Methods and tools for identifying serotypes of streptococcus pneumoniae
Patent Information
- Application Number
- CN202610677002.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-15
- Publication Date
- 2026-10-27
- Estimated Expiration
- 2046-05-15
Smart Images
Figure CN122214521B_ABST
Abstract
Description
Technical Field
[0001] This application generally relates to the field of bacteriology, and more specifically to methods and tools for identifying serotypes of Streptococcus pneumoniae. Background Technology
[0002] Streptococcus pneumoniae ( Streptococcus pneumoniae Streptococcus pneumoniae, belonging to the genus Streptococcus, is one of the earliest known Gram-positive bacteria in humans. It is present in the upper respiratory tract of healthy children and adults, causing a range of infections including meningitis, sepsis, pneumonia, sinusitis, and otitis media. Children under 2 years of age and adults ≥65 years of age are particularly susceptible. Pneumococcal disease is the leading cause of death in children under five years of age and is a serious global public health problem. The pathogenicity of Streptococcus pneumoniae is related to various virulence factors, such as the capsule, cell wall, and its polysaccharide components. Capsular polysaccharide (CPS) is a major antigenic component of the extracellular wall of Streptococcus pneumoniae, possessing anti-phagocytic activity, and is one of the main virulence factors, as well as a basis for serotyping.
[0003] New methods for serological typing of Streptococcus pneumoniae are still needed. Summary of the Invention
[0004] In a first aspect, this application provides a method for identifying serotypes 20A and 20B of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: acquiring NGS sequencing data of Streptococcus pneumoniae in the biological sample; and identifying serotypes 20A and 20B of Streptococcus pneumoniae according to the following steps using PneumoCaT and Serocall software: extracting sequencing data of the whaF gene from the NGS sequencing data and comparing the sequencing data of the whaF gene with the reference sequence of SEQ ID NO: 1; and extracting sequencing data of the wzd gene from the NGS sequencing data and comparing the sequencing data of the wzd gene with the reference sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
[0005] Secondly, this application provides a method for identifying serotypes 15B and 15C of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: acquiring NGS sequencing data of Streptococcus pneumoniae in the biological sample; and identifying serotypes 15B and 15C of Streptococcus pneumoniae using Serocall software according to the following steps: extracting sequencing data of the wciZ gene from the NGS sequencing data; and comparing the sequencing data of the wciZ gene with the reference sequence of SEQ ID NO: 4.
[0006] Thirdly, this application provides a method for identifying serotypes 09A and 09V of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: acquiring NGS sequencing data of Streptococcus pneumoniae in the biological sample; and identifying serotypes 09A and 09V of Streptococcus pneumoniae using Serocall software according to the following steps: extracting sequencing data of the wcjE gene from the NGS sequencing data; and comparing the sequencing data of the wcjE gene with the reference sequence of SEQ ID NO: 5.
[0007] Fourthly, this application provides a method for identifying the serotype of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: (a) obtaining Sanger sequencing data of capsular polysaccharide-specific PCR products of Streptococcus pneumoniae in the biological sample, and comparing the sequencing data with one or more reference sequences obtained from the NCBI database; (b) obtaining NGS sequencing data of biological samples for which no typing result was obtained after the comparison in step (a), and using PneumoCaT software to determine whether the biological sample contains a single serotype of Streptococcus pneumoniae; (c) obtaining NGS sequencing data of biological samples that were determined to contain multiple serotypes of Streptococcus pneumoniae in step (b), and performing typing using SeroCall software; (d) obtaining NGS sequencing data of biological samples for which no typing result was obtained in step (c), and performing typing according to the methods described in the first, second and / or third aspects.
[0008] Fifthly, this application provides a computer-readable medium storing a plurality of instructions, which, when executed, cause a computer system to perform the method described in any one of the first to fourth aspects.
[0009] Sixthly, this application provides a computer product that includes the computer-readable medium described in the fifth aspect. Attached Figure Description
[0010] Figure 1 The PCR amplification results of capsular polysaccharides from 24 BEI Resources standard strains are shown, all of which amplified specific bands. Figures 1-24 represent the PCR amplification results of capsular polysaccharides from different genotypes of BEI Resources standard strains.
[0011] Figure 2 The PCR results of 8 positive and 2 negative strains of the national reference material are shown. Among them, 1-8 are positive samples of Streptococcus pneumoniae, 9 is the control sterile saline sample, 10 is group A streptococcus, and 11 is streptococcus (group C). Only Streptococcus pneumoniae amplified specific bands, while non-streptococcus pneumoniae and saline did not amplify specific bands.
[0012] Figure 3A(including serial numbers 1 to 18) and Figure 3B (Including serial numbers 19 to 31) show the NGS typing results of BEI Resources standard strains, indicating that the typing method of this application is effective for... Figure 3A and Figure 3B The BEI Resources samples of 24 Streptococcus pneumoniae strains shown were correctly typed, whether as single strains or mixed strains.
[0013] Figure 4 An exemplary operation flow of the method provided in this application is shown. Detailed Implementation
[0014] The conventional methods for identifying the serotype of Streptococcus pneumoniae are the capsular swelling method, multiplex PCR, and NGS.
[0015] The capsule swelling method is the gold standard, but it has the following disadvantages: 1) It is cumbersome to operate, requiring manual preparation of antiserum-bacterial mixtures, which takes 2-4 hours per sample; 2) It is serum-dependent, relying on high-quality antiserum (covering 90+ serotypes), and antibodies for some rare serotypes are difficult to obtain; 3) It has a certain degree of subjectivity, and the interpretation of results depends on experience, and false negatives are prone to occur in low-concentration samples; 4) It cannot distinguish subtypes, such as the cross-reactivity between 19A and 19F, which requires additional verification, and the resolution is limited.
[0016] Multiplex PCR is fast, efficient, and inexpensive, but it has limitations such as being unable to detect rare or newly emerging serotypes; primer crossover for some serotypes; and the risk of false negatives, such as capsule gene mutations that may cause primer failure.
[0017] NGS (Neuro-Gastroscopy) offers high resolution, distinguishing all known serotypes through CPS gene cluster and whole-sequence analysis; it can also detect recombinant or newly emerging variants; and it has automation potential, with bioinformatics workflows supporting batch analysis. Currently, the PneumoCaT software in NGS can isolate single isolates (pure strains), but it still cannot identify certain serotypes (e.g., serotypes 20A and 20B are both classified as serotype 20), and it cannot accurately identify and quantify serotypes in mixed samples; if multiple serotypes are found in a sample, it is reported as "mixed," without specifying the specific serotype, failing to meet the requirements for mixed sample detection. SeroCall software can be used for whole-genome sequencing for Streptococcus pneumoniae identification and relative abundance quantification, and can detect mixed serotypes, but it still cannot identify specific serotypes, such as being unable to distinguish between serotypes 15B and 15C, and misreading 09V as 09A.
[0018] PneumoCaT is a comprehensive serotype prediction tool for Streptococcus pneumoniae. It can accurately identify serotypes based on genome assembly or raw sequencing data by in-depth analysis of capsular polysaccharide loci. It can be obtained from https: / / github.com / ukhsa-collaboration / PneumoCaT.
[0019] SeroCall is a high-throughput serotyping tool designed specifically for large-scale sequencing data. It can accurately resolve multiple serotypes and their relative abundance in mixed bacterial strain samples and can be obtained from https: / / github.com / knightjimr / serocall.
[0020] Currently, pneumococcal typing largely relies on traditional multiplex nested PCR. While this method has low reagent costs, it suffers from low throughput, complexity, and time consumption. A single reaction can only detect a limited number of known serotypes, the operation is cumbersome, and manual labor is heavy. Furthermore, it cannot type mixed strains. The NGS method, while more expensive in terms of reagents and sequencing costs, offers high throughput. A single run can simultaneously obtain the gene sequences of all pneumococci in a sample, not limited to a preset serotype, enabling rapid and batch sample analysis.
[0021] Therefore, it is urgent to build upon previous research based on specific circumstances. [1,2,3] To establish and improve a new serotyping method for Streptococcus pneumoniae.
[0022] This application provides a novel serological typing method for Streptococcus pneumoniae to achieve effective identification of Streptococcus pneumoniae (independent strains or mixed strains).
[0023] In a first aspect, this application provides a method for identifying serotypes 20A and 20B of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: acquiring NGS sequencing data of Streptococcus pneumoniae in the biological sample; and identifying serotypes 20A and 20B of Streptococcus pneumoniae according to the following steps using PneumoCaT and Serocall software: extracting sequencing data of the whaF gene from the NGS sequencing data and comparing the sequencing data of the whaF gene with the reference sequence of SEQ ID NO: 1; and extracting sequencing data of the wzd gene from the NGS sequencing data and comparing the sequencing data of the wzd gene with the reference sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
[0024] In some embodiments of the first aspect, the method includes extracting sequencing data of the whaF gene from the NGS sequencing data, the sequencing data of the whaF gene including sequencing data at position 872-873 bp of the whaF gene, and then comparing the sequencing data with a reference sequence of SEQ ID NO: 1 to determine whether base A is present at position 872-873 bp of the whaF gene of Streptococcus pneumoniae.
[0025] In some embodiments of the first aspect, the method includes extracting sequencing data of the wzd gene from the NGS sequencing data, the sequencing data of the wzd gene including sequencing data at the 367 bp position of the wzd gene, and then comparing the sequencing data with a reference sequence of SEQ ID NO: 2 or SEQ ID NO: 3 to determine whether the wzd gene of the Streptococcus pneumoniae has a base G or T at the 367 bp position.
[0026] The difference between serotypes 20A and 20B lies in the whaF and wzd genes. First, in the whaF gene, 20A has an "A" insertion at position 872-873 bp, causing a reading frame shift and rendering the gene a pseudogene; while 20B has no insertion at this position, remaining a normally functioning gene. Second, in the wzd gene, at position 367, 20A has a G base, while 20B has a T base. Based on this, this application provides exemplary reference sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 for these two genes and determines the genotyping rules, thereby successfully genotyping 20A and 20B.
[0027] Secondly, this application provides a method for identifying serotypes 15B and 15C of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: acquiring NGS sequencing data of Streptococcus pneumoniae in the biological sample; and identifying serotypes 15B and 15C of Streptococcus pneumoniae using Serocall software according to the following steps: extracting sequencing data of the wciZ gene from the NGS sequencing data; and comparing the sequencing data of the wciZ gene with the reference sequence of SEQ ID NO: 4.
[0028] In some embodiments of the second aspect, the method includes extracting sequencing data of the wciZ gene from the NGS sequencing data, the sequencing data of the wciZ gene including sequencing data at position 412-417 bp of the wciZ gene; and comparing the sequencing data with the reference sequence of SEQ ID NO: 4 to determine whether there is a deletion / inactivation mutation in the wciZ gene of Streptococcus pneumoniae at position 412-417 bp.
[0029] The main difference between serotypes 15B and 15C lies in the wciZ gene, specifically at the 412-417 bp position. For serotype 15B, wciZ is a functional gene, intact at the 412-417 bp position; while for serotype 15C, wciZ is a pseudogene, meaning there is a deletion / inactivation mutation at the 412-417 bp position, preventing the protein from performing its normal function.
[0030] Thirdly, this application provides a method for identifying serotypes 09A and 09V of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: acquiring NGS sequencing data of Streptococcus pneumoniae in the biological sample; and identifying serotypes 09A and 09V of Streptococcus pneumoniae using Serocall software according to the following steps: extracting sequencing data of the wcjE gene from the NGS sequencing data; and comparing the sequencing data of the wcjE gene with the reference sequence of SEQ ID NO: 5.
[0031] In some embodiments of the third aspect, the method includes extracting sequencing data of the wcjE gene from the NGS sequencing data, the sequencing data of the wcjE gene including sequencing data at the 722 bp position of the wcjE gene; and comparing the sequencing data with a reference sequence of SEQ ID NO: 4 to determine whether the wcjE gene of the Streptococcus pneumoniae contains the base G at the 722 bp position.
[0032] In some scenarios where Serocall software identifies hybrid strains, 09V is misread as 09A. The main difference between 09A and 09V lies in the wcjE gene. 09A has a deletion at the 722 bp position of the wcjE gene, while 09V does not have this deletion, and the base at that position is G.
[0033] Fourthly, this application provides a method for identifying the serotype of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: (a) obtaining Sanger sequencing data of capsular polysaccharide-specific PCR products of Streptococcus pneumoniae in the biological sample and comparing the sequencing data with one or more reference sequences obtained from the NCBI database; (b) obtaining NGS sequencing data of biological samples for which no typing result was obtained after the comparison in step (a) and using PneumoCaT software to determine whether the biological sample contains a single serotype of Streptococcus pneumoniae; (c) obtaining NGS sequencing data of biological samples that were determined to contain multiple serotypes of Streptococcus pneumoniae in step (b) and performing typing using SeroCall software; and (d) obtaining NGS sequencing data of biological samples for which no typing result was obtained in step (c) and performing typing according to the methods described in the first, second and / or third aspects.
[0034] Exemplary NCBI database reference sequences include, but are not limited to:
[0035] The URL for the NCBI database is: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.
[0036] A step-by-step detection strategy is adopted. For example, in the initial stage, a lower-cost first-generation sequencing technology (Sanger sequencing) is used to perform preliminary analysis of capsular polysaccharide-specific PCR products. If a clear genotyping result cannot be obtained at this stage, a higher-cost NGS technology is then initiated, thereby achieving effective cost control. Alternatively, NGS technology can be run directly.
[0037] Implementations of the methods provided in this application can be implemented by one or more computer systems or apparatuses programmed with computer-executable instructions, which may be contained in a non-transitory computer-readable medium. When executed by a processor, the processor executes the instructions to cause the computer system or apparatus to perform one or more functions of the embodiments of this application. The programming techniques, computer languages, apparatuses, and computer-readable media required to achieve this are known in the art.
[0038] Computer-readable media are data storage devices that can store data and are read by computer systems. Examples of computer-readable media include read-only memory, random access memory, CD-ROM, magnetic tape, optical data storage devices, etc. Computer-readable media can be geographically localized or distributed across networked computer systems, allowing computer-readable code to be stored and executed in a distributed manner.
[0039] Computer implementations of the methods described in this application typically involve interfaces, memory, and processors. The types and arrangements of the interfaces, memory, and processors can vary depending on the implementation.
[0040] The interface may include a software interface that communicates with an end user's computing device via the Internet. The interface may also include a physical electronic device configured to receive requests or queries from a device that transmits digital and / or analog information.
[0041] Depending on the specific implementation, any suitable processor type can be used, including, for example, a microprocessor, a programmable logic controller, or a field-programmable logic array. Furthermore, any conventional computer architecture (including, for example, memory, mass storage devices, a processor (CPU), a graphics processing unit (GPU), read-only memory (ROM), and random access memory (RAM) typically connected to the system bus of a data processing device) can be used in computer implementations of the methods of this application.
[0042] The memory can be implemented as ROM, RAM, or a combination thereof, or simply as a general-purpose memory unit. Software modules for executing the methods of this application can be stored in memory and then retrieved and processed by a processor to implement specific tasks or functions. Similarly, one or more steps of the methods of this application can be encoded as program components, stored as executable instructions in memory, and then retrieved and processed by a processor. User input devices, such as keyboards, mice, or other clicking devices, can be connected to a PCI (Peripheral Component Interconnect) bus. If needed, the software can provide an environment representing programs, files, options, etc., through icons, menus, and dialog boxes displayed graphically on a computer monitor screen.
[0043] The computer implementation of the method of this application can be adapted to any type of end-user computing device, including computing devices that communicate via a network connection. The computing device can display graphical interface elements for performing various functions of the method of this application. For example, the computing device can be a server, desktop computer, portable computer, notebook computer, tablet computer, personal digital assistant (PDA), PDA phone, or smartphone, etc. The computing device can be implemented using any suitable combination of hardware and / or software configured for wired and / or wireless communication. For example, in cases where remote control of the system is required, communication can be made via a network.
[0044] If a network connection is required, the method of this application can be adapted to any type of network. The network can be a single network or a combination of multiple networks. For example, the network may include the Internet and / or one or more intranets, terrestrial networks, wireless networks, and / or other suitable types of communication networks. In another example, the network may include a wireless telecommunications network (e.g., a cellular telephone network) suitable for communicating with other communication networks (e.g., the Internet). For example, the network may include a computer network utilizing the TCP / IP protocol (including TCP / IP-based protocols such as HTTP, HTTPS, or FTP).
[0045] Fifthly, this application provides a computer-readable medium storing a plurality of instructions, which, when executed, cause a computer system to perform the method described in any one of the first to fourth aspects.
[0046] Sixthly, this application provides a computer product that includes the computer-readable medium described in the fifth aspect.
[0047] Example
[0048] The following embodiments are intended to illustrate the methods of this application and are not intended to limit the scope of protection of this application.
[0049] Example 1. Serotyping of Streptococcus pneumoniae
[0050] 1. Sample DNA extraction
[0051] Use the bacterial genomic DNA extraction kit (DP302, Tiangen Biotech; other equivalent Gram-positive bacterial nucleic acid extraction kits can also be used) and follow the instructions in the table below to extract bacterial genomic DNA from nasopharyngeal swab samples.
[0052]
[0053] 2. Capsular polysaccharide PCR amplification
[0054] 3. Database construction
[0055] After bacterial genome extraction, transposon-linked beads (TNB) were mixed with the DNA and incubated to achieve DNA fragmentation. Adapters were then added to the ends of the fragmented DNA. PCR amplification was performed using N5 (N5XX) and N7 (N7XX) sequences to obtain a library for high-throughput sequencing. The key reagent used in this study was the TruePrep Flexible DNA Library Prep Kit for Illumina (TD504, Vazyme). The library construction was performed according to the following procedure, following the manufacturer's instructions.
[0056] 3.1. DNA Fragmentation
[0057] 3.2. PCR enrichment
[0058] 3.3. Amplified product length sorting, sequencing, and genotyping
[0059] Please refer to the table below for the specific procedures.
[0060]
[0061] The following section will further explain the fractal strategy used in this method: Serotyping strategies for serotypes 20A and 20B A. Update the database The reference genome sequences of 20A and 20B were added to the PneumoCaT and Serocall software databases, respectively. The GenBank number for the 20A sequence is JQ653094, and the GenBank number for the 20B sequence is JQ653093.
[0062] B. Develop classification standards
[0063] The difference between serotypes 20A and 20B lies in the whaF and wzd genes. First, in the whaF gene, 20A has an "A" insertion at position (872, 873), causing a reading frame shift and rendering the gene a pseudogene; while 20B has no insertion at this position and remains a normally functioning gene. Second, in the wzd gene, at position 367, 20A has a G base, while 20B has a T base.
[0064] Accordingly, this application provides exemplary reference sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 for these two genes.
[0065] C. Judgment Criteria
[0066] Based on the above criteria, if the whaF gene is in a pseudogene state and the base at position 367 is G, it is classified as 20A; if the whaF gene is in a normal functional gene state and the base at position 367 is T, it is classified as 20B.
[0067] Serotyping strategies for serotypes 15B and 15C
[0068] The main difference between 15B and 15C lies in the wciZ gene, specifically at the 412-417 bp position. For serotype 15B, wciZ is a functional gene, intact at the 412-417 bp position; while for serotype 15C, wciZ is a pseudogene, meaning it contains a deletion / inactivation mutation at this position, preventing the protein from performing its normal function. Therefore, for samples with a Serocall typing of 15B / 15C, the following fine typing can be performed: A. Target serotype specificity analysis For serogroups containing 15B and 15C, the raw sequencing data were re-aligned to the serogroup-specific reference sequence (SEQ ID NO: 4) using Bowtie2 software. Base distribution files (mpileup format) were generated for each site, and low-quality sites were filtered out: sites with a match of more than 75% to the reference sequence and an insertion mutation rate of less than 25% were retained.
[0069] B. wciZ gene mutation detection
[0070] B1. Gene Coverage Analysis
[0071] All site information of the wciZ gene region was extracted, and gene coverage (coverage sites / total gene length), average sequencing depth, depth distribution range and average base quality value were calculated. The coverage threshold was set at 70%, and genes below this threshold were considered to be possibly missing.
[0072] B2. Examine for inactivation mutations
[0073] Determining the functional status of the wciZ gene using open reading frame (ORF) analysis: First, reading frame correction is performed to ensure translation accuracy. The start position of the gene-covered region is detected to determine if it is a codon boundary (a multiple of 3). If it is not a codon boundary, the reading frame is adjusted by removing 1-2 start bases. Second, a consensus sequence of the gene is constructed, translated into a protein sequence, and the longest open reading frame (ORF) is compared with the known functional ORF of the gene reference sequence. The comparison result serves as the criterion for determining the gene's functional status.
[0074] B3. Judgment Criteria
[0075] Condition 1: The length of the ORF is within ±15 amino acids of the length of the functional ORF, and it completely contains the functional ORF; Condition 2: If the translated protein sequence contains ≤1 stop codon, the constructed consistent sequence is considered to have ≥90% similarity to the reference sequence. 15B criterion: One of the above two conditions must be met; 15C criterion: Not satisfying any one of the above conditions; Mixed infection determination: When the deletion ratio at sites 412-417 is between 20% and 80%, it is determined to be a mixed infection of 15B and 15C.
[0076] Uncertainty determination: When the coverage of the wciZ gene is <70% or the depth of key sites is insufficient, the report is uncertain and interpreted as 15B / 15C.
[0077] Genotyping strategies for serotypes 09A and 09V
[0078] In certain scenarios where Serocall software identifies hybrid strains, 09V is misread as 09A. The main difference between 09A and 09V lies in the wcjE gene. 09A has a deletion at the 722 bp position of the wcjE gene, while 09V does not have this deletion, and the base at that position is G. Therefore, for samples classified as 09A or 09V by Serocall, the following fine-grained genotyping can be performed: A. Target serotype specificity analysis For serogroups containing 09A and 09V, the raw sequencing data were re-aligned to the serogroup-specific reference sequence (SEQ ID NO: 5) using Bowtie2 software. Base distribution files (mpileup format) were generated for each site, and low-quality sites were filtered out: sites with a match of more than 75% to the reference sequence and an insertion mutation rate of less than 25% were retained.
[0079] B. wcjE gene mutation detection
[0080] B1. Gene Coverage Analysis
[0081] All site information of the wcjE gene region was extracted, and gene coverage (coverage sites / total gene length), average sequencing depth, depth distribution range, and average base quality value were calculated. The coverage threshold was set at 70%, and genes below this threshold were considered to be possibly missing.
[0082] B2. Inspect specific frameshift mutations
[0083] The wcjE gene sequence without deletion at position 722 was used as a reference sequence. The aforementioned sequencing data was compared with the reference sequence, and the reads aligned to that position were summarized and statistically analyzed. The statistical results were used as the criteria for determining the gene's functional status.
[0084] B3. Judgment Criteria
[0085] The wcjE gene has a high-quality G base match at position 722, which indicates it is a functional gene and is identified as 09V. The wcjE gene at position 722 is a high-frequency deletion (>75%), marked as a pseudogene, and identified as 09A; The wcjE gene at position 722 is a mid-frequency deletion (20%-75%), marked as a mixed state, and identified as 09A / 09V.
[0086] 3.4. Process Summary
[0087] Methodological Description: Capsular polysaccharide (CPS) is a major virulence factor of Streptococcus pneumoniae and is used to classify more than 90 different serotypes of Streptococcus pneumoniae. First, CPS-specific primers were used to amplify the metagenomic DNA of human nasopharyngeal swabs. When specific bands appeared in the PCR products, first-generation Sanger sequencing was performed to determine the pneumonia serotype. If no bands appeared or Sanger sequencing failed, WGS sequencing was performed. Pure strain typing was first performed using the PneumoCaT workflow; if unsuccessful, the SeroCall workflow was used for mixed strain typing. For 20A / B, 15B / C, and 09A / 09V, the final typing results were obtained through comprehensive analysis using updated databases or the self-built typing module described above.
[0088] Sequence Description
[0089] References
[0090] 1. Nagaraj G, Ganaie F, Govindan V, Ravikumar KL. Development ofPCRSeqTyping-a novel molecular assay for typing of Streptococcus pneumoniae.Pneumonia (Nathan). 2017 May 25;9:8. doi: 10.1186 / s41479-017-0032-3. PMID:28702310; PMCID: PMC5471960
[0091] 2. Kapatai G, Sheppard CL, Al-Shahib A, Litt DJ, Underwood AP,Harrison TG, Fry NK. 2016. Whole genome sequencing of Streptococcuspneumoniae: development, evaluation and verification of targets for serogroupand serotype prediction using an automated pipeline. PeerJ 4:e2477, 1-35
[0092] 3.Knight J, Dunne E, Mulholland E, Saha S, Satzke C, Tothpal A,Weinberger D. Determining the Serotype Composition of Mixed Samples ofPneumococcus using Whole Genome Sequencing. Microbial Genomics 2021;7:000494,1-10。
Claims
1. A computer-implemented method for identifying serotypes 20A and 20B of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: Receive NGS sequencing data of Streptococcus pneumoniae in the biological sample; as well as Run the PneumoCaT and Serocall software to identify Streptococcus pneumoniae serotypes 20A and 20B according to the following steps: Sequencing data of the whaF gene was extracted from the NGS sequencing data, and the sequencing data of the whaF gene was compared with the reference sequence of SEQ ID NO: 1; as well as Sequencing data of the wzd gene was extracted from the NGS sequencing data, and the sequencing data of the wzd gene was compared with the reference sequence of SEQ ID NO: 2 or SEQ ID NO:
3.
2. The method according to claim 1, comprising: Sequencing data of the whaF gene was extracted from the NGS sequencing data. This whaF gene sequencing data includes the sequencing data at position 872-873 bp of the whaF gene. The sequencing data was then compared with the reference sequence of SEQ ID NO: 1 to determine whether base A is present at position 872-873 bp of the *Streptococcus pneumoniae* whaF gene. Sequencing data of the wzd gene was extracted from the NGS sequencing data. The sequencing data of the wzd gene included the sequencing data at the 367 bp position of the wzd gene. Then, the sequencing data was compared with the reference sequence of SEQ ID NO: 2 or SEQ ID NO: 3 to determine whether the wzd gene of the Streptococcus pneumoniae contained the base G or T at the 367 bp position.
3. A computer-implemented method for identifying the serotype of Streptococcus pneumoniae in a biological sample containing Streptococcus pneumoniae, comprising: (a) Receive Sanger sequencing data of capsular polysaccharide-specific PCR products of Streptococcus pneumoniae in the biological sample and one or more reference sequences in the NCBI database, and compare the sequencing data with the one or more reference sequences; (b) Receive NGS sequencing data of biological samples that did not obtain typing results after alignment in step (a), and run PneumoCaT software to determine whether the biological sample contains a single serotype of Streptococcus pneumoniae. (c) For NGS sequencing data of biological samples identified in step (b) as containing multiple serotypes of Streptococcus pneumoniae, run SeroCall software for typing; (d) For NGS sequencing data of biological samples for which no typing results were obtained in step (c), perform typing according to the method of claim 1 or 2.
4. The method according to any one of claims 1-3, wherein The biological samples are bacterial powder, bacterial cultures, swab samples, sputum samples, urine samples, blood samples, or pleural effusion samples; and / or The biological samples were derived from individuals suffering from pneumococcal-related diseases, selected from meningitis, sepsis, pneumonia, sinusitis, and otitis media.
5. A computer-readable medium storing a plurality of instructions, which, when executed, cause a computer system to perform the method of any one of claims 1-4.
6. A computer product comprising the computer-readable medium of claim 5.