Probe set for targeted detection of pathogenic microorganisms and application

Abstract

The invention provides a pathogenic microorganism probe set, a kit and application, free nucleic acid fragments of four bacteria and two fungi can be specifically captured and enriched, the defect that sequencing data are mainly human due to the fact that mNGS detection is extremely high in human host proportion and low in microorganism content in the conventional technology is overcome, and compared with mNGS, the detection result is more accurate, and the detection accuracy is higher. The detection sequence number and proportion of the pathogenic microorganisms are increased, the detection signal of the pathogenic microorganisms is efficiently amplified, the detection sensitivity is improved, and the detection cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and particularly to a kit and application for targeted detection of the genomes of pathogenic microorganisms. Background Art

[0002] The detection of pathogenic microorganisms has always been an important problem in infection diagnosis. For a long time, microbial culture remains the gold standard for pathogen detection. Clinical applications also include antigen, antibody detection, PCR detection, as well as staining, smear, microscopy, etc. However, the long time and low positive rate are the drawbacks of traditional detection methods and the main reasons for the difficulty in clinical diagnosis.

[0003] As an example of the detection of pathogenic microorganisms, bloodstream infection (BSI) is a severe systemic infectious disease, which can cause shock, disseminated intravascular coagulation (DIC), and multiple organ dysfunction syndrome (MODS) in severe cases. Moreover, the incidence of bloodstream infection shows an increasing trend year by year, and the fatality rate remains high, seriously affecting the recovery and prognosis of patients. Common pathogenic microorganisms causing bloodstream infection include, for example, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Candida albicans, and Candida tropicalis, etc.

[0004] In recent years, the development of metagenomics (meta-genomic next-generation sequencing, mNGS) has provided new ideas for pathogen detection. Metagenomics, also known as the environmental genome or metagenome of pathogenic microorganisms, refers to the sum of all microbial genetic materials in a specific tissue or sample, which includes the genomes of currently culturable and unculturable microorganisms in vitro. With its characteristics of being non-preferential, fast (24h), and highly sensitive, metagenomics can detect all sequences present in a biological sample at one time, including bacteria, fungi, viruses, and parasites. However, as a newly developed detection method, there are still many problems to be solved in metagenomics. For example, due to the extremely high proportion of human host in mNGS and the characteristics of biological samples with low microbial content, the sequencing data is mainly human, resulting in the fact that the true pathogens will be masked by a large number of human sequences, with low sensitivity, easy to cause false negatives, weak specificity, and easy to bring out a large number of background microorganisms, and its clinical positive coincidence rate needs to be improved; moreover, in metagenomic sequencing, due to the extremely low signal of pathogenic microorganism sequences, a high requirement for the amount of detection data is needed, increasing the cost of sequencing.

[0005] Pathogenic microorganism detection includes culture and non-culture auxiliary detection methods. Among them, the culture method is the gold standard for diagnosing bloodstream infections, but it has disadvantages such as a long diagnostic cycle and a low positive detection rate. Currently, auxiliary methods such as next-generation sequencing (NGS) have been gradually applied clinically to further improve the prognosis of patients. However, problems such as high cost and difficult interpretation also limit its application scope in clinical practice.

[0006] Therefore, a detection method with high sensitivity, short cycle, relatively low price, and simple interpretation is needed. Summary of the Invention

[0007] To solve at least one of the above technical problems, the present disclosure provides a probe set, a kit, and an application for detecting pathogenic microorganisms.

[0008] According to the first aspect of the present disclosure, a probe set for detecting pathogenic microorganisms is provided, and the probe set specifically captures the following pathogenic microorganisms: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Candida albicans, and Candida tropicalis.

[0009] In some embodiments, the probe set includes one or more of the sequences shown in Table 1.

[0010] In some embodiments, the probe set contains any one of the nucleotide sequences shown in SEQ ID NO: 1-60 or a nucleotide sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity thereto.

[0011] In some embodiments, the probe set contains a probe set for detecting Staphylococcus aureus, and contains any one of the nucleotide sequences shown in SEQ ID NO: 1-10 or a nucleotide sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity thereto.

[0012] In some embodiments, the probe set contains a probe set for detecting Staphylococcus epidermidis, and contains any one of the nucleotide sequences shown in SEQ ID NO: 11-20 or a nucleotide sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity thereto.

[0013] In some embodiments, the probe set contains a probe set for detecting Enterococcus faecalis, and contains any one of the nucleotide sequences shown in SEQ ID NO: 21 to 30 or a nucleotide sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity therewith.

[0014] In some embodiments, the probe set contains a probe set for detecting Enterococcus faecium, and contains any one of the nucleotide sequences shown in SEQ ID NO: 31 to 40 or a nucleotide sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity therewith.

[0015] In some embodiments, the probe set contains a probe set for detecting Candida albicans, and contains any one of the nucleotide sequences shown in SEQ ID NO: 41 to 50 or a nucleotide sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity therewith.

[0016] In some embodiments, the probe set contains a probe set for detecting Candida tropicalis, which contains any one of the nucleotide sequences shown in SEQ ID NO: 51-60 or a nucleotide sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity therewith.

[0017] In some embodiments, the length of the probes in the probe set is selected from 100-150 bp, and the GC content of the probes in the probe set is selected from 30%-70%, preferably 45%-60%.

[0018] In some embodiments, the length of the probes in the probe set is selected from 100 bp, 101 bp, 102 bp, 103 bp, 104 bp, 105 bp, 106 bp, 107 bp, 108 bp, 109 bp, 110 bp, 111 bp, 112 bp, 113 bp, 114 bp, 115 bp, 116 bp, 117 bp, 118 bp, 119 bp, 120 bp, 121 bp, 122 bp, 123 bp, 124 bp, 125 bp, 126 bp, 127 bp, 128 bp, 129 bp, 130 bp, 131 bp, 132 bp, 133 bp, 134 bp, 135 bp, 136 bp, 137 bp, 138 bp, 139 bp, 140 bp, 141 bp, 142 bp, 143 bp, 144 bp, 145 bp, 146 bp, 147 bp, 148 bp, 149 bp or 150 bp.

[0019] In some embodiments, the probe can be biotin-modified or have any other common modification in the art. In some embodiments, after the probe is biotin-modified, the probe sequence enriched with the target sequence in the sample can be captured and enriched by streptavidin magnetic beads, and then the captured sample is eluted at high temperature with a washing solution. After removing the non-specific capture sequence, PCR specific amplification is carried out and then sequenced on a machine, so as to achieve the purpose of rapid, highly sensitive and specific enrichment detection of the pathogenic microorganism target sequence in the sample.

[0020] In some embodiments, the probes in the probe set are in the form of single-stranded DNA, double-stranded DNA or RNA probes.

[0021] According to a second aspect of the present disclosure, there is provided a kit for detecting pathogenic microorganisms, the kit comprising the probe set described in the first aspect.

[0022] In some embodiments, the kit can be used for natural samples or standards.

[0023] In some embodiments, the kit can be used for the detection of tissue samples or body fluid samples.

[0024] In some embodiments, the body fluid samples include: saliva, whole blood, serum, plasma, milk, urine, lumbar or ventricular CSF, lymph fluid, prostatic fluid, semen, sputum, feces, tears, tumor cells, bronchoalveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion and ascites, amniotic fluid, peritoneal fluid, aqueous humor, vitreous humor, vaginal discharge and their processed products.

[0025] In some embodiments, the tissue samples include tissues, paraffin sections and their processed products.

[0026] According to a third aspect of the present disclosure, there is provided a method for detecting pathogenic microorganisms using the probe set described in the first aspect or the kit described in the second aspect, the method comprising the following steps:

[0027] 1) Extract nucleic acids from the sample,

[0028] 2) Construct a library for the extracted nucleic acids,

[0029] 3) Hybridization capture the library using the probe described in the second aspect to obtain a capture product,

[0030] 4) Sequence the capture product and perform data analysis.

[0031] In some embodiments, step 1) includes removing host nucleic acids.

[0032] In some embodiments, step 3) further includes amplifying the capture product.

[0033] According to a fourth aspect of the present disclosure, there is provided the use of the probe set described in the first aspect in the preparation of a kit for detecting pathogenic microorganisms.

[0034] In some embodiments, the kit can be used for natural samples or standards.

[0035] In some embodiments, the kit can be used for the detection of tissue samples or body fluid samples.

[0036] In some embodiments, the body fluid sample includes: saliva, whole blood, serum, plasma, milk, urine, lumbar or ventricular CSF, lymph fluid, prostatic fluid, semen, sputum, feces, tears, tumor cells, bronchoalveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion, amniotic fluid, peritoneal fluid, aqueous humor, vitreous humor, vaginal discharge, and their processed products.

[0037] In some embodiments, the tissue sample includes tissue, paraffin sections, and their processed products.

[0038] In some embodiments, the pathogenic microorganism includes at least one of the following microorganisms: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Candida albicans, and Candida tropicalis.

[0039] Through screening, the present disclosure has obtained a set of probe groups capable of efficiently and highly specifically detecting pathogenic microorganisms, which can specifically capture and enrich free nucleic acid fragments of 4 bacteria and 2 fungi. The number of probes and the capture of free nucleic acid fragments are technical defects of the primer method for specific capture, and it overcomes the defect that the proportion of human host in the prior art of conventional technology is extremely high and the content of microorganisms is low, resulting in sequencing data mainly dominated by humans. This enables the true pathogens to be captured and enriched from a large number of human sequences. Compared with mNGS, it increases the number and proportion of detected sequences of pathogenic microorganisms, efficiently amplifies the detection signal of pathogenic microorganisms, improves the detection sensitivity, and reduces the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 FIG. shows a schematic flowchart for detecting pathogens according to an embodiment of the present disclosure.

[0041] Figure 2 FIG. shows the result of detecting the library fragment size of the pathogen library using a Qsep biofragment analyzer according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0043] Definition

[0044] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention pertains. For the purpose of interpreting this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0045] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents.

[0046] The expression "about" used herein is as understood by those of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If those of ordinary skill in the art do not understand the use of this term according to the context in which it is used, "about" will mean a specific value plus or minus 10%.

[0047] In the present disclosure, the term "RNA", the full name is "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 bases, ribose, and phosphoric acid. Among them, there are 4 types of bases: adenine (A), guanine (G), uracil (U), and cytosine (C).

[0048] In the present disclosure, the term "DNA", the full name is "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 bases, deoxyribose, and phosphoric acid. Among them, there are 4 types of bases: adenine (A), guanine (G), thymine (T), and cytosine (C).

[0049] In the present disclosure, the term "probe" refers to an oligonucleotide capable of binding to a target nucleic acid of a complementary sequence through one or more types of chemical bonds (usually through complementary base pairing, usually through hydrogen bond formation). Depending on the stringency of the hybridization conditions, the probe may bind to a target sequence that lacks complete complementarity to the probe sequence. There may be any number of base pairs that interfere with the hybridization between the target sequence and the single-stranded sequence described herein. However, if the number of mutations is so large that hybridization does not occur even under the least stringent hybridization conditions, the sequence is not a complementary target sequence. The probe may be single-stranded, or partially single-stranded and partially double-stranded. The strandedness of the probe is described by the structure, composition, and nature of the target sequence. The probe may be directly labeled with biotin that can be subsequently bound by, for example, a streptavidin complex, or indirectly labeled. In some embodiments, the term "probe" includes an oligonucleotide strand, or an oligonucleotide strand complementary thereto.

[0050] In the present disclosure, the term "probe set" generally refers to a collection of more than one probe that localizes and / or quantifies 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 a segment of oligonucleotide, such as a single-stranded DNA molecule or RNA segment.

[0051] In the present disclosure, the term "sequence identity" refers to the "percent sequence identity" or "identity percentage" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences within a comparison window, taking into account 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 is present in both the target sequence and the reference sequence. Since gaps are not nucleotides, gaps present in the target sequence are not counted. Similarly, gaps present in the reference sequence are not counted since target sequence nucleotides are counted and reference sequence nucleotides are not. At least 60% sequence identity includes contiguous segments having sequence identity of 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% over the entire length of the sequence. Methods for aligning comparison 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, which present detailed considerations of sequence alignment methods and homology calculations.

[0052] In the present disclosure, "alignment" refers to the process of comparing a read or tag to 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 location within the reference sequence.

[0053] In the present disclosure, the term "hybridization" or "specific hybridization" refers to the ability of a molecule to bind, duplex, or hybridize only to a specific polynucleotide sequence under stringent conditions, as when the sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[0054] 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. It is known that an adenine base in a first region of a polynucleotide can form specific hydrogen bonds ("base pairs") with a base (if the base is thymine or uracil) in a second region of a polynucleotide that is antiparallel to the first region. Similarly, it is known that a cytosine base in a first polynucleotide strand can base pair with a base (if the 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 can base pair 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 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.

[0055] In the present disclosure, the term "stringent hybridization conditions" refers to 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. The 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 the 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, washed with 0.2×SSC and 0.1% SDS at 65 °C.

[0056] In the present disclosure, the term "sequencing" refers to a technique 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 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 HiSeq 2000, HiSeq 2500, HiSeq 4000, HiSeq X10, NovaSeq 6000, etc.

[0057] 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.

[0058] Examples and accompanying drawings are provided below to assist in understanding the present invention. It should be understood that these examples and drawings are only for illustrating the present invention and do not constitute any limitation. The actual protection scope of the present invention 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 invention.

[0059] Example 1. Probe set for detecting pathogenic microorganisms

[0060] The nucleotide sequence information of the probe set for detecting pathogenic microorganisms is shown in Table 1. The probe set is used to detect Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Candida albicans, and Candida tropicalis.

[0061] Table 1. Probe sequences for detecting pathogenic microorganisms

[0062]

[0063] Table 1 (continued)

[0064]

[0065] Table 1 (continued)

[0066]

[0067] Table 1 (continued)

[0068]

[0069] Example 2. Detection of the detection limit of the simulated sample

[0070] 1. Preparation of the simulated mixed sample

[0071] Human A549 cells were mixed with the following pathogenic microorganisms respectively: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Candida albicans, and Candida tropicalis to construct a simulated mixed sample. The above pathogenic microorganisms were purchased from Beina Biology. Each simulated mixed sample contained 1.14×10 A549 cells4 per mL, the prepared pathogenic microorganism mixture was serially diluted 2-fold three times with sterile PBS, and an equal amount was added to A549 cells to finally obtain a serially diluted pathogenic mock sample, where the concentration of pathogenic microorganisms is shown in Table 2.

[0072] Table 2. Pathogenic microorganisms and concentrations in the simulated mixed samples

[0073]

[0074] 2. Detection

[0075] The pathogenic microorganisms were detected according to the following method, and the process is shown in Figure 1 , including the following steps:

[0076] 1) Plasma separation and extraction of plasma-free nucleic acids

[0077] Take 280 μL of each serially diluted pathogenic mock sample and extract nucleic acids using the QIAamp Viral RNA Mini Kit (Qiagen).

[0078] 2) Library construction

[0079] Use the Hieff C37P4 OnePot cDNA&gDNA Library Prep Kit (Yeasen) to construct a library, and the library fragments are 150 - 200 bp. After purification with Hieff NGS DNA Selection Beads (Yeasen), the library concentration is quantitatively detected by qubit.

[0080] 3) Liquid-phase hybridization capture

[0081] Quantitatively take 25 ng of each library, mix it with a probe set (sequences shown in Table 1) for detecting pathogenic microorganisms labeled with biotin, and use Hybrid Capture Reagents kit (NanoAnda) for hybridization. Through the biotin-streptavidin reaction, the target fragments are anchored on the streptavidin-coated magnetic beads, and the target library fragments are obtained after washing.

[0082] 4) Library amplification

[0083] Use the system and program shown in Table 3 to amplify the captured product by PCR, and the number of cycles is 16.

[0084] Table 3. Library amplification experiment system and program

[0085]

[0086] 5) Sequencing

[0087] The SE100 sequencing results were obtained using a Geneplus 100 sequencer, and the capture library was sequenced at a data volume of 2M for each sample.

[0088] 6) Data splitting and bioinformatics analysis

[0089] The off-machine data was analyzed using a bioinformatics analysis process to output the microbial detection results.

[0090] 3. Detection results

[0091] The off-machine data was analyzed using a bioinformatics process to count the number of high-quality reads per M data volume (Target_RPMCR) in each target region of the sample, and the results are shown in Table 4.

[0092] Table 4. Detection results of pathogenic microorganisms in simulated mixed samples

[0093]

[0094] Note: "-" indicates not detected.

[0095] As can be seen from the above table, the kit of the present invention has a sensitivity of up to 12.5 copy / mL for Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium at a data volume of 2M; and a sensitivity of up to 25 copy / mL for Candida albicans and Candida tropicalis.

[0096] Example 3. Detection of clinical samples

[0097] Twenty-three clinical samples were obtained from a hospital. Library construction was carried out in the same manner as in Example 2. One copy of the library before capture (the library constructed corresponding to step 2 of the detection method in Example 2) was retained as the mNGS library. The result graph of detecting the library fragment size using a Qsep biofragment analyzer is shown in Figure 2 , and the other copy was hybrid capture using the probe set for detecting pathogenic microorganisms described in the present disclosure (the sequences are shown in Table 1) in the same manner as in Example 2 to obtain a hybrid capture library. For each sample, an mNGS library and a hybrid capture library were prepared simultaneously. The sequencing volume of the mNGS library was 33M, and the sequencing volume of the hybrid capture library was 2M. Bioinformatics analysis was carried out in the same manner as in Example 1, and the results are shown in Table 5, where RPMCR is the sequence per million mapped reads.

[0098] Table 5. Detection results of pathogenic microorganisms in clinical samples

[0099]

[0100] As can be seen from the above table, all 23 positive clinical samples can be accurately detected by the detection method of the present invention, and the detection sensitivity reaches 100% (23 / 23), which is much higher than the sensitivity of mNGS, 65.22% (15 / 23). The probe set of the present invention has a good capture effect on the target microorganism.

[0101] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A probe set for detecting pathogenic microorganisms, wherein the probe set contains any one of the nucleotide sequences shown in SEQ ID NO: 1 to 60 or a nucleotide sequence having at least 60% sequence identity therewith.

2. The probe set according to claim 1, wherein, the probe set contains: a probe set for detecting Staphylococcus aureus, containing any one of the nucleotide sequences shown in SEQ ID NO: 1 to 10 or a nucleotide sequence having at least 60% or more sequence identity therewith; and / or, the probe set contains a probe set for detecting Staphylococcus epidermidis, containing any one of the nucleotide sequences shown in SEQ ID NO: 11 to 20 or a nucleotide sequence having at least 60% or more sequence identity therewith; and / or, the probe set contains a probe set for detecting Enterococcus faecalis, containing any one of the nucleotide sequences shown in SEQ ID NO: 21 to 30 or a nucleotide sequence having at least 60% or more sequence identity therewith; and / or, the probe set contains a probe set for detecting Enterococcus faecium, containing any one of the nucleotide sequences shown in SEQ ID NO: 31 to 40 or a nucleotide sequence having at least 60% or more sequence identity therewith; and / or, the probe set contains a probe set for detecting Candida albicans, containing any one of the nucleotide sequences shown in SEQ ID NO: 41 to 50 or a nucleotide sequence having at least 60% or more sequence identity therewith; and / or, the probe set contains a probe set for detecting Candida tropicalis, containing any one of the nucleotide sequences shown in SEQ ID NO: 51 to 60 or a nucleotide sequence having at least 60% or more sequence identity therewith.

3. The probe set according to claim 1, wherein, the probe set is modified with biotin.

4. A kit for detecting pathogenic microorganisms, wherein, the kit includes the probe set according to any one of claims 1 to 3.

5. A method for detecting pathogenic microorganisms using the probe set according to any one of claims 1 to 3 or the kit according to claim 4, the method comprising the following steps: 1) Extract nucleic acid from a sample, 2) Construct a library for the extracted nucleic acid, 3) Use the probe set to perform hybridization capture on the library to obtain a capture product, 4) Sequence the capture product and perform data analysis.

6. Use of the probe set according to any one of claims 1 to 3 in the preparation of a kit for detecting pathogenic microorganisms.

7. The use according to claim 6, wherein, the kit is for natural samples or standards.

8. The use according to claim 6, wherein, The kit is used for the detection of tissue samples or body fluid samples.

9. According to the application described in claim 6, wherein, the body fluid samples include: saliva, whole blood, serum, plasma, milk, urine, lumbar or ventricular CSF, lymph fluid, prostatic fluid, semen, sputum, feces, tears, tumor cells, bronchoalveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion and ascites, amniotic fluid, peritoneal fluid, aqueous humor, vitreous humor, vaginal discharge and their processed products; and / or, the tissue samples include tissues, paraffin sections and their processed products.

10. According to the application described in claim 6, wherein, the kit is used for detecting at least one of the following pathogenic microorganisms: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Candida albicans and Candida tropicalis.

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