Method for obtaining respiratory tract multi-pathogen whole genome by using probe hybridization capture technology, probe group and kit

Abstract

The invention discloses a method, a probe set and a kit for obtaining a respiratory tract multi-pathogen whole genome by using a probe hybridization capture technology, primers designed in a targeted manner can effectively perform WGS genome almost full-length amplification on respiratory tract multi-pathogens; in the design, the parameters of the primers are strictly limited, so that the mixed system of the primers can be effectively amplified under the same condition in one step.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method, a probe set and a kit for obtaining the whole genome of multiple respiratory pathogens by using probe hybridization capture technology. Background Art

[0002] Respiratory multi-pathogens refer to multiple pathogens that infect the respiratory tract simultaneously or successively, causing respiratory diseases such as acute upper respiratory tract infection, bronchitis or pneumonia. These pathogens may include bacteria, fungi, viruses, mycoplasmas and chlamydia. Respiratory multi-pathogen detection uses PCR amplification technology to detect whether there are respiratory pathogens in the sample, and cannot directly obtain the whole genome of the pathogen. If you want to obtain the whole genome of respiratory multi-pathogens, you need to conduct pathogen sequencing experiments on each detected pathogen separately. Most pathogens can be sequenced by amplicon sequencing like influenza viruses; however, amplicon sequencing is not suitable for rhinoviruses, which have many types.

[0003] Probe capture technology, especially probe capture sequencing, is a high-throughput sequencing technology with extensive and in-depth principles and applications. The core principle of probe capture sequencing is to use specific nucleic acid probes to efficiently and specifically enrich the target area, and then read it through a sequencer to obtain high-quality sequencing data of the target area. In addition, there is a probe capture method based on liquid phase hybridization capture technology, which uses the principle of complementary hybridization of nucleic acid molecule bases to design molecular probes modified with biotin. After the probe hybridizes and binds to the target area, the probe is adsorbed by streptavidin magnetic beads, thereby capturing the target area. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0005] The present invention provides a probe group for detecting multiple respiratory pathogens, wherein the probe group is a nucleotide sequence shown in SEQ ID NO: 1-5000.

[0006] Furthermore, the probes in the probe set consist of two parts: a specific sequence for specifically binding to the target DNA region, and a common sequence for subsequent amplification.

[0007] Furthermore, the probe is labeled with a fluorescent dye.

[0008] Furthermore, the fluorescent dye labels include FAM, HEX, TAMRA, ROX, TET, JOE, Cy3, Cy5, VIC, Cy5.5, Texas Red, NED, FAM-dT, TAMRA-dT, CY3-dT, CY5-dT, HEX-dT, and FITC.

[0009] Furthermore, the probe includes a quencher group.

[0010] Furthermore, the quenching group includes BHQ1, TAMRA, BHQ2, Rhodamine 6G, Rhodamine B, BHQ3, Dabcyl, Eclipse, MGB, BHQ1-dT, and BHQ2-dT.

[0011] Furthermore, the probe is labeled with one or more modifying groups.

[0012] Furthermore, the modification groups include amine groups, phosphate groups, biotin, biotin-TEG, and C3-spacer.

[0013] In the present invention, modified nucleotides include but are not limited to 2'-modified nucleotides or 5-methylcytosine. Among them, 2'-modified nucleotides include but are not limited to 2'-O-methyl modified nucleotides, 2'-fluorine modified oligonucleotides; 5-methylcytosine includes but is not limited to 5-methyl-deoxycytosine; 5-methyl-deoxycytosine includes but is not limited to 5-Me-dC, 5-methyl-2'-deoxycytosine. In some embodiments, the primer may include two or more modified nucleotides. Two or more modified nucleotides may have the same or different modifications. In some embodiments, the primer set may include one or more 5-methylcytosines, and 5-methylcytosine includes. The primer set may have 0, 1, 2, 3, 4, 5, 6, 7, 8 or more 2'-O-methyl modified nucleotides, 2'-fluorine modified oligonucleotides, 5-methylcytosine or a combination thereof.

[0014] In the primers of the present invention labeled with a labeling substance, the labeling substance may be directly bound to the primer or may be bound via a linker. As a linker, any linker commonly used in the field may be used, specifically, for example, preferably a nucleic acid of 1 to 3 bases, more preferably a DNA of 1 to 3 bases, further preferably a DNA of 2 bases, and particularly preferably the 2 bases of adenine (A)-adenine (A).

[0015] Furthermore, the respiratory multi-pathogens include coronavirus (229E / HKU1 / NL63 / OC43), enterovirus (A / B / C / D group), influenza virus (A / B), bocavirus, human adenovirus (B / C / D / E group), metapneumovirus, parainfluenza virus (type 1 / 2 / 3 / 4), rhinovirus (A / B / C), astrovirus, mumps virus, measles virus, rubella virus, new coronavirus, syncytial virus, varicella-zoster virus, SARS virus, norovirus, dengue virus, and rotavirus.

[0016] The present invention provides a product for detecting multiple respiratory pathogens, and the product comprises the above-mentioned probe group.

[0017] Furthermore, the products include nucleic acid membrane strips, test kits, and chips.

[0018] Furthermore, the kit also includes reagents required for hybridization capture, buffer and amplification enzyme required for PCR amplification.

[0019] Furthermore, the kit also contains a reaction system required for reverse transcription.

[0020] Furthermore, the reaction system required for the reverse transcription includes random primers.

[0021] Furthermore, the kit also includes a double-stranded cDNA synthesis kit, specifically Random Primer, Reaction Mix 1, Enzyme Mix 1, Reaction Mix 2, Enzyme Mix 2, EDTA, RNase I, and Nuclease-free water.

[0022] Furthermore, the kit also includes a library construction kit, specifically Frag / AT Enzymes, Frag / AT Buffer, Universal Adapters, Ligation Master Mix, Index, Amplification Mix, and Nuclease-free water.

[0023] Furthermore, the kit also includes a rapid hybridization capture kit A, specifically Probe Panel, Universal Blockers, Blocker Solution, Hybridization Mix, Enhancer, Amp Primers, Amplification Mix, and Nuclease-free water.

[0024] Furthermore, the kit also includes a rapid hybridization capture kit B, specifically Streptavidin Binding Beads, Fast Binding Buffer, Fast Wash Buffer 1, Wash Buffer 2, and DNA Purification Beads.

[0025] In some embodiments, the primer or probe is present in a suitable dosage or is provided in one or more containers, or is fixed on a matrix, wherein the primer or probe can be provided as a suspension in an aqueous solution, or, for example, as a freeze-dried or lyophilized powder. On the other hand, as a container for carrying the primer or probe, it can be any conventional container that can accommodate the provided form, such as a microcentrifuge tube, an ampoule or a bottle.

[0026] The present invention provides a method for detecting multiple respiratory pathogens, which comprises the steps of adding the aforementioned probe set into the same container and detecting the whole genome of multiple respiratory pathogens by hybridization capture of the sample.

[0027] Further, the method comprises the following steps:

[0028] 1) Collect samples and prepare them into a tag library;

[0029] 2) Add the aforementioned probe set, Universal Blocker, and Blocker Solution to the library;

[0030] 3) Add DNA purification beads, mix well and incubate at room temperature for 5 minutes, place in a magnetic field and discard the supernatant, wash with ethanol, and repeat once or multiple times;

[0031] 4) Place in a magnetic field to remove excess ethanol, and immediately add Hybridization Mix preheated to 65°C to resuspend;

[0032] 5) After brief centrifugation, add Enhancer;

[0033] 6) Heat at 95°C for 5 minutes, then immediately heat at 60°C for 2 hours;

[0034] 7) Add Streptavidin Binding Beads and Fast Binding Buffer in a volume ratio of 1:2, mix well, place in a magnetic field and discard the supernatant, repeat once or multiple times;

[0035] 8) Add Fast Binding Buffer to resuspend the magnetic beads and hybridization is complete;

[0036] 9) After the hybridization is completed, the product is immediately transferred to a container containing washed Streptavidin Binding Beads, and the ambient temperature of the product is kept greater than or equal to 60° C. and mixed;

[0037] 10) Mix the solution in step 9 at a mixing speed of 600 rpm for 30 min, centrifuge, change the container, let it stand in a magnetic field, and discard the supernatant;

[0038] 11) Add Fast Wash Buffer 1 preheated at 68°C, mix well, incubate at 68°C for 5 min, place in a magnetic field and discard the supernatant, repeat once or more;

[0039] 12) Add Wash Buffer 2 preheated at 48°C, mix well, centrifuge, incubate at 48°C for 5 min, place in a magnetic field and discard the supernatant, repeat once or more;

[0040] 13) After aspirating Wash Buffer 2, immediately add Nucleasa-free water, mix well and incubate;

[0041] 14) Perform PCR amplification and magnetic bead purification;

[0042] 15) Sequencing analysis.

[0043] Furthermore, the PCR amplification is multiplex PCR amplification.

[0044] Furthermore, the amplification reaction is performed under similar amplification conditions.

[0045] Furthermore, the samples include human samples and environmental samples, and the human samples are in vitro human samples.

[0046] Furthermore, the human sample includes one or a combination of tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, tissue culture fluid, tissue extract, homogenized tissue, tumor tissue, cell extract, alveolar lavage fluid, nasopharyngeal / oropharyngeal swab, and sputum.

[0047] Furthermore, the human samples include alveolar lavage fluid, nasopharyngeal / oropharyngeal swabs, and sputum.

[0048] Furthermore, the environmental sample includes a water source.

[0049] Furthermore, the water source includes sewage.

[0050] Furthermore, the sewage includes sewage from wastewater treatment plants, rivers, and sewer systems.

[0051] Furthermore, the method is a method for non-diagnostic purposes.

[0052] Furthermore, the collection of samples to prepare a tag library includes the following specific steps: sample preparation, and tag library preparation.

[0053] Further, sample preparation includes:

[0054] Extract nucleic acid according to the corresponding requirements and steps in the commercial viral nucleic acid extraction kit.

[0055] Double-stranded cDNA synthesis was performed according to the corresponding requirements and steps of the commercial double-stranded cDNA synthesis kit, and the double-stranded cDNA product was purified using 1.2 times the volume of DNA purification beads.

[0056] Further, the purification of the double-stranded cDNA product specifically comprises the following steps:

[0057] 1) Add DNA Purification Beads to the double-stranded cDNA product, mix well, and incubate at room temperature for 5 minutes; place in a magnetic field and discard the supernatant;

[0058] 2) Add 80% ethanol in a magnetic field, discard the supernatant after 30 seconds, and repeat once or more;

[0059] 3) Absorb all ethanol in the magnetic field, add water, mix well and incubate at room temperature for 5 minutes;

[0060] 4) Place in a magnetic field to obtain a product.

[0061] Furthermore, the tag library preparation includes: using the corresponding requirements and steps in the library construction kit to perform endonuclease shearing, adding universal adapters and magnetic bead purification, PCR amplification and purification.

[0062] Further, the PCR amplification and magnetic bead purification in step 14 includes performing PCR amplification using the PCR amplification components in the hybrid capture kit A, and,

[0063] Magnetic bead purification steps: 1) Add DNA purification beads, mix well, and incubate at room temperature for 5 minutes;

[0064] 2) Place in a magnetic field and discard the supernatant, add 80% ethanol, discard the supernatant after 30 seconds, and repeat once or more;

[0065] 3) Remove all ethanol in a magnetic field and dry at room temperature for 5 minutes;

[0066] 4) Remove from the magnetic field, add water, mix well, and incubate at room temperature for 2 minutes.

[0067] In the present invention, amplification refers to the generation of multiple copies of a polynucleotide or a portion of a polynucleotide, usually starting from a small amount of polynucleotide (e.g., a single polynucleotide molecule), and the amplified product obtained is also called an amplicon, which is usually detectable. The amplification of polynucleotides includes a variety of chemical and enzymatic processes.

[0068] The present invention embodiment part provides the exemplary embodiment of amplification condition.However, the term "amplification condition" used herein relates to the temperature and / or incubation time that are suitable for obtaining the detectable amount of target.Therefore, the term "similar amplification condition" means that if desired, each target can be assayed at a similar temperature.The term "similar amplification condition" also refers to if desired, each target can be assayed under similar incubation time.In some cases, the term "similar amplification condition" also relates to the number of amplification cycles.However, it is well known in the art that the number of cycles is not always strict.For example, some samples can be removed or left before other samples for additional amplification cycles.In other cases, the term "similar amplification condition" also relates to the properties of the buffer used and the amplification reagent (enzyme, nucleotide, salt, etc.).The term "similar amplification condition" also refers to that conditions (e.g., time, buffer, number of cycles, temperature, etc.) can be slightly changed or can be the same.

[0069] The present invention provides a library for detecting multiple respiratory pathogens, wherein the library comprises the probe set described above.

[0070] The present invention provides the use of the aforementioned probe set in preparing products for amplifying or detecting multiple respiratory pathogens.

[0071] The present invention provides the aforementioned probe set and the use of the aforementioned product in detecting multiple respiratory pathogens for non-diagnostic purposes.

[0072] Advantages and beneficial effects of the present invention:

[0073] This product combines enzyme fragmentation technology with hybrid capture target enrichment, which can shorten the workflow time and generate accurate and reliable analysis results for target enrichment resequencing. Biotin-labeled specific probes are used to hybridize with sample DNA / cDNA, and streptavidin magnetic beads are used to adsorb hybrid fragments to capture the whole genome of respiratory multi-pathogens. Subsequently, high-throughput sequencing is used to obtain the whole genome sequence information of respiratory multi-pathogens. The library obtained by this kit can be directly connected to the Illumina sequencing platform for second-generation sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1It is a detection principle diagram of the present invention for detecting multiple respiratory pathogens. DETAILED DESCRIPTION

[0075] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0076] Detection principle: This product combines enzyme fragmentation technology with hybrid capture target enrichment, which can shorten the workflow time and generate accurate and reliable analysis results for target enrichment resequencing. Use biotin-labeled specific probes to hybridize with sample DNA / cDNA, and use streptavidin magnetic beads to adsorb hybrid fragments to capture the whole genome of various respiratory pathogens. Subsequently, high-throughput sequencing is used to obtain the whole genome sequence information of various respiratory pathogens. The library obtained by this kit can be directly connected to the Illumina sequencing platform for second-generation sequencing. Figure 1 shown.

[0077] Example 1 Kit for detecting multiple respiratory pathogens

[0078] Main components: as shown in Table 1.

[0079] Table 1

[0080]

[0081]

[0082]

[0083]

Storage and transportation conditions

[0084] Transportation: It must be transported under low temperature or frozen conditions. Dry ice cannot be used during transportation of Rapid Hybridization Capture Kit B.

[0085] Storage: Store the corresponding kit at -20℃ or 4℃ according to the storage conditions in the above table. Try to avoid repeated freezing and thawing of the reagents.

[0086] Validity period: one year

[0087]

Applicable instruments

[0088] This kit can be used on common PCR instruments such as ABI and Bio-Rad.

[0089]

Equipment Consumables

[0090] Vortex mixer, rotary mixer / oscillator, centrifuge, PCR instrument, Qubit quantifier, pipette, 1.5ml centrifuge tube, PCR tube, etc. Specific instruments include Qubit with Thermo catalog number Q33238 TM 4 Fluorometers with WiFi

[0091] Quantitative equipment; Bio-RAD T100 thermal cycler PCR amplification and incubation equipment with item number 1861096; Qilin Bell BE-1100 four-dimensional rotating mixer magnetic bead mixing and streptavidin magnetic bead binding reaction equipment with item number BE-1100.

[0092]

Sample requirements

[0093] Samples: alveolar lavage fluid, nasopharyngeal / oropharyngeal swabs, sputum, etc. The above samples can be used for whole genome probe hybridization capture of multiple respiratory pathogens after nucleic acid extraction.

[0094] Example 2 Method for detecting multiple respiratory pathogens

[0095] 1. Sample preparation

[0096] 1. Sample nucleic acid extraction

[0097] Extract according to the corresponding requirements and steps in the viral nucleic acid extraction kit. The final elution volume is recommended to be 20-50μl. Thermo Fisher MagMAX is recommended. TM Virus / Pathogen Nucleic Acid Isolation Kit (Cat. No. A42352) was used for nucleic acid extraction.

[0098] If the extracted viral nucleic acid is not used for subsequent experiments, it should be stored at -80℃ and avoid repeated freezing and thawing. It should be thawed on ice or at 4℃ before use.

[0099] 2. Double-stranded cDNA synthesis

[0100] Place the components of the double-stranded cDNA synthesis kit on ice or thaw at 4°C, and centrifuge instantly to collect the reagents on the tube wall. After centrifugation, place them on ice for later use.

[0101] Add the components in Table 2 to an RNase-free PCR tube on ice.

[0102] Table 2

[0103] Reagents volume Random Primer 1μl RNA template 13μl Total volume 14μl

[0104] Gently mix the reaction solution in Table 2, centrifuge briefly, and then incubate according to the procedure in Table 3.

[0105] Table 3

[0106] temperature time 85℃ Heat cover 65℃ 5min 4℃ Hold

[0107] After the incubation, place the reaction solution on ice and add the reagents in Table 4.

[0108] Table 4

[0109] Reagents volume Previous step reaction solution 14μl Reaction Mix 1 5μl Enzyme Mix 1 1μl Total volume 20μl

[0110] Mix the reaction solution in Table 4, centrifuge briefly, and incubate according to the procedure in Table 5.

[0111] Table 5

[0112] temperature time 105℃ Heat cover 25℃ 10min 50℃ 30min 85℃ 5min 4℃ Hold

[0113] Place the reaction solution in Table 5 on ice and add the reagent in Table 6.

[0114] Table 6

[0115] Reagents volume Previous step reaction solution 20μl Nuclease-free water 55μl Reaction Mix2 20μl Enzyme Mix 2 5μl Total volume 100μl

[0116] Gently mix the reaction solution in Table 6, centrifuge briefly, and incubate according to the procedure in Table 7.

[0117] Table 7

[0118] program temperature time - OFF Heat cover 1 16℃ 60min 2 4℃ Hold

[0119] After the incubation, add 6 μl EDTA to the reaction solution on ice and mix gently to terminate the reaction.

[0120] Add 10 μl RNase I to the reaction solution, mix gently, and incubate at room temperature for 5 min.

[0121] If no subsequent experiments are to be performed, store at -20°C and place on ice or thaw at 4°C before use.

[0122] 3. Double-stranded cDNA purification

[0123] Use 1.2 times the volume of DNA Purification Beads to purify the double-stranded cDNA product. DNA Purification Beads need to be equilibrated at room temperature for 30 minutes and shaken to mix, otherwise it will affect the purification recovery rate. The specific steps of magnetic bead purification are as follows:

[0124] 1) Add 140 μl DNA Purification Beads to the RT-PCR amplification product, mix by pipetting, and incubate at room temperature for 5 min.

[0125] 2) Transfer to a magnetic rack and place for 2 minutes to ensure that the liquid is clear, then discard the supernatant.

[0126] 3) Place on a magnetic rack and add 200 μl of freshly prepared 80% ethanol, wait for 30 seconds, and discard the supernatant.

[0127] 4) Repeat step 3 above once.

[0128] 5) On the magnetic stand, use a 10μl pipette to aspirate the residual ethanol at the bottom of the PCR tube and dry it at room temperature (if the air is dry, the drying time can be appropriately reduced, but be careful not to over-dry to cause cracks in the magnetic beads). When the surface of the magnetic beads is no longer reflective, elution can be performed.

[0129] 6) Add 22 μl of water, mix gently, transfer the PCR tube to the PCR plate, and incubate at room temperature for 5 min.

[0130] 7) Transfer the PCR tube to a magnetic rack and place it for 2 minutes to ensure that the liquid is clear, and then pipette 20 μl of the supernatant into a clean PCR tube.

[0131] 8) Use Qubit to measure the concentration of purified ds cDNA.

[0132] If no subsequent experiments are to be performed, store at -20°C and place on ice or thaw at 4°C before use.

[0133] 2. Library Preparation

[0134] 1. Endonuclease interruption

[0135] 1) Thaw the components of the library preparation kit on ice or at 4°C. Mix the reagents by flicking gently, centrifuge briefly to collect the reagents on the tube wall, and place on ice for later use. Equilibrate the DNA Purification Beads at room temperature for 30 minutes.

[0136] 2) Add the components in Table 8 to a PCR tube on ice.

[0137] Table 8

[0138] Reagents volume cDNA / DNA 50-200ng Frag / AT Buffer 4μl Frag / AT Enzymes 6μl Nuclease-free water to50μl

[0139] 3) Gently mix the reaction solution, centrifuge it momentarily, and place it on ice.

[0140] 4) Incubate according to the procedure in Table 9. After the temperature of the PCR instrument drops to 4°C, place the sample in the PCR instrument, skip procedure 1, and proceed to the subsequent procedure.

[0141] Table 9

[0142] program temperature time - 85℃ Heat cover 1 4℃ Hold 2 30℃ 10min 3 65℃ 30min 4 4℃ Hold

[0143] 2. Add universal adapter and magnetic bead purification

[0144] 1) Add the components in Table 10 to the PCR tube containing the fragmented cDNA on ice.

[0145] Table 10

[0146]

[0147]

[0148] The amount of Universal Adapters used is related to the amount of cDNA input during fragmentation.

[0149] If the cDNA input amount is less than 100 ng, use 2.5 μl Universal Adapters. If the cDNA input amount is greater than 100 ng, use 5 μl Universal Adapters.

[0150] 2) Gently mix the above reaction solution, centrifuge it instantaneously to collect the reagent on the tube wall, and incubate it according to the procedure in Table 11:

[0151] Table 11

[0152] program temperature time 1 OFF Heat cover 2 20℃ 15min 3 4℃ Hold

[0153] 3) Vortex the DNA Purification Beads until mixed.

[0154] 4) Add 60 μl DNA Purification Beads to the PCR tube after incubation, mix well by pipetting or flicking the PCR tube, and incubate at room temperature for 5 minutes.

[0155] 5) Transfer the PCR tube to a magnetic rack and place it for 2 minutes to ensure that the liquid is clear, then discard the supernatant.

[0156] 6) Place on a magnetic rack and add 200 μl of freshly prepared 80% ethanol, wait for 30 seconds, and discard the supernatant.

[0157] 7) Repeat step 6 above once.

[0158] 8) On the magnetic stand, use a 10μl pipette to aspirate the residual ethanol at the bottom of the PCR tube and dry it at room temperature (if the air is dry, the drying time can be appropriately reduced, but be careful not to over-dry to cause cracks in the magnetic beads). When the surface of the magnetic beads is no longer reflective, elution can be performed.

[0159] 9) Remove the PCR tube from the magnetic stand, add 17 μl of water, mix well with a pipette, and incubate at room temperature for 5 minutes.

[0160] 10) Place the PCR tube on a magnetic rack. After the liquid is clarified, pipette 15 μl of the supernatant (containing the cDNA library) into a clean PCR tube.

[0161] 3. PCR amplification and purification

[0162] 1) Add the components in Table 12 to a PCR tube.

[0163] Table 12

[0164] Reagents volume cDNA library 15μl Amplification Mix 25μl index 10μl Total volume 50μl

[0165] 2) After gently mixing the reaction solution in Table 12, centrifuge it momentarily and perform amplification according to the procedure in Table 13.

[0166] Table 13

[0167]

[0168] 3) Vortex the DNA Purification Beads until mixed.

[0169] 4) Add 50 μl DNA Purification Beads to the PCR tube, mix well by pipetting or vortexing, and incubate at room temperature for 5 minutes.

[0170] 5) Transfer to a magnetic rack and place for 2 minutes to ensure that the liquid is clear, then discard the supernatant.

[0171] 6) Place on a magnetic rack and add 200 μl of freshly prepared 80% ethanol, wait for 30 seconds, and discard the supernatant.

[0172] 7) Repeat step 6 above once.

[0173] 8) On the magnetic stand, use a 10μl pipette to aspirate the residual ethanol at the bottom of the PCR tube and dry it at room temperature (if the air is dry, the drying time can be appropriately reduced, but be careful not to over-dry to cause cracks in the magnetic beads). When the surface of the magnetic beads is no longer reflective, elution can be performed.

[0174] 9) Remove the PCR tube from the magnetic stand, add 22 μl of water, mix well with a pipette, and incubate at room temperature for 5 min.

[0175] 10) Place the PCR tube on a magnetic stand. After the liquid is clarified, pipette 20 μl of the supernatant (containing the amplified tag library) into a clean PCR tube.

[0176] 11) Use Qubit to measure the concentration of the purified tag library.

[0177] If no subsequent experiments are to be performed, store at -20°C and place on ice or thaw at 4°C before use.

[0178] 3. Hybridization Capture

[0179] 1. Magnetic bead concentration and hybridization

[0180] 1) Thaw the components of Hybridization Capture Kit A on ice or at 4°C, and centrifuge to collect the reagents on the tube wall. The DNA Purification Beads need to be equilibrated at room temperature for 30 minutes and fully shaken to mix. The Hybridization Mix needs to be preheated at 65°C in a metal bath for later use.

[0181] 2) Dilute the amplified tag library to 50 ng / μl.

[0182] 3) Add the reagents in Table 14 to each library to prepare a prehybridization solution.

[0183] Table 14

[0184]

[0185] 4) Mix by flicking the PCR tube with your finger and centrifuge briefly to reduce air bubbles in the PCR tube.

[0186] 5) Add 45 μl DNA Purification Beads to the prehybridization solution, mix well by pipetting or vortexing, and incubate at room temperature for 5 minutes.

[0187] 6) Transfer to a magnetic rack and place for 2 minutes to ensure that the liquid is clear, then discard the supernatant.

[0188] 7) Place on a magnetic rack and add 200 μl of freshly prepared 80% ethanol, wait for 30 seconds, and discard the supernatant.

[0189] 8) Repeat step 7 above once.

[0190] 9) On the magnetic stand, use a 10 μl pipette to aspirate the residual ethanol at the bottom of the PCR tube, and immediately add 10 μl of Hybridization Mix preheated to 65°C to resuspend the magnetic bead pellet.

[0191] After vortexing the Hybridization Mix, use it immediately and do not allow it to cool to room temperature. The Hybridization Mix is ​​a viscous reagent, so pipette slowly to ensure accurate volumes.

[0192] In this step, the amount of magnetic beads used is relatively small, so there is no need to dry them separately at room temperature. Be careful not to over-dry the magnetic beads.

[0193] 10) Briefly centrifuge the PCR tube to ensure there are no bubbles in the tube, then add 15 μl Enhancer.

[0194] 11) Carry out the reaction according to the procedure in Table 15. After the temperature of the PCR instrument rises to 95°C, place the sample into the PCR instrument, skip procedure 1, and proceed to the subsequent procedure.

[0195] Table 15

[0197]

[0198] Make sure the PCR tubes are tightly sealed to prevent evaporation during incubation.

[0199] Before proceeding, verify that Program 2 on the thermal cycler is started.

[0200] 2. Targeted capture with streptavidin magnetic beads

[0201] 1) Take out each component of the hybridization capture kit B and equilibrate it at room temperature for at least 30 minutes.

[0202] 2) Ensure that there are no crystals in Fast Binding Buffer, Fast Wash Buffer 1, and Wash Buffer 2 before use. Vortex the pre-equilibrated Streptavidin Binding Beads until mixed.

[0203] 3) Add 100 μl of Streptavidin Binding Beads to a 1.5 ml centrifuge tube. Prepare one centrifuge tube for each hybridization reaction.

[0204] 4) Add 200 μl Fast Binding Buffer to the centrifuge tube and mix well by pipetting.

[0205] 5) Allow the tube to sit on the magnetic rack for 1 minute, then aspirate and discard the supernatant. Be careful not to break up the magnetic beads. Remove the tube from the magnetic rack.

[0206] 6) Repeat steps 4 and 5 above twice, for a total of three washes.

[0207] 7) After the third wash, remove the supernatant and add 200 μl Fast Binding Buffer to resuspend the magnetic beads.

[0208] 8) After the hybridization reaction is completed, open the cover of the PCR instrument and quickly transfer all the hybridization reaction solutions (magnetic bead concentration and hybridization, step (11) product) to the corresponding centrifuge tube containing Streptavidin Binding Beads (cleaned). Mix the reagents by pipetting and flicking.

[0209] Rapid transfer directly from the 60°C thermal cycler is a critical step to reduce off-target reactions. Do not remove the hybridization reaction tubes from the thermal cycler or cool them below 60°C before transferring the solution to the washed Streptavidin Binding Beads.

[0210] 9) Mix the hybridization reaction solution and Streptavidin Binding Beads at room temperature for 30 minutes using a rotary mixer or oscillator at a speed that allows the solution to mix well (600 rpm).

[0211] Do not vortex. Vigorous mixing is not necessary.

[0212] 10) During the incubation of the hybridization reaction solution and magnetic beads, add 100 μl Fast Wash Buffer 1 to the PCR tube or 8-row strip according to the number of samples, mark it and place it in the PCR instrument, preheat it to 68°C, and set the temperature of the hot cover to 68°C. One sample requires 2 tubes of 100 μl Fast Wash Buffer 1.

[0213] 11) Similarly, aliquot Wash Buffer 2 and preheat to 48°C in a PCR instrument with the heated lid set to 48°C. One sample requires three tubes of 100 μl Wash Buffer 2.

[0214] 12) Remove the centrifuge tube containing the hybridization reaction solution and Streptavidin Binding Beads from the mixer and centrifuge briefly to ensure that all the solution is at the bottom of the centrifuge tube.

[0215] 13) Transfer all the solution in the centrifuge tube to a clean PCR tube.

[0216] 14) Let the PCR tube stand on the magnetic rack for 1 minute, aspirate and discard the supernatant. Do not break up the magnetic bead pellet.

[0217] 15) Remove the PCR tube from the magnetic stand and add 100 μl of 68°C preheated Fast Wash Buffer 1. Mix well by pipetting.

[0218] Transfer Fast Wash Buffer 1 directly from the 68°C thermal cycler.

[0219] 16) Place the PCR tube on a PCR instrument and incubate at 68°C for 5 minutes.

[0220] 17) Let the PCR tube stand on the magnetic rack for 1 minute, aspirate and discard the supernatant. Do not break up the magnetic bead pellet.

[0221] 18) Repeat the above steps (15-17) once, washing twice in total using Fast Wash Buffer 1.

[0222] 19) Remove the tube from the magnetic rack and add 100 μl of 48°C preheated Wash Buffer 2. Mix well by pipetting, then centrifuge briefly to ensure that all the solution is at the bottom of the tube.

[0223] Quickly transfer Wash Buffer 2 directly from the 48°C thermal cycler.

[0224] 20) Place the PCR tube on a PCR instrument and incubate at 48°C for 5 minutes.

[0225] 21) Let the PCR tube stand on the magnetic rack for 1 minute, aspirate and discard the supernatant. Do not disrupt the magnetic bead pellet.

[0226] 22) Repeat steps 19-21 above twice, washing a total of three times using Wash Buffer 2.

[0227] 23) After the last wash, use a 10μl pipette to remove the remaining Wash Buffer 2. Proceed immediately to the next step. Do not allow the beads to dry out.

[0228] Before removing the supernatant, briefly centrifuge the magnetic bead pellet to collect the supernatant at the bottom of the tube or microplate and place it back on the magnet.

[0229] 24) Remove the centrifuge tube from the magnetic stand, add 45 μl of water, and mix and incubate the solution (hereinafter referred to as streptavidin-bound magnetic bead suspension) by pipetting.

[0230] IV. Post-capture amplification and purification

[0231] 1. Post-capture PCR amplification

[0232] 1) Thaw the PCR amplification components in the Hybridization Capture Kit A on ice or at 4°C and centrifuge briefly to collect the reagents on the tube wall. Equilibrate the DNA Purification Beads at room temperature for 30 minutes.

[0233] 2) Transfer 22.5 μl of the streptavidin-bound magnetic bead suspension to a 0.2 ml thin-walled PCR tube. Place on ice until used in the next step. If the streptavidin-bound magnetic bead suspension settles, use a pipette to mix it.

[0234] Store the remaining 22.5 μl of water / streptavidin-bound magnetic bead suspension at -20 °C for future use.

[0235] 3) Add the reagents in Table 16 to the PCR tube containing the streptavidin-bound magnetic bead suspension to prepare a PCR mixture. Mix by pipetting.

[0236] Table 16

[0237]

[0238] 4) Centrifuge the tube briefly, transfer it to a thermal cycler and start the cycling program in Table 17.

[0239] Table 17

[0240]

[0241] If no subsequent experiments are to be performed, store at -20°C and place on ice or thaw at 4°C before use.

[0242] 2. Post-capture magnetic bead purification

[0243] 1) Vortex DNA Purification Beads until mixed.

[0244] 2) Add 90 μl DNA Purification Beads to the PCR tube, mix well by pipetting or vortexing, and incubate at room temperature for 5 minutes.

[0245] 3) Transfer to a magnetic rack and place for 2 minutes to ensure that the liquid is clear, then discard the supernatant.

[0246] 4) Place on a magnetic rack and add 200 μl of freshly prepared 80% ethanol, wait for 30 seconds, and discard the supernatant.

[0247] 5) Repeat step 3 above once.

[0248] 6) On the magnetic stand, use a 10μl pipette to aspirate the residual ethanol at the bottom of the PCR tube, dry at room temperature for 5 minutes (if the air is dry, the drying time can be appropriately reduced, and do not over-dry to cause cracks in the magnetic beads). When the surface of the magnetic beads is no longer reflective, elution can be performed.

[0249] 7) Remove the test tube from the magnetic stand, add 32 μl of water, mix well with a pipette, and incubate at room temperature for 2 minutes.

[0250] 8) Transfer 30 μl of supernatant (containing the enriched library) to a clean PCR tube.

[0251] 9) Use Qubit to measure the concentration of the purified capture library.

[0252] Experimental results: The detection results of multiple respiratory pathogens are shown in Table 18. The cDNA concentration in Table 18 is the experimental result of sample preparation in Experimental Process 1, the library concentration is the experimental result of library preparation in Experimental Process 2, and the post-capture library concentration is the experimental result of hybrid capture in Experimental Process 3 and amplification and purification in Experimental Process 4.

[0253] Sample amplification efficiency: 1. All samples were successfully amplified, and the cDNA concentration ranged from 0.1ng / ul to 15.8ng / ul, showing different abundances of the original material. 2. The library concentration ranged from 74.8ng / ul to 272.8ng / ul, indicating that the amplification process was effective and that the amplification efficiency varied between samples.

[0254] 2. Capture efficiency: 1. The concentration of the library after capture ranged from 33.2ng / ul to 68.8ng / ul, reflecting the different efficiency of the capture step for different viruses. 2. Although the original cDNA concentration was low (such as COVID-19, dengue type 1, and rhinovirus), the concentration of the library after capture was still sufficient for subsequent sequencing analysis.

[0255] 3. Sequencing quality: 1. The ratio of Q20 and Q30 bases in all samples is high, indicating good sequencing quality. 2. The coverage is close to or reaches 100%, indicating that the sequencing depth is sufficient, indicating that the primers designed in this experiment can effectively amplify almost the entire length of the target pathogen genome, and the amplification efficiency is stable, which can fully cover the target area.

[0256] Table 18

[0257]

[0258]

[0259] The sequences of the probe sets SEQ ID NO: 1-5000 used in the present invention are shown in Table 19.

[0260] Table 19

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

Claims

1. A probe group for detecting multiple respiratory pathogens, wherein the probe group is a nucleotide sequence shown in SEQ ID NO: 1-5000.

2. The probe set according to claim 1, wherein the probe in the probe set consists of two parts: Specific sequences, used for specific binding to the target DNA region, and, common sequences, used for subsequent amplification; Preferably, the probe is labeled with a fluorescent dye; Preferably, the fluorescent dye label includes FAM, HEX, TAMRA, ROX, TET, JOE, Cy3, Cy5, VIC, Cy5.5, Texas Red, NED, FAM-dT, TAMRA-dT, CY3-dT, CY5-dT, HEX-dT, FITC; Preferably, the probe comprises a quencher group; Preferably, the quenching group includes BHQ1, TAMRA, BHQ2, Rhodamine 6G, Rhodamine B, BHQ3, Dabcyl, Eclipse, MGB, BHQ1-dT, BHQ2-dT; Preferably, the probe is labeled with one or more modifying groups; Preferably, the modifying groups include amine groups, phosphate groups, biotin, biotin-TEG, and C3-spacer; preferably, the respiratory multi-pathogens include coronaviruses (229E / HKU1 / NL63 / OC43), enteroviruses (group A / B / C / D), influenza viruses (A / B), bocaviruses, human adenoviruses (group B / C / D / E), metapneumoviruses, parainfluenza viruses (type 1 / 2 / 3 / 4), rhinoviruses (A / B / C), astroviruses, mumps viruses, measles viruses, rubella viruses, new coronaviruses, syncytial viruses, varicella-zoster viruses, SARS viruses, noroviruses, dengue viruses, and rotaviruses.

3. A product for detecting multiple respiratory pathogens, said product comprising the probe set according to claim 1 or 2; preferably, said product comprises a nucleic acid membrane strip, a test kit, and a chip; Preferably, the kit further comprises reagents required for hybridization capture, buffer and amplification enzyme required for PCR amplification; Preferably, the kit further comprises a reaction system required for reverse transcription; Preferably, the reaction system required for the reverse transcription includes random primers; Preferably, the kit further comprises a double-stranded cDNA synthesis kit, specifically Random Primer, Reaction Mix 1, Enzyme Mix 1, Reaction Mix 2, Enzyme Mix 2, EDTA, RNase I, and Nuclease-free water; Preferably, the kit further comprises a library construction kit, specifically Frag / AT Enzymes, Frag / AT Buffer, Universal Adapters, Ligation Master Mix, Index, Amplification Mix, Nuclease-free water; Preferably, the kit further comprises a rapid hybridization capture kit A, specifically comprising a Probe Panel, Universal Blockers, Blocker Solution, Hybridization Mix, Enhancer, Amp Primers, Amplification Mix, and Nuclease-free water; Preferably, the kit further comprises a rapid hybridization capture kit B, specifically Streptavidin Binding Beads, Fast Binding Buffer, Fast Wash Buffer 1, Wash Buffer 2, and DNA Purification Beads.

4. A method for detecting multiple respiratory pathogens, the method comprising the steps of adding the probe set according to claim 1 or 2 into the same container and detecting the whole genome of multiple respiratory pathogens by hybridization capture of the sample; Preferably, the method comprises the following steps: 1) Collect samples and prepare them into a tag library; 2) adding the probe set, Universal Blocker and Blocker Solution of claim 1 or 2 to the library; 3) adding DNA purification beads, mixing and incubating at room temperature for 5 minutes, placing in a magnetic field and discarding the supernatant, washing with ethanol, and repeating once or multiple times; 4) Place in a magnetic field to remove excess ethanol, and immediately add Hybridization Mix preheated to 65°C to resuspend; 5) After brief centrifugation, add Enhancer; 6) Heat at 95°C for 5 minutes, then immediately heat at 60°C for 2 hours; 7) Add Streptavidin Binding Beads and Fast Binding Buffer in a volume ratio of 1:2, mix well, let stand in a magnetic field, discard the supernatant, and repeat once or more; 8) Add FastBindingBuffer to resuspend the magnetic beads, and hybridization is completed; 9) After the hybridization is completed, the product is immediately transferred to a container containing washed Streptavidin Binding Beads, and the ambient temperature of the product is kept greater than or equal to 60° C. and mixed; 10) Mix the solution in step 9 at a mixing speed of 600 rpm for 30 min, centrifuge, change the container, let it stand in a magnetic field, and discard the supernatant; 11) Add Fast Wash Buffer 1 preheated at 68°C, mix well, incubate at 68°C for 5 min, place in a magnetic field and discard the supernatant, repeat once or more; 12) Add Wash Buffer 2 preheated at 48°C, mix well, centrifuge, incubate at 48°C for 5 min, place in a magnetic field and discard the supernatant, repeat once or more; 13) After aspirating Wash Buffer 2, immediately add Nucleasa-free water, mix well and incubate; 14) Perform PCR amplification and magnetic bead purification; 15) Sequencing analysis.

5. The method according to claim 4, wherein the PCR amplification is multiplex PCR amplification; Preferably, the amplification reaction is performed under similar amplification conditions; Preferably, the sample comprises a human sample and an environmental sample, and the human sample is an ex vivo human sample; preferably, the human sample comprises one or a combination of tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, tissue culture fluid, tissue extract, homogenized tissue, tumor tissue, cell extract, alveolar lavage fluid, nasopharyngeal / oropharyngeal swab, and sputum; Preferably, the human body sample includes alveolar lavage fluid, nasopharyngeal / oropharyngeal swab, and sputum; Preferably, the environmental sample comprises a water source; Preferably, the water source comprises sewage; Preferably, the sewage includes sewage from wastewater treatment plants, rivers, and sewer systems; Preferably, the method is a method for non-diagnostic purposes.

6. The method according to claim 4, wherein the collecting samples and preparing the tag library comprises the following specific steps: sample preparation, and tag library preparation; Preferably, sample preparation includes: Extract nucleic acid according to the corresponding requirements and steps in the commercial viral nucleic acid extraction kit. Double-stranded cDNA synthesis was performed according to the corresponding requirements and steps in the commercial double-stranded cDNA synthesis kit. The double-stranded cDNA product was purified using 1.2 volumes of DNA purification beads; Preferably, the purification of the double-stranded cDNA product specifically comprises the following steps: 1) Add DNA Purification Beads to the double-stranded cDNA product, mix well, and incubate at room temperature for 5 minutes; place in a magnetic field and discard the supernatant; 2) Add 80% ethanol in a magnetic field, discard the supernatant after 30 seconds, and repeat once or more; 3) Absorb all ethanol in the magnetic field, add Nucleasa-free water, mix well and incubate at room temperature for 5 minutes; 4) Place in a magnetic field to obtain a product; Preferably, the tag library preparation includes: performing endonuclease shearing according to the corresponding requirements and steps in the library construction kit, adding universal adapters and magnetic bead purification, PCR amplification and purification.

7. The method of claim 4, wherein the PCR amplification and magnetic bead purification in step 14 comprises performing PCR amplification using the PCR amplification components in the hybrid capture kit A, and, Magnetic bead purification steps: 1) Add DNA purification beads, mix well, and incubate at room temperature for 5 minutes; 2) Place in a magnetic field and discard the supernatant, add 80% ethanol, discard the supernatant after 30 seconds, and repeat once or more; 3) Remove all ethanol in a magnetic field and dry at room temperature for 5 minutes; 4) Remove from the magnetic field, add water, mix well, and incubate at room temperature for 2 minutes.

8. A library for detecting multiple respiratory pathogens, the library comprising the probe set according to claim 1 or 2.

9. Use of the probe set according to claim 1 or 2 in preparing products for amplifying or detecting multiple respiratory pathogens.

10. Use of the probe set according to claim 1 or 2, or the product according to claim 3 in detecting multiple respiratory pathogens for non-diagnostic purposes.

Images