Probe set and method for detecting streptococcus pneumoniae serotype and application

Abstract

The invention provides a probe set for detecting streptococcus pneumoniae serotype, a method and application. The probe set provided by the invention can rapidly identify streptococcus pneumoniae and classify the capsule serotype of streptococcus pneumoniae, and is suitable for serotype replacement and vaccine action monitoring.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid sequencing. Specifically, the present invention relates to a probe set, method and application for detecting serotypes of Streptococcus pneumoniae. Background Art

[0002] Streptococcus pneumoniae (referred to as pneumococcus) is a Gram-positive facultative anaerobic bacterium. Pneumococcus can cause severe pneumococcal disease (PD), such as upper respiratory tract infection, otitis media, pneumonia, bacteremia, meningitis, etc. Bacteremia and meningitis caused by pneumococcus are also called invasive pneumococcaldisease (IPD), with high mortality and serious sequelae. Pneumococcus is the main pathogen of community-acquired pneumonia and one of the main causes of infection and death in children under 5 years old and people over 60 years old. Pneumococcal disease has a serious disease burden, and the WHO lists pneumococcal vaccine as a very high priority vaccine.

[0003] Streptococcus pneumoniae is a gram-positive coccus that colonizes the upper respiratory tract mucosa, and children are more susceptible to colonization. The bacteria can cause community-acquired pneumonia (CAP), bacteremia, and meningitis (the latter two are collectively referred to as invasive pneumococcal disease, IPD). Other infections include otitis media, sinusitis, peritonitis, suppurative arthritis, and occasionally endocarditis.

[0004] Capsular polysaccharide (CPS) on the surface of Streptococcus pneumoniae is of great significance: ⑴ It is an important virulence and pathogenic factor. The virulence of Streptococcus pneumoniae also varies due to its different components. ⑵ Capsular polysaccharide can prevent the cell wall from binding to host antibodies, thereby escaping the attack of the host immune system. ⑶ Capsular polysaccharide is immunogenic and is the basis of serotyping. Each serotype corresponds to a capsular polysaccharide with different chemical and structural properties. The induced antibodies can prevent Streptococcus pneumoniae infection, which is the basis of current vaccines. There are regional differences in the distribution of Streptococcus pneumoniae serotypes. Studies have shown that serotypes 19A, 7F and 3 are the main types in Western Europe, 35B is the main serotype in the United States, 19A, 3 and 22F are the main serotypes in Canada, and 6B, 23F and 19F are the main serotypes prevalent in Japan. The distribution of serotypes is also related to factors such as the site of strain isolation, population race, age, type of disease caused, vaccine use and time. With the use of vaccines, the colonization rate of pneumococcal serotypes contained in the vaccine in the nasopharynx has generally decreased, the infection caused by common pneumococcal serotypes has decreased, and the previously rare serotypes have gradually become popular, changing the prevalence and epidemiology of pneumococci. This phenomenon is called "serotype replacement". Surveillance studies after the widespread use of vaccines have shown that the colonization and pathogenicity of non-vaccine serotypes (NVT) of pneumococcal pneumonia have increased, and their proportion in clinical isolates has increased. Genes encoding different serotypes of pneumococci can undergo gene-level recombination and conversion, and turn to encode another capsule serotype, thereby escaping the effect of the vaccine. In addition, pneumococci can undergo effective intra-species and inter-species DNA recombination, which in turn leads to changes in capsule composition, serotype, antibiotic resistance and virulence factors. With the increasing number of IPD caused by NVT, many countries have responded to serotype replacement by introducing pneumococcal vaccines with more valent serotypes, making the task of vaccine research and development more arduous. It is particularly important to continue monitoring the serotypes of Streptococcus pneumoniae, the changes in serotypes after vaccine protection, and the resulting changes in drug resistance. Summary of the invention

[0005] In order to solve at least one of the above problems, the present disclosure provides a probe library for detecting 90 serotypes of Streptococcus pneumoniae based on tNGS technology, as well as a supporting kit and method, so as to achieve rapid identification of Streptococcus pneumoniae and typing of its capsular serotype, which is suitable for serotype replacement and vaccine effect monitoring.

[0006] According to one aspect of the present disclosure, a genetic marker for detecting Streptococcus pneumonia is provided, the marker comprising any one or more of the following genes: wchB, wchC, wchD, wchG, wchH, wchl, wchE, gaIU, pgmHG261, wciK, wciM, whaC, whaD, whaE, HG262, HG263, wciQ, wciR, wciS, wciT, HG265, HG266, IrpwcilB, whaJ, whaF, HG267, wciG, tnp, wzg, wzh, wzd or wze.

[0007] According to yet another aspect of the present disclosure, a probe set for detecting Streptococcus pneumoniae is provided.

[0008] In some embodiments, the probe set is used to detect the gene markers disclosed herein.

[0009] In some embodiments, the probe set has a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1 to 155, or a nucleotide sequence having at least 85% sequence identity thereto, or a complementary sequence thereof.

[0010] According to yet another aspect of the present disclosure, a composition is provided, comprising the probe set.

[0011] According to yet another aspect of the present disclosure, a kit for detecting Streptococcus pneumoniae is provided, the kit comprising the probe set.

[0012] According to another aspect of the present disclosure, a method for detecting Streptococcus pneumoniae using the probe group, the composition and / or the kit of the present disclosure is provided, and the method comprises using the probe group, the composition and / or the kit to detect Streptococcus pneumoniae.

[0013] In some embodiments, the method comprises the following steps:

[0014] 1) Extract nucleic acid from the sample,

[0015] 2) constructing a library of the extracted nucleic acid,

[0016] 3) Probe hybridization capture of target nucleic acid,

[0017] 4) Detecting the captured products.

[0018] The nucleic acid in step 1) includes DNA or RNA,

[0019] In some embodiments, the nucleic acid in step 1) includes nucleic acid of a pathogenic microorganism.

[0020] In some embodiments, step 1) further comprises removing nucleic acids of human origin,

[0021] In some embodiments, step 3) further comprises amplifying the captured product.

[0022] In some embodiments, the detection in step 4) includes PCR detection, fluorescent quantitative PCR detection, ARMS-PCR detection or sequencing.

[0023] According to another aspect of the present disclosure, provided is the use of the gene markers and / or the probe set described in the present disclosure in preparing a kit for detecting Streptococcus pneumoniae.

[0024] In some embodiments, the pneumococcus comprises one or more of the following serotypes: type 1, type 2, type 3, type 4, type 5, type 6A, type 6B, type 7A, type 7B, type 7C, type 7F, type 8, type 9A, type 9L, type 9N, type 9V, type 10A, type 10B, type 10C, type 10F, type 11A, type 11B, type 11C, type 11D, type 11F, type 12A, type 12B, type 12F, type 13, type 14, type 15A, type 15B, type 15C, type 15F, type 16A, type 16F, type 17A, type 17F, type 18A, type 18B, type 18C, type 18F, type 19A , 19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F and 48.

[0025] In some embodiments, the kit is used for the detection of bacterial culture or body fluid samples.

[0026] In some embodiments, the bacterial culture comprises a solid medium and / or a liquid medium culture product.

[0027] In some embodiments, the body fluid sample includes: blood, plasma, serum, bronchoalveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab and / or cerebrospinal fluid.

[0028] In some embodiments, the tissue sample comprises lung tissue and / or paraffin sections.

[0029] Beneficial effects:

[0030] The probe set provided by the present disclosure can detect and identify 90 serotypes of Streptococcus pneumoniae. After verification by clinical samples, the probe set of the present invention has good sensitivity and specificity and can meet the requirements of clinical detection. The probe set provided by the present disclosure can quickly identify Streptococcus pneumoniae and type its 90 capsule serotypes, which can meet the detection needs of serotype replacement and vaccine effect monitoring. DETAILED DESCRIPTION

[0031] According to databases and literature reports, the present invention determines 90 serotypes as shown in Table 1 as the detection range of the probe.

[0032] Table 1. 90 S. pneumoniae serotypes

[0033]

[0034]

[0035] The present invention screens the above-mentioned pneumococcal serotypes to obtain specific genes as shown in Table 2 and conserved genes as shown in Table 3, designs these genes respectively, and obtains 155 probes as shown in Table 16 by screening a large number of probe groups, so as to improve the sensitivity and accuracy of pneumococcal serotype typing.

[0036] Table 2. Specific genes of Streptococcus pneumoniae serotypes

[0037] Serotype Unique gene markers 1 wchB, wchC, wchD 2 wchG, wchH, wchl 3 wchE, gaIU, pgm 4 HG261, wciK, wciM 5 whaC, whaD, whaE 6A HG262 6B HG263 8 wciQ, wciR, wciS, wciT, HG265, HG266 14 Irp 20 wcilB、whaJ、whaF 24F HG267 33F wciG*

[0038] Table 3. Conserved genes among S. pneumoniae serotypes

[0039] 90 serotypes shared gene markers tnp wxya w wzd w

[0040] definition

[0041] 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 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.

[0042] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0043] 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.

[0044] 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).

[0045] 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).

[0046] In the present disclosure, the term "probe" refers to an oligonucleotide that can bind 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 with the probe sequence. There may be any number of base pairs that will interfere with the hybridization between the target sequence described herein and the single-stranded sequence. 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 properties of the target sequence. The probe may be directly labeled or indirectly labeled with biotin that is later bound by, for example, a streptavidin complex. In some embodiments, the term "probe" comprises an oligonucleotide chain, or an oligonucleotide chain complementary thereto. The appropriate length of the probe depends on the desired use of the probe, typically 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 TaqMan probe, an MGB probe, and a hybridization probe.

[0047] In the present disclosure, the term "probe set" generally refers to a collection of more than one probe, which achieves the location and / or quantification of the target nucleic acid by recognizing and binding (by hybridization pairing) the target sequence. Each probe in the probe combination is generally an oligonucleotide, such as a single-stranded DNA molecule or RNA.

[0048] In the present disclosure, the term "gene marker" refers to a molecular indicator having a specific biological property, biochemical characteristic or aspect, which can be used to determine the presence or absence and / or the severity of a particular disease or condition.

[0049] In the present disclosure, the term "marker" refers to a parameter associated with one or more biological molecules (ie, a "genetic marker"), such as a nucleic acid produced naturally or artificially.

[0050] The terms "hybridization" or "binding" used in the present disclosure refer to the pairing (including partial complementarity) of complementary (including partially complementary) polynucleotide chains. Hybridization and the strength of hybridization (for example, the strength of binding between polynucleotide chains) are affected by many factors well known in the art, including the degree of complementarity between polynucleotides, the stringency of related conditions, which are affected by conditions such as salt concentration, the melting temperature (Tm) of the formed hybrid, the presence of other components, the molar concentration of the hybridized chain, and the G:C content of the polynucleotide chain. When a polynucleotide chain is said to be "hybridized" with another polynucleotide chain, it means that there is a certain complementarity between the two polynucleotide chains, or the two polynucleotide chains form a hybrid under high stringency conditions. When a polynucleotide chain is said not to hybridize with another polynucleotide chain, it means that there is no sequence complementarity between the two polynucleotide chains, or the two polynucleotide chains do not form a hybrid under high stringency conditions.

[0051] The term "complementary" as used in the present disclosure 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 chain can form a specific hydrogen bond ("base pair") with the base of the second region of the polynucleotide chain that is antiparallel and complementary 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 and complementary 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 and complementary manner with the second region of the same or another different polynucleotide, the two regions are complementary. Therefore, the two complementary polynucleotide chains do not need to base pair at every nucleotide position. "Complementary" means that the first polynucleotide chain is 100% or "completely" complementary to the second polynucleotide chain, and therefore base pairing is formed at every nucleotide site. "Complementary" also means that the first polynucleotide strand is not 100% complementary (eg, 90%, or 80% or 70% complementary) and contains mismatched nucleotides at one or more nucleotide positions. In one embodiment, two complementary polynucleotide strands are capable of hybridizing to each other under high stringency hybridization conditions.

[0052] The term "stringent hybridization conditions" as used in the present disclosure 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.

[0053] In the present application, the term "sequence identity" refers to the "percentage of sequence identity" or "percentage of sequence identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences in the comparison window, taking into account the additions or deletions (i.e., gaps) that must be introduced for the optimal comparison of the two sequences. A matching position is any position in which the same nucleotides are present in both the target sequence and the reference sequence. Since the gap is not a nucleotide, the 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 gap present in the reference sequence is not counted. In some embodiments, at least 85% sequence identity includes 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% of the total length of the sequence having a continuous segment with sequence identity.

[0054] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences, to obtain the matching position number, the matching position number is divided by the total number of positions in the comparison window, and the result is multiplied 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 protein and nucleotide sequences. The alignment method of the comparison sequence is well known in the art, for example, by existing software BLASR, DALIGN, BLAST, BLAST-2, ALIGN, ALIGN-2BWA, BOWTIE or Megalign ((DNASTAR)) etc.

[0055] As used herein, the term "sequencing" refers to the process of determining 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, pyrosequencing, capillary electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel signature sequencing, emulsion PCR, low denaturing 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, sequencing by synthesis, 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.

[0056] Examples are provided below to help understand the present invention. However, it should be understood that these examples are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be noted that for those skilled in the art, several variations and improvements made on the basis of this invention conception all belong to the protection scope of the present invention. The reagents used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0057] Example

[0058] Example 1: A method for detecting Streptococcus pneumoniae and its serotypes

[0059] 1. DNA Extraction

[0060] ① Alveolar lavage fluid, sputum and swab fluid were collected according to Magnetic Pathogen Microorganism DNA / RNA Kit Extraction Reagent Instructions: This kit uses a combination of chemical and mechanical lysis methods to extract DNA and RNA, and uses silica-based magnetic beads to quickly separate and purify nucleic acids. dsDNAHS Assay Kit, RNA HSAssay Kit was used for nucleic acid quantification.

[0061] ②Whole blood and plasma were divided into Circulating Nucleic Acid Kit Extraction Reagent Instructions, using column extraction method to concentrate and purify free DNA and RNA in samples, using dsDNA HS Assay Kit, RNA HSAssay Kit was used for nucleic acid quantification.

[0062] 2. cDNA Synthesis

[0063] Nucleic acid was extracted from blood and respiratory samples and made up to 15 μL with nuclease-free water. The ds-cDNA Synthesis Kit is used for cDNA synthesis, including RNA denaturation and first-strand and second-strand synthesis.

[0064] 3. Library Construction

[0065] Using Hieff C37P4OnePot cDNA&gDNA Library Prep Kit was used for library construction.

[0066] ① Fragmentation, end repair and adding "A"

[0067] Prepare fragmentation, end repair and "A" reaction solutions according to Table 4

[0068] Table 4. Fragmentation, end repair and "A" reaction system

[0069] Components Single reaction volume (μL) Second strand cDNA 36 Nuclease-free water 10 Smearase Buffer 10 Smearase 5 Total volume 61

[0070] The reaction solution was mixed by oscillation and centrifuged immediately, and then placed in a PCR instrument and set to the reaction program shown in Table 5.

[0071] Table 5. Fragmentation, end repair and "A" addition reaction procedures

[0072] temperature time 4℃ 1min 30℃ 20min 72℃ 20min 4℃ Keep

[0073] *Set the heated lid temperature to 80°C.

[0074] ②Connector connection

[0075] Prepare the adapter ligation reaction solution according to Table 6.

[0076] Table 6. Linker ligation reaction system

[0077]

[0078]

[0079] The reaction solution was mixed by oscillation and centrifuged instantaneously, placed in a PCR instrument, and the reaction program shown in Table 7 was set to perform adapter ligation.

[0080] Table 7. Adapter ligation reaction procedure

[0081] temperature time 20℃ 15min 4℃ Keep

[0082] After the ligation reaction, the adapter ligation product was purified using magnetic beads and finally dissolved back into 22 μL of nuclease-free water.

[0083] ③Introduction of index by pre-capture PCR (Non-C-PCR)

[0084] Prepare the library amplification reaction solution according to Table 8.

[0085] Table 8. Library amplification reaction system

[0086] Components Single reaction volume (μL) F / R primer (15 μM) 5 2×UltimaHF Amplification Master Mix 25 Adapter-ligated library 20 Total volume 50

[0087] The reaction solution was mixed by oscillation and centrifuged briefly, placed in a PCR instrument, and set to the reaction program shown in Table 9.

[0088] Table 9. Pre-capture PCR reaction program

[0089]

[0090] After the reaction, the PCR product was purified using magnetic beads and finally dissolved back into 27 μL Hyb buffer.

[0091] The purified product was treated with dsDNAHS Assay Kit was used for quantification and Labchip fragment quality control.

[0092] 4. Target sequence enrichment

[0093] ① Hybridization

[0094] After the library quality control is qualified, the enrichment probe and hybridization elution reagent are used to perform hybridization capture elution.

[0095] Prepare the hybridization reaction solution according to Table 10.

[0096] Table 10. Hybridization reaction system

[0097] Components Single reaction volume (μL) library 26 Cot-1DNA 5 Sealing agent 2 Probe 1 Total volume 34

[0098] The reaction solution was mixed by oscillation and centrifuged briefly, placed in a PCR instrument, and set to the reaction program shown in Table 11.

[0099] Table 11. Hybridization reaction program

[0100] temperature time 95℃ 30s 65℃ 1h 65℃ Keep

[0101] *Set the heated lid temperature to 100°C

[0102] ②Magnetic bead capture

[0103] After the reaction is completed, streptavidin magnetic beads are added to capture the PCR product, and the capture program is set according to Table 12.

[0104] Table 12. Capture procedures

[0105] temperature time 65℃ 15min 65℃ Keep

[0106] *Set the temperature of the heated lid to 70°C and resuspend and mix once every 7 minutes during incubation at 65°C.

[0107] ③ Elution

[0108] After the incubation is completed, perform the hot elution and room temperature elution procedures according to Table 13.

[0109] Table 13. Elution system

[0110]

[0111] After discarding the eluate, add 20 μL of nuclease-free water to resuspend the magnetic beads.

[0112] ④ Amplification of hybrid capture products

[0113] Prepare the amplification reaction solution according to Table 14, add it to the magnetic bead suspension in the previous step, vortex to mix, and centrifuge briefly.

[0114] Table 14. Hybridization capture product amplification reaction system

[0115] Components Single reaction volume (μL) 2 × KAPA HiFi Hot Start Ready Mix 25 Primer F / R 2.5 Total volume 27.5

[0116] Place the PCR tube in a PCR instrument, set the reaction program shown in Table 15, and perform library amplification.

[0117] Table 15. Hybridization capture product amplification reaction program

[0118]

[0119]

[0120] *Set the heated lid temperature to 105°C.

[0121] ⑤ Purification of amplified products

[0122] Add 60 μL of magnetic beads to purify the PCR product, and finally dissolve it back into 22 μL TE.

[0123] The purified product was treated with Quantification was performed using the dsDNA HS Assay Kit.

[0124] 5. Sequencing

[0125] Gene+seq 100 sequencer was used for sequencing, and the sequencing experiment was performed according to the operating instructions provided by the manufacturer. The sequencing data of the captured library was analyzed using the bioinformatics analysis process to obtain the results of Streptococcus pneumoniae detection.

[0126] The hybridization step used a probe for detecting Streptococcus pneumoniae as described in Table 16.

[0127] Table 16. Probes for detecting Streptococcus pneumoniae provided by the present disclosure

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] Example 2 Serotype detection experiment of strain samples

[0139] 1. Methods

[0140] 1.1 Sample selection

[0141] Pneumococcal strains were isolated and cultured from clinical samples of the lower respiratory tract (sputum, bronchoalveolar lavage fluid) of patients with pneumococcal disease, and WGS sequencing was performed on the strains. The sequencing results were analyzed using PneumoKity and seroba software to identify the serotype of the strain. According to the serotype identification results, one strain of each serotype was selected for the detection of this methodology. The selected strains are shown in Table 17.

[0142] Table 17. Strain information

[0143] Strain number WGS results 1 19A 2 9A 3 19F 4 22F 5 11F 6 37 7 5 8 3 9 35C 10 23F 11 8 12 6B 13 20 14 6C 15 23A 16 6A / 6B 17 15A 18 15C

[0144] 1.2 Sequencing

[0145] Take 400 μL of each of the 18 different serotypes of Streptococcus pneumoniae liquid samples, break the cell wall with a homogenizer, and extract and purify DNA and RNA with a nucleic acid extraction or purification reagent. Use the probe set provided by the present disclosure, use the library construction, target sequence enrichment, and sequencing method described in Example 1 for detection, and sequence according to the required data volume of 5M for each sample.

[0146] 2. Detection and serotyping results of Streptococcus pneumoniae in strain samples.

[0147] The library after probe group capture was analyzed by bioinformatics process, and the number of sample detection sequences was counted. The results are shown in Table 18.

[0148] Table 18. Sequencing results of bacterial samples

[0149]

[0150]

[0151] From the above results, it can be seen that the method has good detection accuracy in the Streptococcus pneumoniae strain sample, wherein the positive coincidence rate (PPA, also known as sensitivity) of serotype detection is 100%, and the negative coincidence rate (NPA, also known as specificity) is 100%. The probe set provided by the present disclosure can not only detect Streptococcus pneumoniae, but also accurately identify different serotypes of Streptococcus pneumoniae.

[0152] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A genetic marker for detecting Streptococcus pneumoniae, the marker comprising any one or more of the following genes: wchB, wchC, wchD, wchG, wchH, wchl, wchE, gaIU, pgmHG261, wciK, wciM, whaC, whaD, whaE, HG262, HG263, wciQ, wciR, wciS, wciT, HG265, HG266, IrpwcilB, whaJ, whaF, HG267, wciG, tnp, wzg, wzh, wzd or wze.

2. A probe set for detecting Streptococcus pneumoniae, wherein the probe set is used to detect the gene marker according to claim 1.

3. The probe set according to claim 2, It is characterized in that The probe set has a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1 to 155, or a nucleotide sequence having at least 85% sequence identity therewith, or a complementary sequence thereof.

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

5. A kit for detecting Streptococcus pneumoniae, comprising the probe set according to claim 2 or 3.

6. A method for detecting Streptococcus pneumoniae using the probe set according to claim 2 or 3, the composition according to claim 4 and / or the kit according to claim 5, the method comprising detecting Streptococcus pneumoniae using the probe set, the composition and / or the kit.

7. Use of the gene marker according to claim 1 and / or the probe set according to claim 2 or 3 in preparing a kit for detecting Streptococcus pneumoniae.

8. The use according to claim 7, It is characterized in that The pneumococcal bacteria include one or more of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 19A, 19B, 19C, 19F, 19A, 19B, 19C, 19F, 19A, 19B, 19C, 19F, 19A, 19 Type B, Type 19C, Type 19F, Type 20, Type 21, Type 22A, Type 22F, Type 23A, Type 23B, Type 23F, Type 24A, Type 24B, Type 24F, Type 25A, Type 25F, Type 27, Type 28A, Type 28F, Type 29, Type 31, Type 32A, Type 32F, Type 33A, Type 33B, Type 33C, Type 33D, Type 33F, Type 34, Type 35A, Type 35B, Type 35C, Type 35F, Type 36, Type 37, Type 38, Type 39, Type 40, Type 41A, Type 41F, Type 42, Type 43, Type 44, Type 45, Type 46, Type 47A, Type 47F, and Type 48.

9. The use according to claim 7, It is characterized in that The kit is used for detecting bacterial culture or body fluid samples.

10. The use according to claim 9, It is characterized in that The bacterial culture includes solid culture medium and / or liquid culture medium culture product; Preferably, the body fluid sample includes: blood, plasma, serum, alveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab and / or cerebrospinal fluid.

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