Lassa virus as shown in SEQ ID NO. 58~60;

[0025] A probe set for capturing Rift Valley fever phlebovirus as shown in SEQ ID NO. 61~63;

[0026] A probe set for capturing Shigella dysenteriae type 1 as shown in SEQ ID NO. 64~66;

[0027] A probe set for capturing Measles virus as shown in SEQ ID NO. 67~69;

[0028] a probe set for capturing Marburg virus as shown in SEQ ID NOs. 70~72;

[0029] a probe set for capturing Neisseria meningitidis as shown in SEQ ID NOs. 73~75;

[0030] a probe set for capturing Nipah virus as shown in SEQ ID NOs. 76~78;

[0031] a probe set for capturing Plasmodium as shown in SEQ ID NOs. 79~81;

[0032] a probe set for capturing Norovirus as shown in SEQ ID NOs. 82~84;

[0033] a probe set for capturing Tick-borne encephalitis virus as shown in SEQ ID NOs. 85~87;

[0034] a probe set for capturing Japanese encephalitis virus as shown in SEQ ID NOs. 88~90;

[0035] a probe set for capturing Entamoeba histolytica as shown in SEQ ID NOs. 91~93;

[0036] a probe set for capturing Yersinia pestis as shown in SEQ ID NOs. 94~96;

[0037] a probe set for capturing Bacillus anthracis as shown in SEQ ID NOs. 97~99;

[0038] a probe set for capturing Smallpox virus as shown in SEQ ID NOs. 100~102;

[0039] a probe set for capturing Venezuelan equine encephalitis virus as shown in SEQ ID NOs. 103~105;

[0040] a probe set for capturing West Nile virus as shown in SEQ ID NOs. 106~108;

[0041] a probe set for capturing Severe acute respiratory syndrome-related virus as shown in SEQ ID NOs. 109~111;

[0042] a probe set for capturing Simianpox virus as shown in SEQ ID NOs. 112~114;

[0043] a probe set for capturing Zika virus as shown in SEQ ID NOs. 115~117;

[0044] a probe set for capturing Middle East respiratory syndrome coronavirus as shown in SEQ ID NOs. 118~120;

[0045] a probe set for capturing human immunodeficiency virus as shown in SEQ ID NO. 121~123; or

[0046] a probe set for capturing novel coronavirus as shown in SEQ ID NO. 124~126.

[0047] Further, the present application provides a pathogen detection composition comprising at least 5 types of targets in the probe set as shown above.

[0048] Further, the present application provides a pathogen detection composition comprising at least 6 types of targets in the probe set as shown above.

[0049] Further, the present application provides a pathogen detection composition comprising at least 7 types of targets in the probe set as shown above.

[0050] Further, the present application provides a pathogen detection composition comprising at least 8 types of targets in the probe set as shown above.

[0051] Further, the present application provides a pathogen detection composition comprising at least 9 types of targets in the probe set as shown above.

[0052] Further, the present application provides a pathogen detection composition comprising at least 10 types of targets in the probe set as shown above.

[0053] Further, the present application provides a pathogen detection composition comprising at least 20 types of targets in the probe set as shown above.

[0054] Further, the present application provides a pathogen detection composition comprising at least 30 types of targets in the probe set as shown above.

[0055] Further, the present application provides a pathogen detection composition comprising at least 42 types of targets in the probe set as shown above.

[0056] Using the composition of the present application, multiple types of targets can be captured and enriched in one tube at the same time, and multiple pathogens and their derived mutant strains can be quickly identified. At the same time, the amount of data required for subsequent sequencing is significantly reduced through enrichment, avoiding the possibility of missing low-load pathogens.

[0057] It should be noted that the skilled person in the art knows that if 42 types of targets can be hybridized and captured in one tube at the same time, any combination of targets in the 42 types of targets can be hybridized and captured in one tube, which is beyond doubt.

[0058] Further, the present application provides a pathogen detection composition comprising:

[0059] a probe set for capturing Ebola as shown in SEQ ID NO. 1~3;

[0060] a probe set for capturing Oropouche virus as shown in SEQ ID NO. 4~6;

[0061] a probe set for capturing Corynebacterium diphtheriae as shown in SEQ ID NO. 7~9;

[0062] a probe set for capturing Bordetella pertussis as shown in SEQ ID NO. 10~12;

[0063] a probe set for capturing Escherichia coli O157:H7 as shown in SEQ ID NO. 13~15;

[0064] a probe set for capturing Salmonella enterica as shown in SEQ ID NO. 16~18;

[0065] a probe set for capturing Dengue fever as shown in SEQ ID NO. 19~21;

[0066] a probe set for capturing Klebsiella pneumoniae as shown in SEQ ID NO. 22~24;

[0067] a probe set for capturing Vibrio parahaemolyticus as shown in SEQ ID NO. 25~27;

[0068] a probe set for capturing Yellow fever virus as shown in SEQ ID NO. 28~30;

[0069] a probe set for capturing Hantavirus as shown in SEQ ID NO. 31~33;

[0070] a probe set for capturing Cholera CTX gene / O1 group / O139 group as shown in SEQ ID NO. 34~36;

[0071] a probe set for capturing Chikungunya virus as shown in SEQ ID NO. 37~39;

[0072] a probe set for capturing Poliovirus as shown in SEQ ID NO. 40~42;

[0073] a probe set for capturing Influenza A virus as shown in SEQ ID NO. 43~45;

[0074] a probe set for capturing Mycobacterium tuberculosis as shown in SEQ ID NO. 46~48;

[0075] a probe set for capturing Staphylococcus aureus as shown in SEQ ID NO. 49~51;

[0076] a probe set for capturing coxsackievirus as shown in SEQ ID NO. 52~54;

[0077] a probe set for capturing Crimean-Congo hemorrhagic fever virus as shown in SEQ ID NO. 55~57;

[0078] a probe set for capturing Lassa virus as shown in SEQ ID NO. 58~60;

[0079] a probe set for capturing Rift Valley fever phlebovirus as shown in SEQ ID NO. 61~63;

[0080] a probe set for capturing Shigella dysenteriae type 1 as shown in SEQ ID NO. 64~66;

[0081] a probe set for capturing Measles virus as shown in SEQ ID NO. 67~69;

[0082] a probe set for capturing Marburg virus as shown in SEQ ID NO. 70~72;

[0083] a probe set for capturing Neisseria meningitidis as shown in SEQ ID NO. 73~75;

[0084] a probe set for capturing Nipah virus as shown in SEQ ID NO. 76~78;

[0085] a probe set for capturing Plasmodium as shown in SEQ ID NO. 79~81;

[0086] a probe set for capturing Norovirus as shown in SEQ ID NO. 82~84;

[0087] a probe set for capturing Tick-borne encephalitis virus as shown in SEQ ID NO. 85~87;

[0088] a probe set for capturing Japanese encephalitis virus type B as shown in SEQ ID NO. 88~90;

[0089] a probe set for capturing Entamoeba histolytica as shown in SEQ ID NO. 91~93;

[0090] a probe set for capturing Yersinia pestis as shown in SEQ ID NO. 94~96;

[0091] a probe set for capturing Bacillus anthracis as shown in SEQ ID NO. 97~99;

[0092] a probe set for capturing Smallpox virus as shown in SEQ ID NO. 100~102;

[0093] a probe set for capturing Venezuelan equine encephalitis virus as shown in SEQ ID NO. 103~105;

[0094] a probe set for capturing West Nile virus as shown in SEQ ID NO. 106~108;

[0095] a probe set for capturing severe acute respiratory syndrome-related virus as shown in SEQ ID NO. 109~111;

[0096] a probe set for capturing simian pox virus as shown in SEQ ID NO. 112~114;

[0097] a probe set for capturing Zika virus as shown in SEQ ID NO. 115~117;

[0098] a probe set for capturing Middle East respiratory syndrome coronavirus as shown in SEQ ID NO. 118~120;

[0099] a probe set for capturing human immunodeficiency virus as shown in SEQ ID NO. 121~123; and

[0100] a probe set for capturing novel coronavirus as shown in SEQ ID NO. 124~126.

[0101] In some specific embodiments, each component of the composition is present in the same package.

[0102] Further, each component of the composition of the present application is present in a mixed form.

[0103] In a second aspect, the present application provides use of the above-mentioned composition for preparing a kit for detecting a pathogen.

[0104] Further, the kit further comprises a capture magnetic bead.

[0105] Further, the capture magnetic bead captures the target pathogenic nucleic acid fragment through a streptavidin-biotin "lock-key" tight structure.

[0106] In a third aspect, the present application provides a kit for detecting a pathogen, comprising the above-mentioned composition.

[0107] Further, the kit further comprises at least one of the following: a reagent required for nucleic acid extraction, a reverse transcription reagent, a library construction reagent, a reagent required for hybridization capture.

[0108] Further, the reagent required for nucleic acid extraction mainly utilizes that under high-salt and denaturing conditions, nucleic acid can be selectively bound to a silica gel membrane.

[0109] Further, the reagents required for reverse transcription are mainly adjusted by adjusting the buffer conditions to eliminate host nucleic acids by nuclease, and then the RNA in the solution is reverse transcribed into cDNA.

[0110] Further, the reagents required for library construction are first cut by fragmentation enzyme to fragment the nucleic acid, and then primers are added for library construction.

[0111] Further, the reagents required for hybrid capture are mainly combined with the target pathogen by probe Mix, and then the target pathogen is captured by the principle of streptavidin-biotin binding, and then the signal is amplified by PCR reaction.

[0112] In a fourth aspect, the present application provides a use of a composition for preparing high-throughput probe capture sequencing for detecting pathogens, wherein the detection comprises:

[0113] 1) extracting or releasing nucleic acids of the sample to be tested;

[0114] 2) processing the nucleic acids of the sample to be tested for first PCR amplification to construct a library;

[0115] 3) using the composition as described above for capture to obtain a capture product; and

[0116] 4) performing second PCR amplification on the capture product, sequencing and analyzing the results.

[0117] Further, the nucleic acids of the sample to be tested include DNA and / or RNA. Further, the RNA is reverse transcribed into cDNA.

[0118] Further, the library construction includes fragmenting the nucleic acids of the sample to be tested. Further, the PCR program for fragmentation is: 4℃, 1min; 30℃, 5min; 72℃, 5min; 4℃, hold.

[0119] Further, the conditions for the first PCR amplification are:

[0120]

[0121] Further, the conditions for the capture are: 98℃, 5min; 60℃, 45min; hold.

[0122] Further, the conditions for the second PCR amplification are:

[0123] BRIEF DESCRIPTION OF DRAWINGS

[0124] Figure 1 Library peak map of the library prepared by the present application;

[0125] Figure 2Coverage map for the novel coronavirus;

[0126] Figure 3 Coverage map for the Chikungunya virus. DETAILED DESCRIPTION

[0127] The present application will be specifically described below with reference to specific embodiments and examples, and the advantages and various effects of the present application will be more clearly presented thereby. It should be understood by those skilled in the art that these embodiments and examples are used to illustrate the present application, but not to limit the present application.

[0128] All embodiments and optional embodiments of the present application can be combined to form new technical solutions, if not specifically stated. All technical features and optional technical features of the present application can be combined to form new technical solutions, if not specifically stated.

[0129] All steps of the present application can be performed in sequence or randomly, if not specifically stated. For example, the method comprises steps (a) and (b), which means that the method can comprise steps (a) and (b) performed in sequence, or steps (b) and (a) performed in sequence. For example, the method can further comprise step (c), which means that step (c) can be added to the method in any sequence, for example, the method can comprise steps (a), (b) and (c), or steps (a), (c) and (b), or steps (c), (a) and (b), etc.

[0130] The terms used in the present application have the commonly understood meanings understood by those skilled in the art, if not specifically stated.

[0131] The terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting of the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising", when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0132] The following describes the embodiments of the present application. The embodiments described below are exemplary and are used to explain the present application, and should not be understood as limiting the present application. If not specified in the embodiments, the techniques or conditions described in the literature in the art or according to the product instructions are used. If not specified, the reagents or instruments used are conventional products that can be obtained on the market.

[0133] Example 1, the primer used in the present application

[0134] The pathogen probe design dynamically judges the binding ability of the probe to the target region through four key hybridization parameters: mismatch tolerance, flanking coverage length, longest common subsequence, and the number of bases of exact match. For the conserved and specific regions of the pathogen of interest, a probe set as used in the present application is designed as shown in Table 1, ultimately achieving high sensitivity and low cost capture of broad-spectrum pathogens.

[0135] Table 1

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] Example 2, method for capturing different targets based on sequencing

[0146] 1. Experimental reagents and instruments

[0147] Reagents required

[0148] Nucleic acid extraction: QIAamp Viral RNA Mini Kit;

[0149] Carrier RNA oligonucleotides; Buffer AVL* denaturing lysis solution; Buffer AW1 protein removal solution; Buffer AW2 ion removal solution; QIAamp Mini Spin Columns / silica gel membrane filter column with adsorption membrane; Collection Tubes / waste collection filter collection tubes; Buffer AVE 0.04% sodium azide.

[0150] Reverse transcription kit: VMRS0107;

[0151] Pre-treatment enzyme DNA hydrolysis enzyme; hybridization probe NS ribosome oligonucleotide probe; reverse transcriptase (Oligo DT) RNA reverse transcriptase; chain enzyme mixture TMgCl2, EDTA, PH=8.5; random hexamer Random hexamer (NNNNNN).

[0152] Transposase method library construction kit: VMRS0108;

[0153] Fragmentation enzyme (Tn5) Tn5 transposase; fragmentation buffer 10 mM Tris, MgCl2, EDTA, PH=8.5; termination solution 100 nM NaCl solution; primer amplification primer; amplification buffer 10 mM Tris, 50 mM KCl, 1.5 mM MgCl2, PH=8.5; amplification enzyme DNA polymerase; NF water nanofiltration water.

[0154] Hybrid capture enrichment kit: VMRS0112-5;

[0155] 0.9x magnetic beads / capture purification magnetic beads Streptavidin-bound magnetic beads, NaCl, sodium azide; enrichment probe MIX Oligonucleotide probe (SEQ01-1260); blocking agent F Oligonucleotide probe blocking Ttruseq adapter; blocking agent Q Oligonucleotide probe blocking human genome repeat region; hybridization mixture S Paraffin oil-water mixture; amplification primer C Hot-start amplification primer; amplification enzyme C Hot-start DNA polymerase.

[0156] Hybrid capture cleaning solution kit: VM028

[0157] Magnetic bead buffer Tris-HCl, EDTA, NaCl; purification magnetic beads Magnetic beads; magnetic bead cleaning solution SSC buffer, SDS.

[0158] 2 Experimental steps

[0159] 2.1 Extraction of nucleic acid

[0160] 1) After centrifugation of the blood sample, the plasma was separated, and the swab sample was thoroughly blown and mixed. Take 200 μL of the sample, add 5.6 μL of Carrier RNA and 560 μL of AVL lysis solution, gently shake for 15 seconds, and stand at room temperature for 10 minutes. (Note: Carrier RNA should not be repeatedly frozen and thawed more than 3 times.)

[0161] 2) After a brief centrifugation of 5 seconds, 560 μL of absolute ethanol was added, gently inverted to mix, avoiding vigorous shaking, and then centrifuged again briefly.

[0162] 3) Transfer 630 μL of the mixture to the adsorption column, centrifuge at 8000 rpm for 1 minute, and transfer all the liquid in batches.

[0163] 4) Add 500 μL Buffer AW1, centrifuge at 8000 rpm for 1 min, discard the collection tube, and replace it with a new 2 mL collection tube.

[0164] 5) Add 500 μL Buffer AW2, centrifuge at 13000 rpm for 3 min, discard the collection tube, and replace it with a new 2 mL collection tube.

[0165] 6) Perform empty spin, centrifuge at 13000 rpm for 1 min.

[0166] 7) Place the adsorption column on a clean 1.5 mL centrifuge tube, open the cap and dry for 1 min.

[0167] 8) Add 70 μL AVE buffer to the center of the membrane, tightly cap the tube and place it for 1 min, centrifuge at 8000 rpm at room temperature for 1 min, and collect the liquid.

[0168] 2.2 RNA hybridization, denaturation and single-strand synthesis

[0169] RNA purification: take 12 μL of the extracted nucleic acid, add 3 μL of pretreated enzyme T, and place it in a PCR instrument for reaction. The reaction system is as follows: 42°C, 10 min; 85°C, 30 s → 4°C, forever, and the hot cover needs to be covered. Add 2 μL of hybridization probe N to the above product, and the hybridization process is as follows: PCR instrument: 75°C, 1 min; 50°C, 2 min; 4°C, forever

[0170] Single-strand synthesis: in the above product, add 2 μL of reverse transcriptase mixture T, 2 μL of chain enzyme mixture T, and 1 μL of random hexamer T, and place it in a PCR instrument. PCR instrument: 25°C, 5 min → 42°C, 10 min → 85°C, 5 min → 4°C, forever, cover the hot cover.

[0171] 2.3 Fragmentation

[0172] Take another part of the nucleic acid, measure the DNA concentration with qubit, dilute it to 1 ng / μl of DNA nucleic acid, and prepare 30 μl in advance, totaling 30 ng. In the above reaction solution, add 30 ng of DNA nucleic acid, 5 μl of fragmentation buffer, and 2 μl of fragmentation enzyme Tn5, and place it in a PCR instrument. PCR program: 4°C, 1 min; 30°C, 5 min; 72°C, 5 min; 4°C, hold.

[0173] 2.4 First round of PCR (library construction)

[0174] For the above products, add 7 μl of primer, 50 μl of amplification buffer T, 1 μl of amplification enzyme T, and 3 μl of NF water. After mixing, place it in the PCR instrument. PCR program: (hot cover) 95°C, 3 min; (98°C, 20 s; 60°C, 15 s; 72°C, 30 s), 15 cycles; 72°C, 5 min; 4°C, hold.

[0175] The library concentration is determined using a commercial kit, and the library peak chart is shown in Figure 1 .

[0176] 2.5 Hybrid capture

[0177] The capture magnetic beads need to be cleaned and resuspended according to the instructions before use.

[0178] - Library pooling: After measuring the library concentration, calculate the volume of the captured library that needs to be put in (the total amount of each library is not more than 2500 ng, the total number of libraries is not more than 25, and the volume of each library is not more than 45 μl). According to the total volume after mixing, add 0.9X capture magnetic beads, and stand at room temperature for 10 min. Place it on the magnetic stand for 10 min until the liquid is clear, and discard the supernatant; add 1 mL of 80% ethanol, shake the tube for 2 weeks, discard the supernatant, and repeat once. Dry at 37°C until the surface is matte. Add 14 μL of NF water (preheated at 60°C for 60 min), mix well by blowing, and elute at 60°C in a constant temperature metal bath for 5 min. Place it on the magnetic stand for 2 min, collect 9 μL of liquid; measure the concentration of the purified product.

[0179] - Configure the hybridization pre-mix: take 7 μl of the purified product, add 2.5 μl of enrichment probe mix (Table 1), 2 μl of blocking agent F, 3.5 μl of blocking agent Q, and 3.5 μl of NF water.

[0180] - Add 25 μL of hybridization mix S (preheated at 65°C for 60 min) and 30 μL of hybridization enhancer to the hybridization pre-mix. Perform PCR. PCR program: 98°C, 5 min; 60°C, 45 min; 60°C, forever; hot cover: 85°C.

[0181] - Capture: Add the resuspended magnetic beads to the hybridization suspension, and incubate for 15 min (2.5 s / revolution) in a standard suspension instrument. Place it on the magnetic stand for 3 min, and discard the supernatant. Add 100 uL of 68°C magnetic bead cleaning solution 1, mix well by blowing, incubate for 5 min, place it on the magnetic stand for 1 min, and discard the supernatant; repeat the above steps once. Transfer to a new tube, add 150 μL of 48°C magnetic bead cleaning solution 2, mix well by blowing, incubate for 1 min, place it on the magnetic stand for 1 min, and discard the supernatant; repeat once (without changing the new tube). Add 50 μL of NF-H2O to the magnetic beads without drying, and mix well by shaking.

[0182] 2.6 Second round of PCR

[0183] Take 12.5 μl of the captured magnetic beads, add 5 μl of adapter conversion amplification primer C, 25 μl of amplification enzyme C, and 7.5 μl of NF water. Follow the PCR program: 95°C for 3 min; (98°C for 10 s, 65°C for 15 s, 72°C for 15 s) for 25 cycles; 72°C for 3 min; 4°C forever.

[0184] 2.7 Library purification and sequencing

[0185] Add 80 μl of purification magnetic beads to the PCR product, shake and stand at room temperature for 5 min, place on the magnetic stand for 3 min, and discard the supernatant. Add 200 μL of 80% ethanol prepared with NF water, rotate the tube for 2 weeks, discard the supernatant, and repeat once. Dry in a metal bath at 37°C until the surface of the magnetic beads is matte, add 50 uL of NF water for elution. Place on the magnetic stand for 2 min, and collect 45 uL of supernatant.

[0186] After the final determination of the concentration, sequencing is performed, and the pre-allocated data amount for each sample is 4M.

[0187] Example 3, detection results of test samples of the composition of the present application

[0188] The 42-pathogen mixed sample detected by the present application was detected using the composition shown in Table 1 according to the method described in Example 2, and the detection results are shown in Table 2. As can be seen from the table, the 42 pathogens of the present application are all captured and finally detected. For example, the coverage of the novel coronavirus and the chikungunya virus is shown in Figure 2 and Figure 3 .

[0189] Table 2

[0190]

[0191]

Claims

1. A pathogen detection composition comprising a probe set as shown below: an Ebola capturing probe set as shown in SEQ ID NO. 1~3; an Oropouche virus capturing probe set as shown in SEQ ID NO. 4~6; a Corynebacterium diphtheriae capturing probe set as shown in SEQ ID NO. 7~9; a Bordetella pertussis capturing probe set as shown in SEQ ID NO. 10~12; an Enterohemorrhagic Escherichia coli O157:H7 capturing probe set as shown in SEQ ID NO. 13~15; a Salmonella enterica capturing probe set as shown in SEQ ID NO. 16~18; a Dengue capturing probe set as shown in SEQ ID NO. 19~21; a Klebsiella pneumoniae capturing probe set as shown in SEQ ID NO. 22~24; a Vibrio parahaemolyticus capturing probe set as shown in SEQ ID NO. 25~27; a Yellow fever virus capturing probe set as shown in SEQ ID NO. 28~30; a Hantavirus capturing probe set as shown in SEQ ID NO. 31~33; a Cholera CTX gene / Ol group / Ol 39 group capturing probe set as shown in SEQ ID NO. 34~36; a Chikungunya virus capturing probe set as shown in SEQ ID NO. 37~39; a Poliovirus capturing probe set as shown in SEQ ID NO. 40~42; an Influenza A virus capturing probe set as shown in SEQ ID NO. 43~45; a Mycobacterium tuberculosis capturing probe set as shown in SEQ ID NO. 46~48; a Staphylococcus aureus capturing probe set as shown in SEQ ID NO. 49~51; a Coxsackievirus capturing probe set as shown in SEQ ID NO. 52~54; a Crimean-Congo hemorrhagic fever virus capturing probe set as shown in SEQ ID NO. 55~57; a Lassa virus capturing probe set as shown in SEQ ID NO. 58~60; a Rift Valley fever phlebovirus capturing probe set as shown in SEQ ID NO. 61~63; a Shigella dysenteriae type 1 capturing probe set as shown in SEQ ID NO. 64~66; a Measles virus capturing probe set as shown in SEQ ID NO. 67~69; a Marburg virus capturing probe set as shown in SEQ ID NO. 70~72; a Neisseria meningitidis capturing probe set as shown in SEQ ID NO. 73~75; a Nipah virus capturing probe set as shown in SEQ ID NO. 76~78; a Plasmodium capturing probe set as shown in SEQ ID NO. 79~81; a Norovirus capturing probe set as shown in SEQ ID NO. 82~84; a Tick-borne encephalitis virus capturing probe set as shown in SEQ ID NO. 85~87; a Japanese encephalitis virus type B capturing probe set as shown in SEQ ID NO. 88~90. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ a probe set for capturing Entamoeba histolytica as shown in SEQ ID NO. 91~93; a probe set for capturing Yersinia pestis as shown in SEQ ID NO. 94~96; a probe set for capturing Bacillus anthracis as shown in SEQ ID NO. 97~99; a probe set for capturing Variola virus as shown in SEQ ID NO. 100~102; a probe set for capturing Venezuelan equine encephalitis virus as shown in SEQ ID NO. 103~105; a probe set for capturing West Nile virus as shown in SEQ ID NO. 106~108; a probe set for capturing Severe acute respiratory syndrome-related virus as shown in SEQ ID NO. 109~111; a probe set for capturing Simianpox virus as shown in SEQ ID NO. 112~114; a probe set for capturing Zika virus as shown in SEQ ID NO. 115~117; a probe set for capturing Middle East respiratory syndrome coronavirus as shown in SEQ ID NO. 118~120; a probe set for capturing Human immunodeficiency virus as shown in SEQ ID NO. 121~123; and a probe set for capturing Novel coronavirus as shown in SEQ ID NO. 124~126.

2. The composition of claim 1, wherein, Each component of the composition is present in a mixed form.

3. Use of the composition of claim 1 or 2 for preparing a kit for detecting pathogens by sequencing.

4. Use according to claim 3, characterized in that, The kit further comprises capture magnetic beads.

5. Use according to claim 3 or 4, characterized in that, The capture magnetic beads capture the target pathogenic nucleic acid fragments through a "lock-and-key" tight structure of streptavidin-biotin.

6. A kit for detecting pathogens by sequencing, comprising the composition of claim 1 or 2.

7. The kit of claim 6, wherein The kit further comprises at least one of the following: reagents required for nucleic acid extraction, reverse transcription reagents, library construction reagents, and hybridization capture required reagents.

8. Use of a composition for the preparation of a high flux probe capture sequencing assay for the detection of a pathogen, wherein, The detection comprises: 1) extracting or releasing nucleic acids of the sample to be tested; 2) processing the nucleic acids of the sample to be tested for first PCR amplification to construct a library; 3) using the composition of claim 1 or 2 for capture to obtain a capture product; and 4) performing second PCR amplification on the capture product, sequencing and analyzing the results.

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