WO2023077488A1 - Combinaison de marqueurs mnp de streptococcus pneumonia, combinaison de paires d'amorces, kit et utilisations associées - Google Patents

Combinaison de marqueurs mnp de streptococcus pneumonia, combinaison de paires d'amorces, kit et utilisations associées Download PDF

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WO2023077488A1
WO2023077488A1 PCT/CN2021/129166 CN2021129166W WO2023077488A1 WO 2023077488 A1 WO2023077488 A1 WO 2023077488A1 CN 2021129166 W CN2021129166 W CN 2021129166W WO 2023077488 A1 WO2023077488 A1 WO 2023077488A1
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streptococcus pneumoniae
mnp
combination
marker
markers
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Chinese (zh)
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高利芬
肖华峰
周俊飞
李论
方治伟
彭海
陈利红
李甜甜
万人静
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江汉大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/14—Streptococcus; Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the embodiment of the present invention relates to the field of biotechnology, in particular to a combination of MNP markers of Streptococcus pneumoniae, a combination of primer pairs, a kit and applications thereof.
  • Streptococcus pneumoniae (Streptococcus pneumonia) is a human pathogenic Gram-positive bacterium that mainly causes lobar pneumonia in humans and is one of the important pathogenic microorganisms of infectious pneumonia; it can also invade other parts of the body and cause secondary Pleurisy, otitis media, mastoiditis, endocarditis and suppurative meningitis. It can be spread by means of droplets, and is more common in winter and early spring, often in parallel with respiratory virus infection. According to epidemiological studies, China is one of the countries with the highest number of deaths of children under 5 years old due to diseases related to Streptococcus pneumoniae infection. Therefore, rapid and accurate diagnosis of Streptococcus pneumoniae infectious diseases is of great clinical significance.
  • Streptococcus pneumoniae is also a commonly used model pathogenic microorganism for laboratory research.
  • As a group creature in the interaction with the host and the environment, the individuals in the group will mutate.
  • this imperceptible variation will cause different laboratories or different periods of the same laboratory with the same named strains to be actually different, resulting in non-reproducible and non-comparable experimental results.
  • the heterogeneity of human Hella cell laboratories has led to a large number of incomparable experimental results and data waste. Therefore, the development of rapid and accurate detection and analysis methods for Streptococcus pneumoniae is of great significance for the clinical diagnosis, disease control monitoring and scientific research of Streptococcus pneumoniae.
  • the classic culture method is the gold standard for detecting Streptococcus pneumoniae, but it is time-consuming and complicated to operate, which is far from meeting the needs of large samples and rapid detection.
  • molecular detection technology has developed rapidly. Fluorescent multiplex PCR technology is one of the most commonly used molecular methods for detecting microorganisms, and it is also one of the detection technologies commonly used in the current national and industry standards for microbial detection.
  • the number of targets detected by it is limited, and mutations cannot be detected, and the detection of a few markers is prone to detection failure.
  • Metagenome sequencing technology is another technology for detecting Streptococcus pneumoniae, but metagenomic sequencing often includes a large amount of host sequencing data, which brings a lot of data waste and background noise, especially when detecting low-abundance bacteria samples. Ultra-deep sequencing, if you want to detect mutations and distinguish variants, will lead to ultra-high sequencing costs.
  • Super multiplex PCR amplification combined with high-throughput sequencing technology can target and enrich target microorganisms in samples with low microbial content, avoiding the large amount of data waste and background noise caused by whole-genome-dependent pathogenic bacteria isolation and culture steps and metagenomic sequencing , Deep sequencing can be achieved at low cost, and base variation can be detected through deep sequencing, and species can be distinguished to the most precise taxonomic level, which has the advantages of less sample requirement, high sensitivity, high accuracy and fine typing.
  • the molecular markers detected by existing targeted detection technologies mainly include SNP and SSR markers.
  • SSR markers are recognized as the most polymorphic markers, but their number is small in microorganisms; SNP markers are huge in number, densely distributed, and are dimorphic markers, and the polymorphism of a single SNP marker is not enough to capture potential alleles in microbial populations genetic diversity. Therefore, the development of novel molecular markers of highly polymorphic Streptococcus pneumoniae and its high-throughput, accurate and sensitive detection technology has become an urgent technical problem to be solved.
  • the invention provides MNP marker combination, primer composition, detection method and application of Streptococcus pneumoniae.
  • the MNP marker is a new type of molecular marker, which refers to a polymorphic marker caused by multiple nucleotide variations in an upper region of the genome.
  • MNP marker and its detection technology MNP marker method have application potential in the detection of Streptococcus pneumoniae, variation monitoring, fingerprint database construction and other aspects.
  • the development, screening and application of MNP markers have a good application basis in plants.
  • the present invention is the first in the field of Streptococcus pneumoniae, and there are no related literature reports.
  • the purpose of the present invention is to provide a Streptococcus pneumoniae MNP marker combination, primer pair combination, kit and application thereof, which can carry out qualitative identification and variation detection of Streptococcus pneumoniae, with multi-target, high-throughput, high sensitivity and precision The typing effect.
  • a MNP marker combination of Streptococcus pneumoniae refers to the MNP marker combination screened on the Streptococcus pneumoniae genome, which is different from other species and has multiple cores within the Streptococcus pneumoniae species.
  • the genome region of nucleotide polymorphism includes 15 markers of MNP-1 to MNP-15 on the reference nucleotide sequence of Streptococcus pneumoniae genome sequence AE007317.
  • the labeled nucleotide sequence of MNP-1 ⁇ MNP-15 is specifically shown as SEQ ID NO.1-SEQ ID NO.15, wherein ID NO.16-SEQ ID NO.30 is the upper primer ID NO .31-SEQ ID NO.45 is the lower primer.
  • Table 1 of the description further explains that the start and end positions of the MNP marker marked in Table 1 are determined based on the reference sequence AE007317.
  • the multiple PCR primer pair combination includes 15 pairs of primers, and the specific primer nucleotide sequence is as SEQ ID NO .16-shown in SEQ ID NO.45.
  • each MNP-labeled primer includes an upper primer and a lower primer, as shown in Table 1 of the specification.
  • a detection kit for detecting the MNP marker combination of Streptococcus pneumoniae includes the primer pair combination.
  • kit also includes a multiplex PCR master mix.
  • the application of the core MNP marker combination of Streptococcus pneumoniae or the primer pair combination or the detection kit in the qualitative detection of Streptococcus pneumoniae of non-diagnostic purpose is provided.
  • Application in the preparation of qualitative detection products for Streptococcus pneumoniae is provided.
  • the construction of the DNA fingerprint database of Streptococcus pneumoniae and the detection of genetic variation of the MNP marker combination of Streptococcus pneumoniae or the combination of multiple PCR primers or the detection kit are provided and typing applications.
  • the DNA of the Streptococcus pneumoniae strain to be tested is carried out by using the primer combination of the present invention for the first round of multiplex PCR amplification, and the number of cycles is not higher than 25; Add sample tags and next-generation sequencing adapters for the second round of PCR amplification; quantify the second-round amplification products after purification; when detecting multiple strains, perform high-throughput sequencing by mixing equal amounts of the second-round amplification products; sequencing The results are compared to the reference sequence of Streptococcus pneumoniae, and the genotype data of the tested strain on the 15 MNP markers are obtained.
  • the quality control scheme and the determination method use the DNA of Streptococcus pneumoniae with known copy number as the detection sample, evaluate the sensitivity, accuracy and specificity of the kit for detecting Streptococcus pneumoniae, and formulate the kit The quality control scheme and determination method for the detection of Streptococcus pneumoniae.
  • the DNA fingerprint database When used in the Streptococcus pneumoniae DNA fingerprint database, all the genotype data of Streptococcus pneumoniae obtained from the reference sequence and the genotype data of the MNP markers of the new type strains obtained by actual measurement are entered into the database file, that is, constitute DNA fingerprinting database of Streptococcus pneumoniae. Therefore, using the primer combination, the DNA fingerprint database can be continuously enriched.
  • the genotype of the strain to be tested in the MNP marker will be compared with the reference sequence library composed of the published reference sequence of Streptococcus pneumoniae and the constructed DNA fingerprint database. Yes, to identify whether the Streptococcus pneumoniae in the sample is an existing strain or a new variant.
  • the detection of genetic variation between strains includes using the MNP primer combination to amplify and sequence the genome DNA of the Streptococcus pneumoniae to be tested, and obtain the genotype data of the respective strains marked by the MNP. Through pairwise comparison, it is analyzed whether there are differences in the main genotypes of the strains to be tested on the MNP markers. If there is a difference, it means that there is variation in the strain to be tested.
  • single-plex PCR can also be used to amplify the 15 markers of the strain to be tested, and then perform Sanger sequencing on the amplified products.
  • test strain After obtaining the sequences, compare the genotypes in pairs. If there are markers with discordant genotypes, it indicates that there is variation among the tested strains. When detecting the genetic variation within the strain, it is judged by statistical model whether the MNP markers in the strain to be tested detect sub-genotypes other than the main genotype. If the test strain has a subgenotype in at least one MNP marker, it is determined that there is genetic variation within the test strain.
  • the present invention has the following advantages:
  • the invention provides a Streptococcus pneumoniae MNP marker combination, primer pair combination, kit and application thereof.
  • MNP markers Compared with traditional SSR markers and SNP markers, MNP markers have the following advantages: (1) Allelic types are abundant, and there are 2 n allele types on a single MNP marker, which is higher than traditional commonly used SSR and SNP markers, which can meet Detection of multi-allelic genotypes that exist widely in microorganisms; (2) strong species discrimination ability, only a small amount of MNP markers can realize species identification, reducing the detection error rate. MNP markers are mainly developed based on reference sequences.
  • the MNP marker combination is amplified by super multiplex PCR through the primer pair combination, and the amplified product is sequenced by the second-generation high-throughput sequencing technology, which has the following detection advantages: (1) Fine typing, using multiplex PCR to detect multiple Streptococcus pneumoniae-specific targets, using next-generation sequencing technology to detect multiple targets, and finely typing Streptococcus pneumoniae according to sequence characteristics, without parallel experiments.
  • the MNP marker combination, primer pair combination and kit of the present invention have the characteristics of multi-target, high throughput, high efficiency, high accuracy and high sensitivity for the identification of Streptococcus phaeresus, meeting the requirements for a large number of samples Streptococcus pneumoniae identification needs; to meet the needs of monitoring genetic variation between and within strains of Streptococcus pneumoniae; to meet the needs of building a standard and shareable DNA fingerprint database of Streptococcus pneumoniae; to meet the needs of precise typing of Streptococcus pneumoniae . Therefore, the 15 MNP marker combinations, primer pair combinations and kits provided by the present invention can provide technical support for scientific research and epidemic strain monitoring of Streptococcus pneumoniae.
  • Figure 1 is a schematic diagram of MNP marker polymorphism
  • Fig. 2 is the screening and primer design flowchart of Streptococcus pneumoniae MNP marker combination
  • Fig. 3 is the detection flowchart of MNP marker combination
  • Example 1 The screening of Streptococcus pneumoniae MNP marker combination and the design of multiple PCR amplification primers
  • the genome sequence information of representative strains of the microbial species to be tested can also be obtained through high-throughput sequencing, where high-throughput sequencing can be whole genome or simplified genome sequencing.
  • high-throughput sequencing can be whole genome or simplified genome sequencing.
  • the genome sequences of at least 10 genetically representative isolates are generally used as references.
  • the step S1 specifically includes:
  • step lengths can also be used when screening on the reference genome with a window of 100-300 bp.
  • the step size is 1 bp, which is conducive to comprehensive screening.
  • the multiple PCR amplification primers labeled with MNP are designed by primer design software.
  • the primer design follows that the primers do not interfere with each other. All primers can be combined into a primer pool for multiple PCR amplification, that is, all designed primers can be used in one amplification reaction. normal expansion.
  • the primers used to identify the MNP marker combination are shown in Table 1.
  • the method for detecting the MNP markers is to amplify all the MNP markers at one time through multiplex PCR, sequence the amplified products through second-generation high-throughput sequencing, analyze the sequencing data, and evaluate the primers according to the detected markers. Combination compatibility.
  • Example 2 MNP markers and kits for performance evaluation and threshold setting for the identification of Streptococcus pneumoniae
  • the Streptococcus pneumoniae DNA of Example 1 was added to human genomic DNA to prepare 1 copy/reaction, 10 copies/reaction and 100 copies/reaction of Streptococcus pneumoniae simulation samples, and an equal volume of sterile water was set as a blank Control, a total of 4 samples.
  • Four replicate libraries were constructed per day for each sample and tested continuously for 3 days, that is, 12 sets of sequencing data were obtained for each sample, as shown in Table 2.
  • the MNP markers and kits were evaluated against Streptococcus pneumoniae The reproducibility, accuracy, and sensitivity of identification, and the establishment of thresholds for contamination of the quality control system and detection of target pathogens.
  • the kit can stably detect more than 7 MNP markers in samples with 10 copies/reaction, and at most 2 MNP markers can be detected in a small number of samples with 0 copies/reaction.
  • the cartridge can clearly distinguish between 10 copies/reaction and 0 copies/reaction samples, with technical stability and detection sensitivity as low as 10 copies/reaction.
  • the sequencing results of 4 sets of sequencing data were obtained for 3 Streptococcus pneumoniae 100 copies/reaction templates, and the results were compared in pairs.
  • the results are shown in Table 3.
  • the number of MNP markers with differences in main genotypes is all 0;
  • the quality control scheme that the present invention adopts is specifically as follows:
  • the amount of sequencing data is greater than 4.5 million bases. The calculation is based on the fact that the number of MNP markers detected in each sample is 15, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 4.5 million bases, most samples can be guaranteed to cover each marker in one experiment. The number of sequencing fragments reaches 1000 times, ensuring accurate analysis of the base sequence of each MNP marker.
  • Blank control noise index P nc/Nc, wherein nc and Nc respectively represent the number of sequenced fragments and the total number of sequenced fragments of Streptococcus pneumoniae in the blank control.
  • the signal index of the test sample S nt/Nt, wherein nt and Nt respectively represent the number of sequenced fragments and the total number of sequenced fragments of Streptococcus pneumoniae in the test sample.
  • the present invention stipulates that when the signal-to-noise ratio is greater than 10 times, it can be determined that the pollution in the detection system is acceptable.
  • the average signal-to-noise ratio of 10 copies of samples and the blank control is 54.8, and the lowest value is 41.4.
  • the signal-to-noise ratio threshold of Streptococcus pneumoniae is 41, that is, when the signal-to-noise ratio of Streptococcus pneumoniae in the sample is greater than 41, and the marker detection rate is greater than or equal to 40%, It was determined that the nucleotides of Streptococcus pneumoniae were detected in the sample. Therefore, the kit provided by the present invention can sensitively detect 10 copies/reaction of Streptococcus pneumoniae.
  • the MNP marker combination, primer pair combination and kit of Streptococcus pneumoniae provided by the present invention can detect Streptococcus pneumoniae with high sensitivity, high accuracy and specificity.
  • the DNA of all strains or samples used to construct the Streptococcus pneumoniae DNA fingerprint database was extracted by conventional CTAB method and commercial kits, and the quality of DNA was detected by agarose gel and ultraviolet spectrophotometer. If the ratio of the absorbance value of the extracted DNA at 260nm to 230nm is greater than 2.0, the ratio of the absorbance value at 260nm to 280nm is between 1.6 and 1.8, the main band of DNA electrophoresis is obvious, and there is no obvious degradation and RNA residue, it means that the genomic DNA has reached Relevant quality requirements, follow-up experiments can be carried out.
  • the constructed MNP fingerprint database is based on the gene sequence of the detected strains, so it is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed, shared, and updateable at any time.
  • Embodiment 4 application in fine typing of Streptococcus pneumoniae
  • the primer combination and MNP marker combination detection method obtain the MNP fingerprint of each bacterial strain; compare the MNP fingerprint of each bacterial strain obtained with the published genomic sequence of Streptococcus pneumoniae and the constructed Streptococcus pneumoniae The MNP fingerprint database is compared; if the genotype is 100% identical to the existing strain, it is judged as a very similar strain of the existing strain, and if there is a main genotype difference in more than one MNP marker, it is judged as a new mutant strain.
  • the detection and typing of the 6 Streptococcus pneumoniae strains are shown in Table 5. There are differences between the 6 Streptococcus pneumoniae S-4 detected and the other 5 in the main genotype of 1 MNP marker, and they are judged as 2 strains .
  • the genotypes of the 5 strains with the same genotype were consistent with the NCTC7465 strain, and they might be the progeny strains of this strain.
  • the strains in S-4 and the reference sequence library were inconsistent in the main genotypes of the two MNP markers, and were determined to be new mutant strains. It can be seen that the resolution of Streptococcus pneumoniae in the method reaches the level of single base, and fine typing of Streptococcus pneumoniae in the sample can be realized.
  • Embodiment 5 detection of genetic variation between bacterial strains of Streptococcus pneumoniae
  • Streptococcus pneumoniae Detection of genetic variation in Streptococcus pneumoniae, including variation between and within strains. Since Streptococcus pneumoniae parasitizes the host, it detects the genetic variation of Streptococcus pneumoniae between hosts and within hosts. The variation between hosts is detected by comparing the main genotypes. By comparing the obtained fingerprints of Streptococcus pneumoniae in pairs, the main genotypes are identified based on the MNP marker method with 100% reproducibility and accuracy. Two strains exist A major genotype difference for one marker can then be detected.
  • the application of the kit to identify genetic variation among strains by detecting MNP markers can be used to ensure the genetic consistency of the same named strains in different laboratories, thereby ensuring the comparability of research results, which is important for Streptococcus pneumoniae. Scientific research is of great significance.
  • Streptococcus pneumoniae mutates in the host or within the population, and when the population is tested for molecular markers, it appears as an allelic type outside the main genotype of the marker.
  • mutant individuals have not yet accumulated, they only account for a very small part of the population, showing low-frequency allele types, which are difficult to detect with existing technologies.
  • Low-frequency allele types are often mixed with technical errors, making them difficult to distinguish with existing techniques.
  • the present invention detects highly polymorphic MNP markers.
  • the technical error rate of MNP markers is significantly lower than that of SNP markers, based on the fact that multiple errors are less likely to occur simultaneously than one error.
  • the invention uses a statistical model to distinguish real sub-allelic genotypes from wrong genotypes caused by technical errors. specifically:
  • the authenticity evaluation of the secondary allelic type in this embodiment is carried out as follows: first, the allelic type with strand preference (the ratio of the number of sequencing sequences covered on the DNA double strand) is excluded according to the following rules: the strand preference is greater than 10 times, or more than 5 times different from the strand preference of the main allele type.
  • the rationale for the statistical model is to assume that the candidate minor allele is the product of the major allele type due to PCR or sequencing errors. Under this assumption, the number of reads supported by the candidate minor allele type (c) conforms to a binomial distribution based on marker sequencing depth and amplicon sequencing error rate e(n), where n refers to the candidate minor allele The number of SNPs that differ between genotypes and major genotypes. If the number of supporting sequencing sequences of the candidate allele type exceeds the critical value, the assumption that the allele type is the main allele type due to PCR or sequencing errors will be overturned, and it will be determined as the real secondary allele type. When there were multiple candidate minor alleles, multiple corrections were performed on the P values of each candidate allele type, and candidate alleles with FDR ⁇ 0.5% were considered true minor allele types.
  • the number of bases that differ between the i-th minor allele type and the main genotype is ni
  • the number of sequenced sequences supported by it is ci
  • the error rate ei is the number of sequenced sequences ci of the minor allele type and the total sequenced sequence of the marker
  • the ratio of the number N, that is, ei ci/N;
  • the critical value of the number of sequencing sequences only when the number of sequencing sequences of the minor allele type exceeds the critical value, it is determined as the real minor allele type (Table 4). When there are multiple candidate minor alleles, the P value of each candidate allele type is multiple-corrected, and the candidate alleles with FDR ⁇ 0.5% are judged to be true minor allele types.
  • the DNA of the S-1 and S-4 strains with inter-strain variation in Table 5 was calculated according to the following 8 ratios: 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/ 100, 5/100, and 7/100 were mixed to prepare artificial heterozygous samples, and each sample was tested 3 times to obtain a total of 24 sequencing data.
  • the heterozygous genotype markers can be detected in 24 artificial heterozygous samples, indicating that the developed MNP marker detection of Streptococcus pneumoniae The applicability of the method in detecting genetic variation in strains.

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Abstract

La présente invention appartient au domaine technique de la biologie moléculaire et divulgue une combinaison de marqueurs MNP de Streptococcus pneumonia, une combinaison de paires d'amorces, un kit et des utilisations associées. La combinaison de marqueurs MNP comprend 15 marqueurs, et les séquences nucléotidiques spécifiques sont telles que représentées dans SEQ ID NO : 1 à SEQ ID NO : 15 ; et la combinaison de paires d'amorces comprend 15 paires d'amorces, et les séquences nucléotidiques spécifiques sont telles que représentées dans SEQ ID NO :16 à SEQ ID NO : 45. La combinaison de marqueurs MNP permet d'identifier spécifiquement le Streptococcus pneumonia et de distinguer finement les différentes souches ; les amorces n'interfèrent pas entre elles et la combinaison des techniques d'amplification multiplex et de séquençage permet de séquencer et d'analyser en une seule fois toutes les combinaisons de marqueurs de plusieurs échantillons.
PCT/CN2021/129166 2021-11-06 2021-11-06 Combinaison de marqueurs mnp de streptococcus pneumonia, combinaison de paires d'amorces, kit et utilisations associées WO2023077488A1 (fr)

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