A probe set, method and application of pathogenic microorganism detection

Abstract

The present invention discloses a probe group, method and application for detecting pathogenic microorganisms, wherein the probe group specifically detects pathogenic microorganisms in a sample. In particular, in the field of central nervous system infection diseases, the defects of conventional central nervous system infection pathogen detection are overcome: pathogen targeted enrichment overcomes the defects of long cycle and low positive rate of traditional detection methods; overcomes the defects of high host ratio, low microbial load and low sensitivity of mNGS; targeted enrichment overcomes the low pathogen load of central nervous system infection itself and the difficulty of detecting some intracellular bacteria. The sensitivity and specificity of pathogen detection are greatly improved, and it has important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular, relates to a probe set, method and application of pathogenic microorganism detection. Background Art

[0002] Central nervous system (CNS) infection is a common, difficult and critical disease. Globally, there are more than 30 million patients with meningitis each year, and the disability and mortality rates of meningitis are high. Because the cerebrospinal fluid is a sterile environment, once the pathogen invades, the disease develops rapidly; the sources of pathogens include bloodstream infection, respiratory infection, focal infection or retrograde infection of the virus through nerves; in bacterial meningitis infection, the pathogen load is less than 103CFU / ml, and the lowest is 40CFU / ml; due to the rapid onset of meningitis, the wide distribution of pathogens, low load and difficulty in sampling, it is difficult to identify the pathogen and the positive rate is low; failure to make a clear diagnosis and standardize treatment is the main reason for the high mortality rate. Currently, there are two types of methods for identifying pathogens of central nervous system infection. One is culture-based methods, such as morphology, cell physiological and biochemical characteristics, bacterial culture typing, mass spectrometry, and automated microbial analysis systems; the other is molecular detection-based methods, such as gene chips (CN102251053A), multiplex PCR (CN113201599A), metagenomics (mNGS), and ultra-multiplex PCR (CN112831604A) that can enrich pathogens and probe capture methods.

[0003] The results obtained from traditional culture are still the gold standard for CNS infection detection, but compared with the culture positivity rate of other systems, especially in cerebrospinal fluid testing, the positivity rate is extremely low, only about 10%; that is, more than 90% of CNS infections cannot be clearly diagnosed by etiology when relying on traditional testing.

[0004] Molecular detection gene chips and specific primer PCR have good specificity and sensitivity; but they also have the defects of low throughput and false positives; combined with specific technologies such as antigen detection, the cause of 50% of patients is still unknown. mNGS detects a wide range of pathogens, which can help detect most pathogens of CNS infection to a certain extent, and assist in clinical rapid diagnosis. However, the application defects of mNGS limit its sensitivity in detecting central nervous system infections. For samples with a high proportion of human hosts and low microbial content, the real pathogens will be masked by a large number of human sequences, which can easily cause false negatives and low sensitivity; mNGS technology has low efficiency in extracting nucleic acids from pathogens and fungi with thicker cell walls, resulting in low detection rate and sensitivity; and the indiscriminate detection of all pathogens makes it difficult to distinguish between background microbial detection and pathogenic bacteria, which brings more confusion to interpretation and clinicians. In addition, mNGS testing is expensive because the sequence signals of pathogenic microorganisms may only account for 1%, requiring the amount of test data to be no less than 20M to ensure a certain positive rate. For low-load sample types such as cerebrospinal fluid, even higher data volumes are required, increasing sequencing costs (Expert consensus on the application of second-generation sequencing of cerebrospinal fluid metagenomics for infectious diseases of the central nervous system).

[0005] The existing methods for detecting pathogens that infect the central nervous system are still mainly insufficient in sensitivity. A highly sensitive detection method for low-load pathogens and intracellular pathogens is needed to improve the clinical positive compliance rate of test samples. The use of methods such as multiplex PCR or liquid hybridization capture to positively enrich pathogenic microorganisms can effectively improve the detection sensitivity. In the former, the multiplex PCR detection cycle is short but the number of primers is limited: the nonlinear growth of dimers limits the multiplex number; therefore, the detectable pathogen range is limited; there is large interference between primers, which makes optimization difficult. Once the detection range changes, the entire reaction system needs to be redesigned. In addition, the repeated fragments introduced by PCR during the library construction process have insufficient fragment diversity and cannot represent the true enrichment of the target area. Once a contaminated fragment is amplified, the abundance of false positive pathogens detected is high, and it will be difficult to distinguish the true pathogen. Summary of the invention

[0006] In order to solve at least one of the above problems, the present disclosure provides a probe set, method and application of pathogenic microorganism detection.

[0007] According to the first aspect of the present disclosure, a probe group for detecting pathogenic microorganisms is provided, wherein the probe group comprises any combination of one or more pathogenic microorganisms in the nucleotide sequence or continuous fragments as shown in SEQ ID NOs: 1 to 30, or a nucleotide sequence having a sequence identity of more than 85% therewith, or a complementary sequence thereof.

[0008] In some embodiments, the probe set comprises any combination of one or more pathogenic microorganisms in the nucleotide sequence or continuous fragments as shown in SEQ ID NO: 1 to 12, or a nucleotide sequence having a sequence identity of more than 85% thereto, or a complementary sequence thereof, and / or,

[0009] The probe group comprises any combination of one or more pathogenic microorganisms in the nucleotide sequences or continuous fragments as shown in SEQ ID NOs: 13 to 21, or a nucleotide sequence having a sequence identity of more than 85% therewith, or a complementary sequence thereof, and / or,

[0010] The probe group comprises any combination of one or more pathogenic microorganisms in the nucleotide sequences or continuous fragments shown in SEQ ID NOs: 22 to 30, or a nucleotide sequence having a sequence identity of more than 85% therewith, or a complementary sequence thereof.

[0011] In some embodiments, the probe group detects one or any combination of two or more of the following 10 pathogenic microorganisms: Klebsiella oxytoca, Streptococcus constellatus, Coxiella burnetii, Mycoplasma pneumoniae, Candida albicans, Blastomyces dermatitidis, Coccidioides immitis, cytomegalovirus, human adenovirus type 7, and lymphocytic choriomeningitis virus.

[0012] In some embodiments, the probe set detects the following four bacteria: Klebsiella oxytoca, Streptococcus constellatus, Coxiella burnetii, or Mycoplasma pneumoniae, and / or,

[0013] The probe set detects the following three fungi: Candida albicans, Blastomyces dermatitidis or Coccidioides immitis, and / or,

[0014] The probe set detects the following three viruses: cytomegalovirus, human adenovirus type 7, or lymphocytic choriomeningitis virus.

[0015] In some embodiments, the probe group detects the following pathogenic microorganisms: one or any combination of two or more pathogenic microorganisms selected from Coxiella burnetii, Mycoplasma pneumoniae, Candida albicans, Blastomyces dermatitidis, and Coccidioides immitis.

[0016] In some embodiments, the probes in the probe set are 100 to 150 bp in length.

[0017] In some embodiments, the probes in the probe set are 120 bp in length.

[0018] In some embodiments, the GC content of the probes in the probe set is 30-70%.

[0019] In some embodiments, the GC content of the probes in the probe set is 45-60%.

[0020] According to a second aspect of the present disclosure, a composition is provided, comprising the probe group according to the first aspect of the present disclosure.

[0021] According to a third aspect of the present disclosure, a kit is provided, the kit comprising the probe set described in the first aspect or the composition described in the second aspect.

[0022] In some embodiments, the kit is used for the detection of tissue or body fluid samples, such as tissue, cells, blood, alveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion or processed products thereof.

[0023] In some embodiments, the kit is used for detecting cerebrospinal fluid samples.

[0024] According to the fourth aspect of the present disclosure, there is provided a non-diagnostic and non-therapeutic detection method for detecting pathogenic microorganisms using the probe group of the first aspect, the composition of the second aspect or the kit of the third aspect, the method comprising hybridization capture of the target nucleic acid by the probe.

[0025] In some embodiments, the method comprises the following steps:

[0026] 1) Extract nucleic acid from the sample,

[0027] 2) constructing a library of the extracted nucleic acid,

[0028] 3) Probe hybridization capture of target nucleic acid,

[0029] 4) Detecting the captured products.

[0030] The nucleic acid in step 1) includes DNA or RNA,

[0031] In some embodiments, the nucleic acid in step 1) includes nucleic acid of a pathogenic microorganism.

[0032] In some embodiments, step 1) further comprises removing nucleic acids of human origin,

[0033] In some embodiments, step 3) further comprises amplifying the captured product.

[0034] In some embodiments, the detection in step 4) includes PCR detection, fluorescent quantitative PCR detection, ARMS-PCR detection or sequencing.

[0035] According to a fifth aspect of the present disclosure, provided is the use of the probe group described in the first aspect, the composition described in the second aspect, the kit described in the third aspect, or the detection method described in the fourth aspect in detecting pathogenic microorganisms.

[0036] In some embodiments, the pathogenic microorganism is a pathogenic microorganism that infects the central nervous system.

[0037] In some embodiments, the central nervous system infection comprises encephalitis, meningitis, or cerebrospinal meningitis.

[0038] In some embodiments, the central nervous system infection comprises a bacterial infection, a fungal infection, or a viral infection.

[0039] In some embodiments, the central nervous system infection comprises a cerebrospinal fluid infection.

[0040] The probe set, method and application of pathogen detection disclosed in the present invention are mainly aimed at patients infected with pathogenic microorganisms, including intracellular bacteria and RNA viruses; a highly sensitive probe set is designed to detect 10 pathogenic microorganisms including 4 bacteria, 3 fungi and 3 viruses, especially Coxiella burnetii, Mycoplasma pneumoniae, Candida albicans, Blastomyces dermatitidis, Coccidioides immitis and other difficult-to-detect bacteria, intracellular bacteria and special pathogens. Through probe hybridization capture technology, the LOD for low-load pathogens can reach a minimum of 20 copies / ml, and the detection of pathogens and viruses in infected samples has an enrichment effect of 10 to 20,000 times.

[0041] Especially in the field of central nervous system infection diseases, it overcomes the defects of conventional central nervous system infection pathogen detection: pathogen targeted enrichment overcomes the defects of traditional detection methods such as long cycle and low positive rate; overcomes the defects of high host ratio, low microbial load and low sensitivity of mNGS; targeted enrichment overcomes the low pathogen load of central nervous system infection itself and the difficulty of detecting some intracellular bacteria, greatly improving the sensitivity and specificity of pathogen detection of central nervous system infection diseases.

[0042] Moreover, the sequencing cost is lower. Pathogen mNGS detection requires 30M data. When the probe group and method disclosed in the present invention are used for targeted probe capture, 1 to 2M data can highly enrich the target pathogen.

[0043] In summary, the probe set, method and application for pathogen detection disclosed in the present invention have important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The abundance map of Klebsiella oxytoca is shown. The horizontal axis is the genome coordinate, the vertical axis is the sequencing depth, the lower right corner is the sequencing data volume, the UMR in the upper right corner is the unique aligned gene sequence, COV is the genome coverage, and DIS is the genome dispersion.

[0045] Figure 2 The abundance map of Streptococcus constellatus detection is shown. The horizontal axis is the genome coordinate, the vertical axis is the sequencing depth, the lower right corner is the sequencing data volume, the UMR in the upper right corner is the unique aligned gene sequence, COV is the genome coverage, and DIS is the genome dispersion.

[0046] Figure 3 The abundance graph of cytomegalovirus detection is shown. The horizontal axis is the genome coordinate, the vertical axis is the sequencing depth, the lower right corner is the sequencing data volume, the UMR in the upper right corner is the unique aligned gene sequence, COV is the genome coverage, and DIS is the genome dispersion. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0048] definition

[0049] 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 belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0050] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0051] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0052] In the present disclosure, the term "sequence" refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, this term includes double-stranded and single-stranded DNA, as well as RNA. The term "nucleic acid" used herein is a single-stranded or double-stranded covalently linked nucleotide sequence, wherein the 3' and 5' ends on each nucleotide are connected by a phosphodiester bond. Polynucleotides can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be synthesized and manufactured in vitro, or isolated from natural sources. Nucleic acids can further include modified DNA or RNA, such as DNA or RNA that has been methylated. According to the principle of base complementarity, when a nucleotide sequence is known, the nucleotide information of its complementary sequence can be obtained accordingly.

[0053] In the present disclosure, the term "complementary" refers to the concept of sequence complementarity between regions of two polynucleotide chains or between two regions of the same polynucleotide chain. It is known that the adenine base of the first region of the polynucleotide can form a specific hydrogen bond ("base pair") with the base of the second region of the polynucleotide that is antiparallel to the first region (if the base is thymine or uracil). Similarly, it is known that the cytosine base of the first polynucleotide chain can base pair with the base of the second polynucleotide chain that is antiparallel to the first region (if the base is guanine). If at least one nucleotide in the first region can base pair with the base of the second region when the first region of the polynucleotide is arranged in an antiparallel manner with the second region of the same or another different polynucleotide, then the two regions are complementary. Therefore, two complementary polynucleotides do not need to base pair at every nucleotide position. "Complementary" means that the first polynucleotide is 100% or "completely" complementary to the second polynucleotide, and therefore base pairing is formed 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 high stringency hybridization conditions.

[0054] In the present disclosure, the term "stringent hybridization conditions" is defined as conditions that can be determined empirically under which a probe or primer specifically hybridizes to a target sequence but not to non-target sequences. The term "stringent conditions" is functionally defined in terms of the hybridization of a nucleic acid probe to a target nucleic acid (i.e., to a specific nucleic acid sequence of interest) by the specific hybridization method discussed in Sambrook et al., 1989, 9.52-9.55. See also Sambrook et al., 1989 9.47-9.52, 9.56-9.58; Kanehisa 1984 Nucl. Acids Res. 12: 203-213; and Wetmur et al. 1968 J. Mol. Biol. 31: 349-370. Appropriate stringent conditions that promote DNA hybridization are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1989, 6.3.1-6.3.6.

[0055] In the present disclosure, "sequence identity" refers to the "percentage of sequence identity" or "percentage of identity" between two polynucleotides, that is, the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide, a gap present in the target sequence is not counted. Similarly, since the target sequence nucleotides are counted and the nucleotides from the reference sequence are not counted, the gaps present in the reference sequence are not counted. At least 85% sequence identity includes a continuous segment having at least 85%, 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.

[0056] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same nucleic acid base occurs in both sequences, to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The determination of the sequence identity percentage between the comparison of the sequence and the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite available on the BLAST website of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses the BLASTN algorithm to compare between two sequences. BLASTN is used for comparing nucleic acid sequences. Other suitable programs are, for example, a part of the EMBOSS suite of Needle, Stretcher, Water or Matcher, bioinformatics programs.

[0057] In the present disclosure, a "fragment" refers to a continuous segment having sequence identity of at least 55%, at least 65%, at least 75%, at least 85%, at least 95% or 100% of the full length of the sequence.

[0058] In the present disclosure, the term "probe" refers to labeling a primer with a capture label or a detection label, and detecting the primer product. The probe sequence is used to hybridize with the sequence generated by the primer sequence, and usually 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 desired use of the probe, usually in the range of 6 to 50 nucleotides. Preferably, the appropriate length of the primer includes 15 to 35 nucleotides. In the present invention, the probe can be one of a TaqMan probe, an MGB probe, and a hybridization probe. For example, the probe is a TaqMan probe, and the 5' end of the labeled probe is a fluorescent luminescent group, which is one of FAM, VIC, TET, JOE, ROX, CY3, CY5, and HEX; the 3' end of the labeled probe is a fluorescent quenching group, which is one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, and NFQ.

[0059] In the present disclosure, the term "nucleic acid" generally refers to a monomeric or polymeric form of nucleotides of any length, which is a deoxyribonucleotide or a ribonucleotide, or an analog or variant thereof. A nucleic acid molecule may include one or more unmodified or modified nucleotides. Nucleic acids may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of nucleic acids: ribonucleic acid (RNA), deoxyribonucleic acid (DNA), coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis (locus), exons, introns, messenger RNA (mRNA), transfer RNA (RNA), ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, complementary deoxyribonucleic acid (cDNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Nucleic acid can comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs, such as peptide nucleic acids (PNA), morpholino and locked nucleic acids (LNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), 2'-fluoro, 2'-OMe and thiophosphorylated DNA. Nucleic acid can include one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) or variants thereof. In some instances, nucleic acid is DNA or RNA or derivatives thereof. Nucleic acid can be single-stranded or double-stranded. Nucleic acid can be circular.

[0060] In the present disclosure, the term "RNA", the full name of "RiboNucleic Acid" (abbreviated as RNA), is one of the four biological macromolecules contained in biological cells. RNA is a macromolecular polymer composed of nucleotides. Nucleotides are composed of bases, ribose and phosphate. There are 4 types of bases: adenine (A), guanine (G), uracil (C) and cytosine (C).

[0061] In the present disclosure, the term "DNA", the full name of which is "deoxyribonucleic acid" (abbreviated as DNA), is one of the four biological macromolecules contained in biological cells, which is 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 functioning of organisms. DNA is a macromolecular polymer composed of deoxynucleotides. Deoxynucleotides are composed of bases, deoxyribose and phosphate. There are four types of bases: adenine (A), guanine (G), thymine (T) and cytosine (C).

[0062] In the present disclosure, the term "target nucleic acid" generally refers to any nucleic acid comprising a sequence to be detected. The target nucleic acid can be DNA or RNA. The target nucleic acid can be naturally occurring or synthetic (such as an amplification product, a vector, etc.). The target nucleic acid can be, but need not be, purified or isolated. Depending on the properties desired to be determined, the target nucleic acid can be derived from plant or animal tissue, or taken from a reaction mixture. The target nucleic acid is not limited in length, although the target nucleic acid can be exposed to a restriction endonuclease before being detected or identified by the method of the present application.

[0063] In this disclosure, "sequencing" refers to the process used to determine the sequence (eg, the identity and order of monomeric units) of a biological molecule, eg, 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 terminator sequencing, whole genome sequencing, hybridization sequencing, pyrophosphate sequencing, capillary electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, low denaturation temperature co-amplification 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, synthesis sequencing, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, DNA nanoball sequencing (DNBSEQ), composite probe anchor polymerase sequencing (cPAS), and combinations thereof. In some embodiments, sequencing can be performed by a genetic analyzer, such as a genetic analyzer commercially available from Illumina, Inc., Pacific Biosciences, Inc., Applied Biosystems / Thermo Fisher Scientific, or Shenzhen MGI Intelligent Manufacturing Technology 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.

[0064] Examples and drawings are provided below to help understand the present disclosure. However, it should be understood that these examples and drawings are only used to illustrate the present disclosure and do not constitute any limitation. The actual scope of protection 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.

[0065] Example 1. Probe design for detecting different pathogens of cerebrospinal fluid infection

[0066] In this embodiment, probes for detecting different pathogens of cerebrospinal fluid infection are designed, as shown in Table 1.

[0067] Table 1. Pathogenic microorganisms and corresponding probe sequences

[0068]

[0069] Table 1 (continued)

[0070]

[0071] Table 1 (continued)

[0072]

[0073] Example 2. The construction experiment of the gene library of cerebrospinal fluid samples includes the following steps:

[0074] Nucleic acid extraction: Nucleic acid extraction kit (Jinjia, KB0055048) was used for nucleic acid extraction, and the extraction was performed according to the instructions: 280ul of cerebrospinal fluid sample was taken, and enzymatic reagent was added to lyse the cells to release nucleic acids; negative reference (human cell A549) and positive reference (human cell A549 + Staphylococcus aureus, purchased from Beina Bio) were also lysed. After conventional reagent adsorption and washing, and elution with waste liquid, the extracted nucleic acid was obtained. The sample nucleic acid was subjected to concentration quality control using a fluorescence meter and an analysis kit: the sample was quantified using an Invitrogen Qubit fluorescence meter and a Qubit dsDNA HS analysis kit, and the total amount of nucleic acid extracted was calculated based on the concentration measurement value and the recovery volume. The concentration quality control standards are shown in Table 2.

[0075] Table 2. Free DNA + RNA extraction concentration quality control standards

[0076] Free DNA extraction concentration judge ≥0.1ng / ul Normal database construction Too low Normal database construction

[0077] The nucleic acid concentration in the cerebrospinal fluid sample may be lower than the detection range of the Qubit fluorometer and will not affect the subsequent library construction.

[0078] 2. Library construction: Library construction kit (Jinjia, KB0055048), operate according to the instructions: take 5ng of nucleic acid extraction product and react in a PCR instrument at 70℃ for 5min to complete RNA denaturation through a one-step method, use the first-chain synthesis reagent, vortex the reaction mixture to mix and centrifuge, set the reaction program according to the instructions to complete the synthesis of the first-chain cDNA and the synthesis of the second-chain cDNA; then perform cDNA&gDNA fragmentation, end repair and dA tail addition; then use the TA connection method to add the adapter; after the connection product is purified by magnetic beads, perform library amplification and library purification and recovery to obtain the intermediate library. The total amount of the library is quality controlled according to the concentration measurement value and the recovery volume.

[0079] 1) See Table 3 for concentration quality control.

[0080] The total amount of library was calculated based on the concentration and recovery volume using the Invitrogen Qubit Fluorometer and Qubit dsDNA HS Assay Kit.

[0081] Table 3. Library concentration quality control standards

[0082] Library yield judge ≥2000ng Normal hybridization 1000ng~2000ng Risk hybrid <1000ng Rebuild the database

[0083] 2) Fragment quality control:

[0084] Use Qsep100 for fragment quality control:

[0085] S2 cartridge; quality inspection samples were diluted with 0.1x Dilution Buffer to a final concentration of 0.5-1.0 ng / μL, with a volume ≥15 μL.

[0086] Qualified: fragment distribution is 150-600bp, the main peak of the library is 250-300bp, and there is no adapter dimer (130-140bp), primer (30-70bp) and large fragment contamination (more than 1000bp).

[0087] 3. Liquid phase hybridization capture

[0088] 1) Library pre-blocking: Dry the library pre-blocking reagent in a vacuum concentrator and set aside;

[0089] 2) Hybridization: prepare hybridization mixture, incubate at room temperature for 10 min, mix well, and place the hybridization mixture PCR tube in a PCR instrument to start the hybridization reaction (95°C denaturation, 30 s; 65°C reaction, 1 h).

[0090] 3) Magnetic bead washing: Add 100ul of washing solution to the PCR tube containing magnetic beads and hybridization reaction solution, shake at 65℃ to mix, centrifuge until the solution is clear, and discard the supernatant.

[0091] 4) Hybridization library amplification: Perform PCR amplification in a PCR reaction system. The amplification procedure is shown in Table 4.

[0092] Table 4. PCR amplification program

[0093]

[0094] 5) Hybridization library quality control:

[0095] Concentration quality control: Take 1ul library sample and use Qubit fluorometer and Qubit dsDNA HS analysis kit to quantify the library. The hybridization library yield quality control standards are shown in Table 5.

[0096] Table 5. Hybridization library yield quality control standards

[0097] Library yield judge ≥550ng Risky machine 110ng~550ng Normal boarding ≤110ng Risky machine

[0098] Fragment quality control: The main peak of qualified fragments is 250-300bp, without adapter dimers (130-140bp), primers (30-70bp) and large fragment contamination (more than 1000bp)

[0099] Example 3. Test of simulated cerebrospinal fluid samples

[0100] The experiment was conducted using low-load pathogens as shown in Table 6 as sample starting amounts, using SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27 probes; nucleic acid extraction, library construction, hybridization capture, and sequencing were performed in a similar manner to Example 2. The sequencing data bioinformatics process was analyzed to obtain the results shown in Table 6.

[0101] Table 6. Low pathogen load input and test results

[0102]

[0103] The test results of cerebrospinal fluid simulation samples are shown in Table 6. The LOD of the present invention for low-load cerebrospinal fluid infection pathogens can reach as low as 20 copies / ml, which meets the detection limit performance requirements for the detection of common cerebrospinal fluid infection pathogens in clinical practice.

[0104] Example 4. Comparative experiment on conventional metagenomic detection and probe hybridization capture detection of difficult-to-detect bacteria in cerebrospinal fluid

[0105] Cerebrospinal fluid samples from three patients with clinical meningitis infection were used, and the probes described in Example 1 were used to perform nucleic acid extraction, library construction, hybridization capture and sequencing in a similar manner to Example 2. The sequencing data bioinformatics process analysis yielded the results shown in Table 7, where the detected abundance graph is shown in Figures 1 to 3 .

[0106] Table 7. Test results of cerebrospinal fluid samples from 3 patients with clinical meningitis infection

[0107]

[0108] As shown in Table 7, compared with the results of cerebrospinal fluid mNGS and probe capture, the present disclosure has an enrichment effect of 10 to 20,000 times for the detection of pathogens and viruses in cerebrospinal fluid infection samples, significantly improving the detection sensitivity of cerebrospinal fluid samples.

[0109] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and all technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A probe set for detecting pathogenic microorganisms of central nervous system infection, the probe set comprising a combination of nucleotide sequences as shown in SEQ ID NOs: 1 to 3 and 7 to 30, or complementary sequences thereof.

2. The probe set according to claim 1, characterized in that: The pathogenic microorganisms detected by the probe group include: Klebsiella oxytoca Klebsiellaoxytoca Coxiella burnetii Coxiella burnetii , Mycoplasma pneumoniae Mycoplasmapneumoniae , Candida albicans Candida albicans , Blastomyces dermatitidis Blastomyces dermatitidis Coccidioides immitis Coccidioidesimmitis , cytomegalovirus, human adenovirus type 7, lymphocytic choriomeningitis virus, or any combination of two or more pathogenic microorganisms.

3. A composition comprising the probe set according to claim 1 or 2. 4 . A kit comprising the probe set according to claim 1 or 2 .

5. A method for detecting pathogenic microorganisms for non-diagnostic purposes using the probe set according to claim 1 or 2, the composition according to claim 3 or the kit according to claim 4, the method comprising the step of hybridizing and capturing the target nucleic acid using the probe.

6. The method according to claim 5, characterized in that The method comprises the following steps: 1) extracting nucleic acid from a sample, 2) constructing a library for the extracted nucleic acid, 3) hybridizing and capturing the target nucleic acid with a probe, and 4) detecting the captured product.

7. The method according to claim 6, characterized in that The nucleic acid in step 1) includes DNA or RNA.

8. The method according to claim 6, characterized in that The nucleic acid in step 1) includes nucleic acid of pathogenic microorganisms.

9. The method according to claim 6, characterized in that The step 1) further comprises removing nucleic acids of human origin.

10. The method according to claim 6, characterized in that The step 3) also includes amplifying the captured product.

11. The method according to claim 6, characterized in that The detection in step 4) includes PCR detection, fluorescence quantitative PCR detection, ARMS-PCR detection or sequencing.

12. Use of the probe set according to claim 1 or 2 and the composition according to claim 3 in preparing a kit for detecting pathogenic microorganisms; the pathogenic microorganisms include: Klebsiella oxytoca Klebsiellaoxytoca Coxiella burnetii Coxiella burnetii , Mycoplasma pneumoniae Mycoplasmapneumoniae , Candida albicans Candida albicans , Blastomyces dermatitidis Blastomyces dermatitidis Coccidioides immitis Coccidioidesimmitis , cytomegalovirus, human adenovirus type 7, lymphocytic choriomeningitis virus, or any combination of two or more pathogenic microorganisms.

13. The use according to claim 12, characterized in that: The pathogenic microorganisms are pathogenic microorganisms that infect the central nervous system.

14. The use according to claim 13, characterized in that The central nervous system infection includes encephalitis, meningitis or cerebrospinal meningitis.

15. The use according to claim 13, characterized in that: The central nervous system infection includes bacterial infection, fungal infection or viral infection.

16. The use according to claim 13, characterized in that: The central nervous system infection includes cerebrospinal fluid infection.

17. The use according to claim 12, characterized in that: The kit is used for detecting tissue or body fluid samples.

18. The use according to claim 17, characterized in that The tissue or body fluid sample includes one or more of the following: tissue, cell, blood, alveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion or processed materials thereof.

19. The use according to claim 12, characterized in that: The kit is used for detecting cerebrospinal fluid samples.

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