Probe set and method for detecting respiratory tract infection pathogenic microorganisms and application of probe set and method

Abstract

The invention provides a probe set and method for detecting respiratory tract infection pathogenic microorganisms and application of the probe set and method. The probe group is high in specificity and sensitivity, capable of accurately capturing target species in a pathogen spectrum and efficiently enriching the target species, meanwhile, few library fragments of non-target species are introduced, pathogenic microorganisms are convenient to identify, the detection sensitivity is high, the accuracy is good, the time cost is saved, the coverage degree is wide, and the application prospect is wide. Various bacteria, fungi, viruses and other pathogenic microorganisms related to respiratory tract infection can be covered.

Description

Technical Field

[0001] The present disclosure relates to the field of genetic engineering technology, and in particular to a probe set, method and application thereof for detecting pathogenic microorganisms of respiratory tract infection. Background Art

[0002] In recent years, with the increase in the elderly population, the longer life expectancy of patients with chronic diseases (diabetes, severe liver and kidney diseases), and the increase in immune-related treatments (transplantation, chemotherapy, use of immunosuppressants), the number of people with low immune function has increased relatively, and the number of infections caused by pathogenic microorganisms including bacteria, viruses, fungi, and parasites has increased, posing a challenge to etiological diagnosis. For a long time, the gold standard for clinical pathogen detection has been microbial culture. Other detection methods such as smear microscopy, antigen-antibody detection, PCR and other molecular biological methods have also been gradually applied to microbial detection. However, the culture method takes a long time and has a low positive rate. The antigen-antibody detection and PCR methods rely on the clinician's pre-judgment and selection of the kit.

[0003] With the development of high-throughput sequencing technology, this technology has also been applied to the detection of clinical microorganisms. mNGS (metagenomics next-generation sequencing), also known as metagenomic second-generation sequencing technology, has the characteristics of no preference and rapidity (<24h). It does not require cultivation and can directly extract nucleic acid sequences of all microorganisms including bacteria, fungi, viruses and parasites from environmental / clinical samples. However, there is also the problem of low sensitivity, which is mainly due to the high proportion of hosts and the ease of bringing out a large number of background microbial signals. To solve this problem, the experimenters have also taken corresponding measures, but with little success. At the same time, the amount of sequencing required to improve the species signal is large, resulting in high sequencing costs, which significantly increases the cost. Therefore, targeted sequencing methods have emerged. Compared with metagenomic sequencing, targeted sequencing can increase the data proportion of target species, reduce background signals and avoid interference while increasing the signal of target species, ensuring the positive rate, thereby reducing the required sequencing amount.

[0004] Current targeted sequencing is mainly divided into targeted sequencing methods based on multiple amplification techniques and targeted sequencing methods based on probe capture. By enriching the target DNA sequence through methods such as multiplex PCR or liquid hybridization capture and then sequencing, the proportion of target sequences in the sequencing data can be increased, thereby improving the detection sensitivity. The method of multiplex PCR is difficult to design due to the limitation of primer dimer formation, and the target range is small. At the same time, compared with the multiplex amplification method, the probe capture method has higher inclusiveness for target sites and can further improve the positive rate. However, how to design targeted capture probes with both high specificity and conservativeness for a wide range of pathogenic microorganisms to accurately detect different pathogenic microorganisms and meet the requirements of different sample types, disease types, and pathogen types is an urgent problem to be solved. Summary of the Invention

[0005] To solve at least one of the above technical problems, the present disclosure provides a probe set, a kit, a detection method, and an application thereof for detecting pathogenic microorganisms in respiratory tract infections.

[0006] According to a first aspect of the present disclosure, there is provided a probe set for detecting pathogenic microorganisms in respiratory tract infections, the probe set comprising any one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 144, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto.

[0007] In some embodiments, the probe set is used for species identification.

[0008] In some embodiments, the bacterial strains include one or more of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Stenotrophomonas maltophilia, Escherichia coli, Enterobacter cloacae, Streptococcus pyogenes, Aspergillus fumigatus, Candida albicans, Human parainfluenza virus, Human adenovirus, Human metapneumovirus, Human bocavirus, Mycobacterium tuberculosis complex, Legionella pneumophila, Listeria monocytogenes, Mycoplasma pneumoniae, Chlamydia psittaci, Influenza A virus H1N1, Influenza B virus, Human Respiratory syncytial virus A, or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0009] In some embodiments, the probe set includes any one or more of the following groups:

[0010] A probe set for detecting Haemophilus influenzae, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 3, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0011] A probe set for detecting Stenotrophomonas maltophilia, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 7-9, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0012] A probe set for detecting Enterobacter cloacae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 13-15, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0013] A probe set for detecting Streptococcus pyogenes, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 25-27, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0014] A probe set for detecting Staphylococcus aureus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 31-33, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0015] A probe set for detecting Klebsiella pneumoniae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 34-36, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0016] A probe set for detecting Acinetobacter baumannii, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 37-39, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0017] A probe set for detecting Escherichia coli, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 40-42, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0018] A probe set for detecting Pseudomonas aeruginosa, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 43-45, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0019] A probe set for detecting Streptococcus pneumoniae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 52-54, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0020] A probe set for detecting Aspergillus fumigatus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 61-63, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0021] A probe set for detecting Candida albicans, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 64-66, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0022] A probe set for detecting influenza A virus H1N1, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 70-72, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0023] A probe set for detecting influenza B virus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 82-84, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0024] A probe set for detecting respiratory syncytial virus type A, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 85-87, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0025] A probe set for detecting parainfluenza virus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 94-96, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0026] A probe set for detecting adenovirus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 97-99, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0027] A probe set for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 112-114, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting human metapneumovirus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 115-117, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0028] A probe set for detecting bocavirus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 118 to 120, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0029] A probe set for detecting Mycobacterium tuberculosis complex, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 121 to 123, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0030] A probe set for detecting Legionella pneumophila, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 130 to 132, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0031] A probe set for detecting Listeria monocytogenes, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 133 to 135, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0032] A probe set for detecting Mycoplasma pneumoniae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 139 to 141, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; and

[0033] A probe set for detecting Chlamydia psittaci, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 142 to 144, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith.

[0034] In some embodiments, the probe set is used for species identification.

[0035] In some embodiments, the bacterial strains include one or more of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Stenotrophomonas maltophilia, Escherichia coli, Moraxella catarrhalis, Enterobacter cloacae, Serratia marcescens, Burkholderia cepacia, Klebsiella oxytoca, Enterobacter aerogenes, Proteus mirabilis, Streptococcus pyogenes, Haemophilus parainfluenzae, Citrobacter freundii, Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Influenza virus, Human Respiratory syncytial virus, Rhinovirus, Human parainfluenza virus, Human adenovirus, coronavirus, Human metapneumovirus, Human bocavirus, Mycobacterium tuberculosis complex, Mycobacterium avium, Mycobacterium intracellulare, LegionellaLegionella pneumophila, Listeria monocytogenes, Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia psittaci.

[0036] In some embodiments, the influenza virus includes Influenza A virus H1N1, Influenza A virus H3N2, Influenza A virus H5N1, Influenza A virus H7N9, Influenza A virus H9N2, or Influenza B virus.

[0037] In some embodiments, the respiratory syncytial virus includes Human Respiratory syncytial virus A and Human Respiratory syncytial virus B.

[0038] In some embodiments, the coronavirus includes Human coronavirus 229E, Human coronavirus HKU1, Human coronavirus NL63, Human coronavirus OC43, or the novel coronavirus SARS-CoV-2.

[0039] In some embodiments, the probe set further includes any one or more of the following groups:

[0040] A probe set for detecting Haemophilus parainfluenzae, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 4-6, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0041] A probe set for detecting Moraxella catarrhalis, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 10-12, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith;

[0042] A probe set for detecting Burkholderia cepacia, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 16 to 18, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0043] A probe set for detecting Klebsiella oxytoca, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 19 to 21, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0044] A probe set for detecting Klebsiella aerogenes, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 22 to 24, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0045] A probe set for detecting Citrobacter freundii, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 28 to 30, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0046] A probe set for detecting Serratia marcescens, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 46 to 48, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0047] A probe set for detecting Proteus mirabilis, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 49 to 51, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0048] A probe set for detecting Cryptococcus neoformans, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 55 to 57, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0049] A probe set for detecting Candida auris, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 58 to 60, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0050] A probe set for detecting influenza A virus H3N2, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 67 to 69, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0051] A probe set for detecting influenza A virus H5N1, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 73 to 75, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0052] A probe set for detecting influenza A virus H7N9, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 76 to 78, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0053] A probe set for detecting influenza A virus H9N2, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 79 to 81, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0054] A probe set for detecting respiratory syncytial virus type B, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 88 to 90, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0055] A probe set for detecting rhinovirus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 91 to 93, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0056] A probe set for detecting coronavirus 229E, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 100 to 102, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0057] A probe set for detecting coronavirus HKU1, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 103 to 105, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0058] A probe set for detecting coronavirus NL63, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 106 to 108, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0059] A probe set for detecting coronavirus OC43, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 109 to 111, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0060] A probe set for detecting Mycobacterium avium, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 124 to 126, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0061] A probe set for detecting Mycobacterium intracellulare, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 127 to 129, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0062] A probe set for detecting Chlamydia pneumoniae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 136 to 138, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto.

[0063] In some embodiments, the probes are modified with biotin. After modification with biotin, the probe sequences that have enriched the microorganisms in the sample are captured and enriched using streptavidin magnetic beads. Subsequently, the captured sample is eluted at high temperature using a washing solution to remove non-specifically captured sequences, and then subjected to PCR specific amplification followed by sequencing on a machine, thereby achieving the purpose of rapidly, highly sensitively and specifically enriching and detecting pathogenic microorganisms in the sample.

[0064] According to a second aspect of the present disclosure, there is provided a kit, which comprises the probe set described in the first aspect.

[0065] According to a third aspect of the present disclosure, there is provided a method for detecting pathogenic microorganisms of respiratory tract infections using the probe set described in the first aspect and / or the kit described in the second aspect.

[0066] In some embodiments, the method comprises the following steps:

[0067] 1) Extract nucleic acid from the sample,

[0068] 2) Construct a library for the extracted nucleic acid,

[0069] 3) Use the probe set to hybridize and capture the target sequence,

[0070] 4) Sequence the captured product and perform analysis.

[0071] In some embodiments, step 1) includes removing host nucleic acid.

[0072] In some embodiments, step 3) further includes amplifying the captured product.

[0073] In some embodiments, the sample includes cell, tissue or body fluid samples.

[0074] In some embodiments, the sample includes tissue, cells, blood, plasma, serum, bronchoalveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, pleural effusion, or a processed product thereof.

[0075] According to a fourth aspect of the present disclosure, there is provided an application of the probe set described in the first aspect in the preparation of a kit.

[0076] In some embodiments, the probe set is used for the detection of bronchoalveolar lavage fluid, sputum, nasopharyngeal swab or oral swab fluid.

[0077] In some embodiments, the probe set is used for detecting one or more of bacteria, fungi, viruses, mycoplasmas or chlamydias.

[0078] In some embodiments, the probe set is used to detect one or more of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Stenotrophomonas maltophilia, Escherichia coli, Moraxella catarrhalis, Enterobacter cloacae, Serratia marcescens, Burkholderia cepacia, Klebsiella oxytoca, Enterobacter aerogenes, Proteus mirabilis, Streptococcus pyogenes, Haemophilus parainfluenzae, Citrobacter freundii, Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Influenza virus, Human Respiratory syncytial virus, Rhinovirus, Human parainfluenza virus, Human adenovirus, coronavirus, Human metapneumovirus, Human bocavirus, Mycobacterium tuberculosis complex, Mycobacterium avium, Mycobacterium intracellulare, LegionellaLegionella pneumophila, Listeria monocytogenes, Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia psittaci.

[0079] In some embodiments, the influenza virus includes Influenza A virus H1N1, Influenza A virus H3N2, Influenza A virus H5N1, Influenza A virus H7N9, Influenza A virus H9N2, or Influenza B virus.

[0080] In some embodiments, the respiratory syncytial virus includes Human Respiratory syncytial virus A and Human Respiratory syncytial virus B.

[0081] In some embodiments, the coronavirus includes Human coronavirus 229E, Human coronavirus HKU1, Human coronavirus NL63, Human coronavirus OC43, or the novel coronavirus SARS-CoV-2.

[0082] The present disclosure provides a set of probe groups capable of detecting pathogenic microorganisms causing respiratory tract infections. The probe groups have high-quality specificity and sensitivity, can accurately capture target species in the pathogen spectrum, efficiently enrich the target species, and at the same time introduce fewer library fragments of non-target species, facilitating the identification of pathogenic microorganisms, with high detection sensitivity, good accuracy, saving time costs, and having a wide coverage, capable of covering various bacteria, fungi, viruses and other pathogenic microorganisms related to respiratory tract infections. Detailed implementation manners

[0083] Respiratory Tract Infections refer to infectious diseases caused by pathogens invading and multiplying in the nasal cavity, pharynx, trachea, bronchi and other parts of the human body. It is a common infectious disease. The pathogens of respiratory tract infections can be divided into types such as bacteria, viruses, fungi, mycoplasma, chlamydia and rickettsia. Traditional isolation culture and serological detection methods are time-consuming and cumbersome, resulting in problems such as untimely detection, low sensitivity and low positive rate.

[0084] Although the metagenomic sequencing method has a wide detection range, there are problems such as complex background bacteria detection, difficult interpretation: it is difficult to detect pathogens with small genomes such as viruses, high detection limits: large sequencing data volume, long analysis time and high sequencing cost.

[0085] Although the tNGS method based on multiplex PCR has the characteristics of high sensitivity and rapidity, due to primer dimers restricting the number of primers added in a system, the target range of pathogen spectra that can be designed by this method is small, and when designing a panel of more species, the number of primer pairs that can be allocated to a single species is small, which cannot guarantee the detection performance.

[0086] Due to the complexity of the identification environment of pathogenic microorganisms, the tNGS based on probe capture puts forward higher requirements for the selection of target regions and probe design. Most traditional probe design methods select some relatively conserved molecular markers as targets for probe design, such as the 16S ribosomal region, etc. However, such molecular markers are often extremely conserved and cannot guarantee good specificity in a complex clinical environment.

[0087] The strategy of tiling probes for the whole genome or whole exome region can obtain signals of more target species, but often requires a large number of probes, with high costs; it is also easy to introduce some non-specific signals.

[0088] Therefore, there is an urgent need for a high-quality probe set for the detection of pathogenic microorganisms in respiratory tract infections.

[0089] Based on the detection of a large number of clinical samples of our company and combined with the microbial database, the genomic sequences of 38 pathogenic microorganisms related to respiratory tract infections that frequently appear clinically (see Table 1) are selected as the target regions, and a probe set is generated by overlapping design for the target regions. The probe length is about 120bp, with nucleotide sequences shown in SEQ ID NO:1~144, and biotin-labeled nucleotide probes are synthesized.

[0090] Table 1. 38 pathogenic microorganisms related to lower respiratory tract infections

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] In a specific embodiment, the method for detecting pathogenic microorganisms in lower respiratory tract infections using the probe set includes the following steps:

[0102] Take a biological fluid sample (bronchoalveolar lavage fluid, sputum, etc.), remove host nucleic acids and then perform lysis to extract pathogenic microorganism DNA.

[0103] Perform library preparation on the extracted DNA, including fragmentation, end repair and addition of "A", then add adapters to the ends of the DNA fragments and amplify them using PCR to obtain a pre-library. The pre-library with qualified quantification and fragment quality control is prepared for hybridization with the probe: Use the probe set for detecting pathogenic microorganisms in lower respiratory tract infections to hybridize with the qualified library, capture the target sequence, elute the non-target sequences and impurities, and then amplify and purify the captured target sequence to obtain a final library. After qualified quantification and quality control, sequence the final library.

[0104] Use bioinformatics tools to analyze the sequencing data to identify pathogenic microorganisms in lower respiratory tract infections. Bioinformatics analysis involves the following steps, including quality control, removal of host sequences, alignment with the reference genome, and species identification.

[0105] To make the objectives, technical solutions and advantages of the present disclosure clearer and more understandable, the following further details the present disclosure in combination with embodiments. The specific embodiments described herein are only used to explain the present disclosure and do not constitute any limitation to the present disclosure. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0106] Definition

[0107] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0108] Unless the context clearly indicates otherwise, the expressions "a" and "an" as used herein include plural referents.

[0109] The expression "about" as used herein is as understood by a person of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If a person of ordinary skill in the art is not aware of the use of this term according to the context in which it is used, "about" will mean at most plus or minus 10% of a particular value.

[0110] In this disclosure, the term "pathogenic microorganism" or "pathogenic microbe" is used to refer to viruses, bacteria, yeasts, and other microorganisms that can cause diseases in a subject.

[0111] In this disclosure, the term "species group" refers to the species group to which the target species belongs. In some embodiments, the target species is a bacterial strain, and the species group includes all subspecies of that bacterial strain. In some embodiments, the target species is a species, and the species group includes all subspecies of that species. In some embodiments, the target species is a virus, and the species group includes all subtypes of that virus.

[0112] In this disclosure, the term "RNA", full name "ribonucleic acid" (RiboNucleic Acid, abbreviated as RNA), is one of the four biological macromolecules contained in biological cells, a type of nucleic acid. RNA is a macromolecular polymer composed of nucleotides. Nucleotides are composed of a base, a ribose, and a phosphate. Among them, there are 4 types of bases: adenine (A), guanine (G), uracil (U), and cytosine (C).

[0113] In this disclosure, the term "DNA", full name "deoxyribonucleic acid" (DeoxyriboNucleic Acid, abbreviated as DNA), is one of the four biological macromolecules contained in biological cells, a type of nucleic acid. DNA carries the genetic information necessary for the synthesis of RNA and proteins and is an essential biological macromolecule for the development and normal operation of organisms. DNA is a macromolecular polymer composed of deoxynucleotides. Deoxynucleotides are composed of a base, a deoxyribose, and a phosphate. Among them, there are 4 types of bases: adenine (A), guanine (G), thymine (T), and cytosine (C).

[0114] In the present disclosure, the term "probe" refers to a primer labeled with a capture label or a detection label to detect a primer product. The probe sequence is used to hybridize with the sequence generated by the primer sequence and generally hybridizes with a sequence that does not include the primer sequence. Similar to the primer sequence, the probe sequence is also labeled with a capture label or a detection label. It should be noted that when the primer is labeled with a capture label, the probe is labeled with a detection label, and vice versa. The appropriate length of the probe depends on the intended use of the probe and is generally in the range of 80 to 200 nucleotides (nt). Preferably, the appropriate length of the primer includes 100 to 150 nucleotides. In the present invention, the probe can be one of a fluorescent probe and a hybridization probe.

[0115] In the present disclosure, the term "probe set" generally refers to a collection of more than one probe, which realizes the localization and / or quantification of a target nucleic acid by recognizing and binding to a target sequence (by means of hybridization pairing). Each probe in the probe combination is usually an oligonucleotide, such as a single-stranded DNA molecule or RNA.

[0116] In the present disclosure, "alignment" refers to the process of comparing a read or tag with a reference sequence and thereby determining whether the reference sequence contains the read sequence. If the reference sequence contains the read, the read can be mapped to the reference sequence, or in some embodiments, to a specific position in the reference sequence.

[0117] In the present disclosure, the term "sequence identity" refers to the "percent sequence identity" or "percent identity" between two polynucleotides, that is, the number of identical matching positions shared by the sequences within a comparison window, taking into account the additions or deletions (i.e., gaps) that must be introduced for the optimal alignment of the two sequences. A matching position is any position where the same nucleotide exists in both the target sequence and the reference sequence. Since a gap is not a nucleotide, the gaps present in the target sequence are not counted. Similarly, since the nucleotides of the target sequence are counted and the nucleotides from the reference sequence are not counted, the gaps present in the reference sequence are not counted. At least 60% sequence identity includes continuous segments having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity over the entire length of the sequence.

[0118] Methods for aligning sequences are well known in the art. Various programs and alignment algorithms are described in "Smith and Waterman, Adv. Appl. Math. 2" 482, 1981; Needleman and Wunsch, J. Mol. Biol. 48" 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85" 2444, 1988; Higgins and Sharp, Gene, 73" 237-44, 1988; Higgins and Sharp, CABIOS 5" 151-3, 1989; Corpet et al., Nuc. Acids Res. 16" 10881-90, 1988; Huang et al., Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24" 307-31, 1994; Altschul et al., J. Mol. Biol. 215" 403-10, 1990, presenting detailed considerations of sequence alignment methods and homology calculations.

[0119] In the present disclosure, the term "hybridization" or "specific hybridization" refers to a molecule binding, duplexing, or hybridizing only with a specific polynucleotide sequence under stringent conditions, which is carried out when the sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[0120] In the present disclosure, the term "complementary" refers to the concept of sequence complementarity between regions of two polynucleotide strands or between two regions of the same polynucleotide strand. An adenine base in a first region of a known polynucleotide is capable of forming a specific hydrogen bond ("base pair") with a base (if that base is thymine or uracil) in a second region of a polynucleotide that is antiparallel to the first region. Similarly, a cytosine base in a first polynucleotide strand is known to base pair with a base (if that base is guanine) in a second polynucleotide strand that is antiparallel to the first region. Two regions are complementary if at least one nucleotide in the first region is capable of base pairing with a base in the second region when the first region of the polynucleotide is arranged antiparallel to the second region of the same or another different polynucleotide. Thus, two complementary polynucleotides do not need to base pair at every nucleotide position. "Complementary" refers to 100% or "perfect" complementarity of a first polynucleotide with a second polynucleotide, and thus base pairing occurs at every nucleotide site. "Complementary" also refers to a first polynucleotide that is not 100% complementary (e.g., 90%, or 80% or 70%, or 60%, or 50% complementary) and contains mismatched nucleotides at one or more nucleotide positions. In one embodiment, two complementary polynucleotides are capable of hybridizing to each other under highly stringent hybridization conditions.

[0121] In the present disclosure, the term "stringent hybridization conditions" refers to the conditions under which a probe hybridizes to its target subsequence, typically in a complex mixture of nucleic acids, but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, under defined ionic strength and pH, the stringent conditions selected are about 5 - 10 °C lower than the thermal melting point (Tm) of a particular sequence. Tm is the temperature (at a specified ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (since the target sequence is in excess, at Tm, 50% of the probe is occupied at equilibrium). Stringent conditions can also be achieved by adding destabilizing agents (such as formamide). For selective or specific hybridization, the positive signal is at least twice the background, preferably 10 times the background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC, and 1% SDS, incubated at 42 °C, or 5×SSC, 1% SDS, incubated at 65 °C, and washed at 65 °C with 0.2×SSC and 0.1% SDS.

[0122] In the present disclosure, the term "sequencing" refers to techniques for determining the sequence (e.g., the identity and order of monomeric units) of a biomolecule, such as a nucleic acid, such as DNA or RNA. Exemplary sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy chain termination sequencing, whole genome sequencing, hybridization sequencing, pyrosequencing, capillary electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired-end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, synthetic sequencing, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa genome analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, DNA nanoball sequencing (DNBSEQ), combinatorial probe anchor synthesis sequencing (cPAS), and combinations thereof. In some embodiments, sequencing can be performed using a genetic analyzer, such as a genetic analyzer commercially available from Illumina, Inc., Pacific Biosciences, Inc., Applied Biosystems / Thermo Fisher Scientific, or BGI Genomics Co., Ltd., etc. For example, BGI DNBseq sequencing platforms such as BGISEQ-500, BGISEQ-50, MGISEQ-2000, MGISEQ-200, DNBSEQ-T7, DNBSEQ-G99, DNBSEQ-T20X2, or Illumina's HiSeq2000, HiSeq2500, HiSeq4000, HiSeqX10, NovaSeq6000, etc.

[0123] In the present disclosure, the term "targeted sequencing" refers to a technique that uses biotin-labeled DNA or RNA probes to capture target fragments in a DNA sample and perform sequencing. The probes can be labeled with biotin. Each nucleotide in the probes of the present invention can be chemically synthesized using, for example, a general DNA synthesizer (e.g., Model 394 manufactured by Applied Biosystems). Any other method well known in the art can also be used to synthesize oligonucleotides, such as probes.

[0124] Examples are provided below to facilitate understanding of the present disclosure. It should be understood that these examples are only used to illustrate the present disclosure, but do not constitute any limitation. The actual protection scope of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0125] Example

[0126] Example 1 A method for detecting pathogenic microorganisms

[0127] The steps are as follows:

[0128] 1. DNA extraction

[0129] The applicable sample ranges include bronchoalveolar lavage fluid, sputum, swab fluid, etc.

[0130] According to the Magnetic Pathogen Microorganism DNA / RNA Kit extraction reagent instructions, use a lysis method that combines chemical and mechanical methods to extract DNA and RNA, use silica-based magnetic beads to rapidly separate and purify nucleic acids, and use dsDNA HS Assay Kit, RNA HS Assay Kit for nucleic acid quantification.

[0131] 2. cDNA synthesis

[0132] Take the extracted nucleic acid and make up to 15 μL with nuclease-free water, and perform cDNA synthesis according to ds-cDNA SynthesisKit, including RNA denaturation, synthesis of the first strand and the second strand.

[0133] 3. Library construction

[0134] Use C37P4 OnePot cDNA&gDNA Library Prep Kit for library construction. It can be understood that the library construction reagent is not limited by the sequencing platform and can be prepared by conventional methods according to needs.

[0135] ① Fragmentation, end repair and addition of "A"

[0136] Prepare the fragmentation, end repair and addition of "A" reaction solution according to Table 2.

[0137] Table 2. Fragmentation, end repair and addition of "A" reaction system

[0138] Component Single reaction volume (μL) Second-strand cDNA 36 Nuclease-free water 10 Smearase buffer 10 Smearase enzyme 5 Total volume 61

[0139] Vortex and mix the reaction solution, then centrifuge it instantaneously. Place it in a PCR instrument and set the reaction program as shown in Table 3.

[0140] Table 3. Fragmentation, End Repair, and A-Tailing Reaction Program

[0141] Temperature Time 4℃ 1 min 30℃ 20 min 72℃ 20 min 4℃ Hold

[0142] * Set the hot lid temperature to 80 °C.

[0143] ② Adapter Ligation

[0144] Prepare the adapter ligation reaction solution according to Table 4.

[0145] Table 4. Adapter Ligation Reaction System

[0146] Component Single reaction volume (μL) A-tailed DNA 61· Ligation Enhancer 30 Novel T4 DNA Ligase 5 Adapter (3 μM) 5 Total volume 101

[0147] Vortex and mix the reaction solution, then centrifuge it instantaneously. Place it in a PCR instrument and set the reaction program as shown in Table 5 for adapter ligation.

[0148] Table 5. Adapter Ligation Reaction Program

[0149] Temperature Time 20℃ 15 min 4℃ Hold

[0150] After the ligation reaction, use magnetic beads to purify the adapter ligation product, and finally resuspend it in 22 μL of nuclease-free water.

[0151] ③ Pre-Capture PCR (Non-C-PCR) to Introduce Index

[0152] Prepare the library amplification reaction solution according to Table 6.

[0153] Table 6. Library Amplification Reaction System

[0154]

[0155] Vortex and mix the reaction solution, then centrifuge it instantaneously. Place it in a PCR instrument and set the reaction program as shown in Table 7.

[0156] Table 7. Library Amplification Reaction Program

[0157]

[0158] After the reaction, use magnetic beads to purify the PCR product, and finally resuspend it in 27 μL of Hyb buffer.

[0159] Quantify the purified product using dsDNA HS Assay Kit and perform fragment quality control with Labchip.

[0160] 4. Target Sequence Enrichment

[0161] ① Hybridization

[0162] After the library quality control is qualified, enrichment probes (sequences with nucleotide sequences shown in SEQ ID NO: 1 to 144, specific information is shown in Table 1) and hybridization elution reagents are used for hybridization capture and elution. Specifically, hybridization capture can be carried out on a single library, or according to the corresponding required library volume input, and the hybridization capture effect can also be achieved.

[0163] Prepare the hybridization reaction solution according to Table 8.

[0164] Table 8. Hybridization reaction system

[0165] Component Single reaction volume (μL) Library 26 Cot-1 DNA 5 Blocker 2 Probe 1 Total volume 34

[0166] After the reaction solution is shaken and mixed evenly, centrifuge it instantaneously, place it in a PCR instrument, and set the reaction program shown in Table 9.

[0167] Table 9. Hybridization reaction program

[0168] Temperature Time 95℃ 30s 65℃ 1h 65℃ Hold

[0169] * Set the hot lid temperature to 100 °C

[0170] ② Magnetic bead capture

[0171] After the reaction is completed, add streptavidin magnetic beads to capture the PCR product, and set the capture program according to Table 10.

[0172] Table 10. Streptavidin magnetic bead capture program

[0173] Temperature Time 65℃ 15 min 65℃ Hold

[0174] * Set the hot lid temperature to 70 °C, and resuspend and mix evenly once every 7 minutes during incubation at 65 °C.

[0175] ③ Elution

[0176] After incubation, perform the hot elution and room temperature elution procedures according to Table 11.

[0177] Table 11. Elution system

[0178]

[0179] After discarding the eluate, add 20 μL of nuclease-free water to resuspend the magnetic beads.

[0180] ③ Amplification of hybridization capture products

[0181] Prepare the amplification reaction solution according to Table 12, add it to the magnetic bead suspension in the previous step, vortex and mix evenly, and centrifuge instantaneously.

[0182] Table 12. Hybridization capture product amplification system

[0183]

[0184] Place the PCR tube of the present disclosure in the PCR instrument of the present disclosure, set the reaction program shown in Table 13, and perform library amplification.

[0185] Table 13. Amplification program for hybridization capture products

[0186]

[0187] * Set the hot lid temperature to 105 °C.

[0188] ④ Purification of amplification products

[0189] Add 60 μL of magnetic beads to purify the PCR products, and finally redissolve them in 22 μL of TE.

[0190] For the purified products, use dsDNA HS Assay Kit for quantification.

[0191] 5. Sequencing on the machine

[0192] Use the Gene+seq 100 sequencer for sequencing on the machine, and perform the sequencing operation on the machine according to the operation manual provided by the manufacturer.

[0193] 6. Analyze the sequencing data of the capture library using the bioinformatics analysis process to obtain the detection results of pathogenic microorganisms.

[0194] Example 2 Accuracy evaluation experiment of simulated samples

[0195] 1. Method

[0196] 1.1 Preparation of simulated samples

[0197] The simulated mixed sample contains human A549 cells, and each simulated mixed sample contains 1×10 5 cells / mL, and finally the concentration of pathogenic microorganisms is shown in Table 14.

[0198] Table 14. Pathogenic microorganisms and concentrations in simulated mixed samples

[0199]

[0200] 1.2 Sequencing detection

[0201] Take 400 μl of each simulated sample, perform cell wall breaking treatment with a homogenizer, and extract and purify DNA and RNA with nucleic acid extraction or purification reagents. Detect using the library construction, target sequence enrichment, and sequencing on the machine methods described in Example 2, and sequence according to the required data volume of 5M for each.

[0202] 2. Detection results of pathogenic microorganisms in simulated samples.

[0203] The captured library was subjected to bioinformatics analysis to count the number of detected sequences in the sample. The results are shown in Table 15, where RPM is the number of sequences per million mapped reads.

[0204] Table 15. Statistical results of sequencing data

[0205] Sample number Pathogenic microorganism RPM S1 Streptococcus pyogenes 1628.103 S2 Candida albicans 2046.484 S3 Escherichia coli 18.673 S4 Streptococcus pneumoniae 889.754 S5 Acinetobacter baumannii 1641.898 S6 Legionella pneumophila 1758.41 S7 Pseudomonas aeruginosa 2071.172 S8 Listeria monocytogenes 4815.381 S9 Stenotrophomonas maltophilia 142.911 S10 Haemophilus influenzae 1175.048 S11 Enterobacter cloacae 158.589 S12 Streptococcus pneumoniae 11258.72 S13 Aspergillus fumigatus 6526.036 S14 COVID-19 2375.037 S15 Influenza A (H1N1) 65.514

[0206] From the above results, it can be seen that this method has good detection accuracy in respiratory simulated samples, and the total detection time is less than 24 h, and the total bioinformatics analysis time of the samples is less than 1 h.

[0207] Example 3. Detection experiment of clinical samples

[0208] 1. Method

[0209] 1.1 Randomly select 18 lower respiratory tract samples (including bronchoalveolar lavage fluid and sputum) with high clinical suspicion of infection.

[0210] 1.2 Library preparation

[0211] Take 400 μl of each clinical sample, perform cell wall breaking treatment with a homogenizer, and extract and purify DNA and RNA with nucleic acid extraction or purification reagents. The library construction and target sequence enrichment methods described in Example 1 were used for the experiment, and the library before hybridization capture (pre-library) and the corresponding captured library (final library) were respectively run on the Gene+seq 100 sequencing platform for NGS sequencing. The pre-library was sequenced according to the required data volume of 50 M per sample, and the final library was sequenced according to the required data volume of 5 M per sample.

[0212] 2. Results

[0213] The pre-library (hereinafter referred to as "mNGS") and the corresponding hybridization-captured final library (hereinafter referred to as "tNGS") were respectively run on the Gene+seq 100 sequencing platform for NGS sequencing, and then bioinformatics analysis was performed to detect the normalized number of sequences (sequences per million mapped reads, RPM) and the enrichment efficiency. The results are shown in Table 16.

[0214] Table 16. Bioinformatics analysis results of sequencing data of mNGS and tNGS

[0215]

[0216]

[0217] In the detection of the above 18 samples, the enrichment multiple of tNGS is as high as 4.7 to 3517.89 times that of mNGS, and the detected RPM of tNGS is significantly higher than that of mNGS; all the target pathogenic microorganisms in Table 16 can be detected by tNGS, while 4 cases are missed by mNGS. From the above results, it can be seen that the performance of hybridization capture is better and the sensitivity is higher than that of the metagenomic method in respiratory samples.

[0218] The technical solutions of the present disclosure are not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solutions of the present disclosure falls within the protection scope of the present disclosure.

Claims

1. A probe set for detecting pathogenic microorganisms in respiratory tract infections, characterized in that, The probe set includes any one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 144, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto.

2. The probe set according to claim 1, wherein The probe set includes any one or more of the following groups: A probe set for detecting Haemophilus influenzae, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 3, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Stenotrophomonas maltophilia, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 7 to 9, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Enterobacter cloacae, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 13 to 15, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Streptococcus pyogenes, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 25 to 27, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Staphylococcus aureus, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 31 to 33, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Klebsiella pneumoniae, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 34 to 36, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Acinetobacter baumannii, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 37 to 39, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Escherichia coli, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 40 to 42, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Pseudomonas aeruginosa, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 43 to 45, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Streptococcus pneumoniae, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 52 to 54, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Aspergillus fumigatus, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 61 to 63, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Candida albicans, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 64 to 66, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting influenza A virus H1N1, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 70 to 72, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting influenza B virus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 82 to 84, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting respiratory syncytial virus A type, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 85 to 87, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting parainfluenza virus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 94 to 96, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting adenovirus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 97 to 99, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting novel coronavirus SARS-CoV-2, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 112 to 114, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting human metapneumovirus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 115 to 117, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting bocavirus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 118 to 120, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Mycobacterium tuberculosis complex, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 121 to 123, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Legionella pneumophila, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 130 to 132, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Listeria monocytogenes, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 133 to 135, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Mycoplasma pneumoniae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 139 to 141, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; and A probe set for detecting Chlamydia psittaci, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 142 to 144, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith.

3. The probe set according to claim 1, characterized in that, The probe set further comprises any one or more of the following groups: A probe set for detecting Haemophilus parainfluenzae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 4 to 6, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting Moraxella catarrhalis, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 10 to 12, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting Burkholderia cepacia, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 16 to 18, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting Klebsiella oxytoca, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 19 to 21, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting Klebsiella aerogenes, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 22 to 24, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting Citrobacter freundii, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 28 to 30, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting Serratia marcescens, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 46 to 48, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting Proteus mirabilis, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 49 to 51, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting Cryptococcus neoformans, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 55 to 57, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting Candida auris, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 58 to 60, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith; A probe set for detecting influenza A virus H3N2, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 67-69, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting influenza A virus H5N1, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 73-75, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting influenza A virus H7N9, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 76-78, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting influenza A virus H9N2, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 79-81, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting respiratory syncytial virus type B, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 88-90, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting rhinovirus, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 91-93, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting coronavirus 229E, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 100-102, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting coronavirus HKU1, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 103-105, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting coronavirus NL63, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 106-108, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting coronavirus OC43, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 109-111, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Mycobacterium avium, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 124-126, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Mycobacterium intracellulare, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 127-129, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Chlamydia pneumoniae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 136 to 138, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto.

4. The probe set according to any one of claims 1 to 3, characterized in that The probe is modified with biotin.

5. A kit for detecting pathogenic microorganisms in respiratory tract infections, characterized in that, The kit comprises the probe set according to any one of claims 1 to 4.

6. A method for detecting pathogenic microorganisms of respiratory tract infections using the probe set according to any one of claims 1 to 4 and / or the kit according to claim 5, the method comprising the following steps: 1) Extract nucleic acid from a sample; 2) Construct a library for the extracted nucleic acid; 3) Hybridization capture of the target sequence using the probe set; 4) Sequence the captured product and perform analysis.

7. Use of the probe set according to any one of claims 1 to 4 in the preparation of a kit.

8. The method according to claim 6 or the application according to claim 7, characterized in that, The sample includes cell, tissue or body fluid samples; Preferably, the sample includes tissue, cells, blood, plasma, serum, bronchoalveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, pleural effusion, or their processed products.

9. The method according to claim 6 or the application according to claim 7, characterized in that, The probe set is used to detect one or more of bacteria, fungi, viruses, mycoplasma or chlamydia.

10. The method according to claim 6 or the application according to claim 7, characterized in that, The probe set is used to detect one or more of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Stenotrophomonas maltophilia, Escherichia coli, Moraxella catarrhalis, Enterobacter cloacae, Serratia marcescens, Burkholderia cepacia, Klebsiella oxytoca, Enterobacter aerogenes, Proteus mirabilis, Streptococcus pyogenes, Haemophilus parainfluenzae, Citrobacter freundii, Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Influenza virus, Human Respiratory syncytial virus, Rhinovirus, Human parainfluenza virus, Human adenovirus, coronavirus, Human metapneumovirus, Human bocavirus, Mycobacterium tuberculosis complex, Mycobacterium aviumavian), Mycobacterium intracellulare, Legionella pneumophila, Listeria monocytogenes, Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia psittaci; Preferably, the influenza virus includes Influenza A virus H1N1, Influenza A virus H3N2, Influenza A virus H5N1, Influenza A virus H7N9, Influenza A virus H9N2 or Influenza B virus; Preferably, the respiratory syncytial virus includes Human Respiratory syncytial virus A and Human Respiratory syncytial virus B; Preferably, the coronavirus includes Human coronavirus 229E, Human coronavirus HKU1, Human coronavirus NL63, Human coronavirus OC43 or novel coronavirus SARS-CoV-2.

Images