CN114277164B - MNP marker combination, primer pair combination, test kit and application of Streptococcus pneumoniae - Google Patents

Abstract

The invention belongs to the technical field of molecular biology, and discloses a MNP (MNP) marker combination, a primer pair combination and a kit of streptococcus pneumoniae and application thereof, wherein the MNP marker combination comprises 15 markers, and a specific nucleotide sequence is shown as SEQ ID NO.1-SEQ ID NO. 15; the primer pair combination comprises 15 pairs of primers, and the specific primer nucleotide sequence is shown as SEQ ID NO.16-SEQ ID NO. 45. The MNP marker combination can specifically identify streptococcus pneumoniae and finely distinguish different strains; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that sequence analysis can be carried out on all the mark combinations of multiple samples at one time; the kit has the advantages of high flux, multiple targets, high sensitivity, high accuracy and culture-free detection, can be applied to detection of large-scale samples, and has important significance for scientific research and monitoring of streptococcus pneumoniae.

Description

A MNP labeling combination, primer pair combination, kit and application thereof for Streptococcus pneumoniae

Technical Field

The embodiments of the present invention relate to the field of biotechnology, and in particular to a MNP labeling combination, a primer pair combination, a kit and applications thereof for Streptococcus pneumoniae.

Background Art

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 purulent meningitis. It can be spread through droplets and is more common in winter and early spring, often in conjunction with respiratory viral infections. Therefore, rapid and accurate diagnosis of pneumococcal infectious diseases is of great clinical significance.

In addition, Streptococcus pneumoniae is also a common model pathogen for laboratory research. As a group organism, individuals in the group will mutate in the interaction with the host and the environment. For laboratory research, this subtle variation will cause the strains with the same name in different laboratories or in different periods of the same laboratory to be actually different, resulting in irreproducible and incomparable 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 Streptococcus pneumoniae detection and analysis methods 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, and is far from meeting the needs of large samples and rapid detection. In recent years, molecular detection technology has developed rapidly. Fluorescence multiplex PCR technology is one of the most commonly used molecular methods for detecting microorganisms, and it is also one of the commonly used detection technologies in the current national and industry standards for microbial detection. However, the number of targets it detects is limited, and it cannot detect mutations. The detection of a few markers is prone to detection failure. Metagenomic 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 testing samples of low-abundance bacteria, ultra-deep sequencing is required. If you want to detect mutations and distinguish variants, it will lead to extremely high sequencing costs.

Super-multiplex PCR amplification combined with high-throughput sequencing technology can enrich target microorganisms in samples with low microbial content, avoiding the large amount of data waste and background noise caused by the whole genome-dependent pathogen isolation and culture steps and metagenomic sequencing. Deep sequencing can be achieved at a low cost. By detecting base variations through deep sequencing, species can be distinguished to the most refined taxonomic level, with the advantages of small sample requirements, 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 and densely distributed, and are dimorphic markers. The polymorphism of a single SNP marker is not enough to capture the potential allele diversity in microbial populations. Therefore, the development of new molecular markers of highly polymorphic Streptococcus pneumoniae and their high-throughput, accurate and sensitive detection technology has become a technical problem that needs to be solved urgently.

The present invention provides a MNP marker combination, a primer combination, a detection method and an application of Streptococcus pneumoniae, wherein the MNP marker is a new type of molecular marker, which refers to a polymorphic marker caused by multiple nucleotide variations in a region of a genome.

In view of the above advantages and characteristics, MNP labeling and its detection technology MNP labeling method has application potential in the detection of Streptococcus pneumoniae, mutation monitoring, fingerprint database construction, etc. The development, screening and application of MNP labeling method have a good application basis in plants. The present invention is the first in the field of Streptococcus pneumoniae and has not been reported in relevant literature.

Summary of the invention

The purpose of the present invention is to provide a MNP marker combination, a primer pair combination, a kit and applications thereof for Streptococcus pneumoniae, which can be used for qualitative identification and variation detection of Streptococcus pneumoniae, and have the effects of multi-target, high throughput, high sensitivity and fine typing.

In order to achieve the above effects, the present invention adopts the following technical solutions:

In a first aspect of the present invention, a MNP marker combination for Streptococcus pneumoniae is provided, wherein the MNP marker combination refers to a genomic region screened on the Streptococcus pneumoniae genome that is distinguished from other species and has multiple nucleotide polymorphisms within the Streptococcus pneumoniae species, including 15 markers MNP-1 to MNP-15 on the Streptococcus pneumoniae genome sequence AE007317 as a reference nucleotide sequence.

In the above technical solution, the labeled nucleotide sequences of MNP-1 to MNP-15 are specifically shown as SEQ ID NO.1-SEQ ID NO.15, wherein ID NO.16-SEQ ID NO.30 are upper primers and ID NO.31-SEQ ID NO.45 are lower primers. Table 1 of the specification further illustrates that the start and end positions of the MNP labels marked in Table 1 are determined based on the reference sequence AE007317.

In the second aspect of the present invention, a multiplex PCR primer pair combination for detecting the MNP marker combination is provided, the multiplex PCR primer pair combination comprises 15 pairs of primers, and the specific primer nucleotide sequences are shown in SEQ ID NO.16-SEQ ID NO.45.

In the above technical solution, each MNP-labeled primer includes an upper primer and a lower primer, as shown in Table 1 of the specification.

In a third aspect of the present invention, a detection kit for detecting the MNP marker combination of Streptococcus pneumoniae is provided, wherein the kit comprises the primer pair combination.

Furthermore, the kit also includes a multiplex PCR premix.

In the fourth aspect of the present invention, the use 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 for non-diagnostic purposes and in the preparation of a qualitative detection product for Streptococcus pneumoniae is provided.

In the fifth aspect of the present invention, there is provided the use of the MNP marker combination of Streptococcus pneumoniae or the multiplex PCR primer pair combination or the detection kit in the construction of a DNA fingerprint database of Streptococcus pneumoniae, genetic variation detection and typing.

In the above application, the primer combination of the present invention is first used to perform a first round of multiplex PCR amplification on the DNA of the strain to be tested, and the number of cycles is not higher than 25; after the amplification products are purified, sample labels and second-generation sequencing adapters based on the second round of PCR amplification are added; the second round of amplification products are purified and quantified; when multiple strains are detected, the second round of amplification products are mixed in equal amounts and then subjected to high-throughput sequencing; the sequencing results are aligned to the reference sequence of the Streptococcus pneumoniae to obtain the genotype data of the strain to be tested on the 15 MNP markers.

When used for qualitative identification of Streptococcus pneumoniae, the number of sequencing sequences of Streptococcus pneumoniae detected in the sample to be tested and the blank control and the number of MNP markers detected are used to determine whether the sample to be tested contains Streptococcus pneumoniae nucleic acid after quality control. The quality control scheme and determination method are to use DNA of Streptococcus pneumoniae with a known copy number as the test sample, evaluate the sensitivity, accuracy and specificity of the kit for detecting Streptococcus pneumoniae, and formulate the quality control scheme and determination method for the kit for detecting Streptococcus pneumoniae.

When used for the DNA fingerprint database of Streptococcus pneumoniae, all the genotype data of Streptococcus pneumoniae obtained from the reference sequence and the genotype data of the MNP marker of the new type strain obtained by actual measurement are entered into the database file, that is, the DNA fingerprint database of Streptococcus pneumoniae is formed. Therefore, the DNA fingerprint database can be continuously enriched by using the primer combination.

When used for typing Streptococcus pneumoniae, the genotype of the strain to be tested obtained at the MNP marker is compared with a reference sequence library consisting of a public reference sequence of Streptococcus pneumoniae and a constructed DNA fingerprint database to identify whether the Streptococcus pneumoniae in the sample is an existing strain or a new variant.

When used for detecting genetic variation of Streptococcus pneumoniae, it includes genetic variation detection between strains and within strains. The genetic variation detection between strains includes using the MNP primer combination to amplify and sequence the genomic DNA of the Streptococcus pneumoniae to be tested, and obtain the genotype data of each strain on the MNP marker. By comparing two by two, analyze whether there is a difference in the main genotype of the strain to be tested on the MNP marker. If there is a difference, it means that the strain to be tested has variation. As an alternative, the 15 markers of the strain to be tested can also be amplified separately by single PCR, and then the amplified products are Sanger sequenced. After obtaining the sequence, the genotypes are compared two by two. If there are markers with inconsistent genotypes, it means that there is variation between the strains to be tested. When detecting genetic variation within the strain, a statistical model is used to determine whether the MNP marker of the strain to be tested detects a secondary genotype other than the main genotype. If the strain to be tested has a secondary genotype in at least one MNP marker, it is determined that there is genetic variation within the strain to be tested.

Compared with the prior art, the present invention has the following advantages:

The present invention provides a Streptococcus pneumoniae MNP labeling combination, a primer pair combination, a kit and applications thereof.

Compared with existing labeling and labeling detection technologies, the technical solution provided by the present invention has the following advantages:

Compared with traditional SSR markers and SNP markers, MNP markers have the following advantages: (1) Rich alleles, with ²ⁿ alleles on a single MNP marker, which is higher than the traditional commonly used SSR and SNP, and can meet the detection of multi-allelic genotypes widely present in microorganisms; (2) Strong species differentiation ability, only a small number of MNP markers are needed to achieve species identification, reducing the detection error rate. MNP markers are mainly developed based on reference sequences. According to the resequencing data of the reported representative strains of Streptococcus pneumoniae, a large number of MNP markers that are distinguished from other species, polymorphic within the species, and conservative on both sides can be mined; the conservative sequences on both sides of the MNP marker can be used to design MNP marker detection primers suitable for multiple PCR amplification; and according to the test results of the standard, a set of MNP markers with the largest polymorphism, the widest coverage, and the best compatible primer combination can be screened.

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 multiple PCR to detect multiple pneumococcal specific targets at one time, using the second-generation sequencing technology to detect multiple targets, fine typing of pneumococcal according to sequence characteristics, and no parallel experiments are required. (2) High efficiency, using sample DNA barcodes to break through the limitation of the number of sequencing samples, and tens of thousands of MNP markers of hundreds of samples can be detected at one time; (3) High sensitivity, using multiple PCR to detect multiple targets at one time, avoiding high false negatives and low sensitivity caused by failure of amplification of a single target; (4) High accuracy, using the second-generation high-throughput sequencer to sequence the amplified product hundreds of times to obtain accurate genotypes. (5) No culture, based on PCR technology to enrich the target, no need to isolate and culture the pathogen.

In view of the above advantages and characteristics, 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 pneumoniae, meeting the needs of identifying Streptococcus pneumoniae in a large number of samples; meeting the needs of monitoring genetic variation between and within strains of Streptococcus pneumoniae; meeting the needs of building a standard and shareable DNA fingerprint database of Streptococcus pneumoniae; and meeting the needs of accurate 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the MNP labeling polymorphism;

FIG2 is a flow chart of the screening and primer design of MNP marker combinations for Streptococcus pneumoniae;

FIG3 is a flow chart of the detection of MNP labeling combinations;

FIG. 4 is a graph showing the relationship between the copy number of Streptococcus pneumoniae in the sample to be tested and the number of valid sequencing sequences.

DETAILED DESCRIPTION

In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific embodiments. The accompanying drawings provide preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

It should be noted that, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be purchased from the market or prepared by existing methods.

Example 1 Screening of Streptococcus pneumoniae MNP marker combinations and design of multiplex PCR amplification primers

S1. Screening of MNP labeling combinations for Streptococcus pneumoniae

Based on the complete or partial genome sequences of 538 different isolates of Streptococcus pneumoniae published online, 15 MNP markers were obtained by sequence alignment. For species for which no genome data exists online, genome sequence information of representative strains of the microbial species to be detected can also be obtained by high-throughput sequencing, where high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure the polymorphism of the markers screened, the genome sequences of at least 10 genetically representative isolates are generally used as references.

The 15 MNP markers screened are shown in Table 1:

Table 1 The starting positions of the MNP markers and detection primers on the reference sequence

The step S1 specifically includes:

Selecting a genome sequence of a representative strain of the Streptococcus pneumoniae as a reference genome, performing sequence alignment between the genome sequence and the reference genome, and obtaining single nucleotide polymorphism markers of each strain of the Streptococcus pneumoniae;

On the reference genome, a window of 100-300 bp is used and a window shift is performed with a step length of 1 bp to screen and obtain multiple candidate MNP marker regions, wherein the candidate MNP marker region contains ≥2 of the single nucleotide variation markers, and the single nucleotide polymorphism markers do not exist in the sequences of 30 bp at both ends;

In the candidate polynucleotide polymorphism marker region, a region with a discrimination degree DP≥0.2 is screened as an MNP marker; wherein DP=d/t, t is the comparison logarithm when all species in the candidate polynucleotide polymorphism marker region are compared pairwise, and d is the sample logarithm of at least two single nucleotide polymorphism differences in the candidate polynucleotide polymorphism marker region.

As an optional implementation, when screening is performed on the reference genome with a window of 100-300bp, other step sizes may also be selected. This implementation adopts a step size of 1bp, which is conducive to comprehensive screening.

S2. Design of primers for multiplex PCR amplification

The MNP-labeled multiple PCR amplification primers are designed by primer design software. The primer design follows the principle that 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 amplified normally in one amplification reaction.

In this embodiment, the primers used to identify the MNP marker combination are shown in Table 1.

S3. Evaluation of detection efficiency of primer combinations

The method for detecting the MNP markers is to amplify all the MNP markers at once by multiplex PCR, sequence the amplified products by second-generation high-throughput sequencing, analyze the sequencing data, and evaluate the compatibility of the primer combination according to the detected markers.

Using the DNA of the Streptococcus pneumoniae ATCC6303 strain with a known copy number provided by the Hubei Provincial Center for Disease Control and Prevention, a simulated Streptococcus pneumoniae sample was prepared, that is, the Streptococcus pneumoniae with a known copy number was added to human genomic DNA (2ng/reaction) to prepare a template of 1000 copies/reaction, and the primer combination was used to detect by the MNP marker detection method, and 4 repeated sequencing libraries were constructed. According to the detection results in the 4 libraries, 15 MNP markers with high species specificity and high intraspecies discrimination provided by the present invention and their detection primer pair combinations with high amplification efficiency and good compatibility were finally screened out.

Example 2 Performance evaluation and threshold setting of the MNP marker and kit for identification of Streptococcus pneumoniae

The pneumococcal DNA of Example 1 was added to human genomic DNA to prepare pneumococcal simulation samples of 1 copy/reaction, 10 copies/reaction and 100 copies/reaction, and an equal volume of sterile water was set as a blank control, for a total of 4 samples. Four replicate libraries were constructed for each sample every day, and the test was continued for 3 consecutive days, that is, 12 sets of sequencing data were obtained for each sample, as shown in Table 2. According to the number of sequencing fragments and the number of MNP markers of pneumococcal MNP markers detected in the blank control and pneumococcal nucleotide standards in 12 repeated experiments, the reproducibility, accuracy and sensitivity of the MNP marker and the kit for pneumococcal identification were evaluated, and the thresholds for quality control system contamination and target pathogen detection were established.

The detection process of MNP labeling is shown in Figure 3.

1. Analysis of the sensitivity and stability of MNP labeling method for detecting Streptococcus pneumoniae

As shown in Table 2, the kit can stably detect more than 7 MNP markers in samples with 10 copies/reaction, and can detect at most 2 MNP markers in a few samples with 0 copies/reaction. The kit can clearly distinguish between samples with 10 copies/reaction and 0 copies/reaction, and has technical stability and a detection sensitivity as low as 10 copies/reaction.

Table 2 Primer compatibility and detection sensitivity analysis of MNP labeling method for Streptococcus pneumoniae

2. Evaluation of the reproducibility and accuracy of the MNP-labeled detection method for qualitative detection of Streptococcus pneumoniae

The sequencing results of 4 sets of sequencing data were obtained for 3 templates of 100 copies/reaction of Streptococcus pneumoniae. The results were compared pairwise, as shown in Table 3. The number of MNP markers with differences in the main genotype was 0. According to the principle that the reproducible genotype between two repeated experiments is considered accurate, the accuracy rate a = 1-(1-r)/2 = 0.5+0.5r, where r represents the reproducibility, that is, the ratio of the number of reproducible markers of the main genotype to the number of common markers. In the reproducibility test of this project, the difference logarithm of the main genotype of MNP markers between different libraries and different sequencing batches of each sample is 0, the reproducibility r = 100%, and the accuracy a = 100%.

Table 3 Analysis of the accuracy of MNP labeling method for Streptococcus pneumoniae

3. Threshold setting for detecting Streptococcus pneumoniae using MNP labeling detection method

Due to the extreme sensitivity of the MNP marker detection method, data contamination during the detection process can easily lead to false positives. Therefore, it is necessary to perform quality control on the generated sequencing data. After quality control, the number of sequencing fragments and the number of detected markers of the MNP marker and internal standard DNA marker of Streptococcus pneumoniae detected in each sample are analyzed.

The quality control scheme adopted by the present invention is as follows:

1) The amount of sequencing data is greater than 4.5 million bases. The calculation is based on the number of MNP markers detected for each sample is 15, and the length of a sequencing fragment is 300 bases. Therefore, when the amount of data is greater than 4.5 million bases, most samples can ensure that the number of sequencing fragments covering each marker reaches 1000 times in one experiment, ensuring accurate analysis of the base sequence of each MNP marker.

2) Determine whether the contamination is acceptable based on the signal index S of Streptococcus pneumoniae in the test sample and the noise index P of Streptococcus pneumoniae in the blank control, where:

The blank control noise index P = nc/Nc, where nc and Nc represent the number of sequencing fragments of Streptococcus pneumoniae and the total number of sequencing fragments in the blank control, respectively.

The signal index S of the test sample is nt/Nt, wherein nt and Nt represent the number of sequenced fragments of Streptococcus pneumoniae and the total number of sequenced fragments in the test sample, respectively.

3) Calculate the detection rate of MNP markers in the test sample, which refers to the ratio of the number of detected markers to the total number of designed markers.

As shown in Table 4, the average signal-to-noise ratio of Streptococcus pneumoniae in 1 copy sample and blank control is 4.3. Therefore, the present invention stipulates that when the signal-to-noise ratio is greater than 10 times, it can be determined that the contamination in the detection system is acceptable.

As shown in Table 4, the average value of the signal-to-noise ratio of the sample with 10 copies and the blank control is 54.8, and the minimum value is 41.4. In the 12 sets of data with 10 copies/reaction, at least 7 MNP markers can be stably detected, accounting for 46.7% of the total markers. Therefore, under the condition of ensuring accuracy, this standard stipulates that the signal-to-noise ratio judgment 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 is determined that the nucleotides of Streptococcus pneumoniae are detected in the sample. Therefore, the kit provided by the present invention can sensitively detect Streptococcus pneumoniae with 10 copies/reaction.

Table 4 Signal-to-noise ratio of Streptococcus pneumoniae in 12 tests for each of the 4 samples

4. Evaluation of the specificity of MNP-labeled detection method for detecting Streptococcus pneumoniae

The DNA of Streptococcus pneumoniae and Mycobacterium tuberculosis, Acinetobacter strains, Bordetella pertussis, Bordetella hominis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Epstein-Barr virus, Haemophilus influenzae, varicella zoster virus, cytomegalovirus, herpes simplex virus, human bocavirus, Klebsiella pneumoniae, Legionella, Moraxella catarrhalis, Pseudomonas aeruginosa, Rickettsia, Staphylococcus aureus, and Streptococcus pyogenes were artificially mixed in equimolar amounts to prepare a mixed template, and a blank template was used as a control. The Streptococcus pneumoniae in the mixed template was detected by the method provided by the present invention, and three repeated experiments were performed. As a result, the sequencing sequences obtained in the three repeated experiments could detect 15 MNP markers of Streptococcus pneumoniae. After analysis according to the quality control scheme and judgment threshold, the nucleic acid of Streptococcus pneumoniae was judged to be positive in all three repeated experiments, indicating that the MNP marker and the kit have high specificity in detecting Streptococcus pneumoniae in complex templates.

In summary, the MNP labeling combination, primer pair combination and reagent kit for Streptococcus pneumoniae provided by the present invention can detect Streptococcus pneumoniae with high sensitivity, high accuracy and specificity.

Example 3 Construction of Streptococcus pneumoniae DNA fingerprint database

The DNA of all strains or samples used to construct the DNA fingerprint database of Streptococcus pneumoniae was extracted using conventional CTAB method, commercial kits, etc., and the quality of DNA was tested by agarose gel and UV spectrophotometer. If the ratio of the absorbance value of the extracted DNA at 260nm and 230nm is greater than 2.0, the ratio of the absorbance value at 260nm and 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 meets the relevant quality requirements and subsequent experiments can be carried out.

The primer combination and MNP marker combination detection method were used to detect 6 progeny strains of a Streptococcus pneumoniae strain provided by the Hubei Provincial Center for Disease Control and Prevention. The samples were named S-1 to S-6, and the average sequencing coverage of each sample reached 2032 times. All 15 MNP markers can be detected for each strain (Table 5). The main genotype of each marker of each strain was obtained after sequence alignment of the sequencing data of the 6 strains, forming the MNP fingerprint map of each strain. The MNP fingerprint maps of strains with different main genotypes were entered into the database to construct the MNP fingerprint database of Streptococcus pneumoniae; the MNP fingerprint maps of Streptococcus pneumoniae detected in the new samples were compared with the constructed MNP fingerprint database, and the MNP fingerprint maps of samples with different main genotypes were entered into the constructed MNP fingerprint database. The constructed MNP fingerprint database is based on the gene sequence of the detected strain, so it is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared and updatable at any time.

Table 5 Detection and analysis of 6 strains of Streptococcus pneumoniae

Example 4: Application in fine typing of Streptococcus pneumoniae

The MNP fingerprint of each strain was obtained by using the primer combination and MNP marker combination detection method; the MNP fingerprint of each strain was compared with the published genome sequence of Streptococcus pneumoniae and the constructed MNP fingerprint database of Streptococcus pneumoniae; strains with 100% identical genotypes to the existing strains were determined to be very similar strains of the existing strains, and strains with major genotype differences in more than one MNP marker were determined to be new variants. The detection and typing of 6 Streptococcus pneumoniae strains are shown in Table 5. The 6 Streptococcus pneumoniae S-4 and the other 5 strains detected had differences in the major genotype of 1 MNP marker and were determined to be 2 strains. The 5 strains with the same genotype were consistent with the genotype of the NCTC7465 strain and may be the progeny of the strain. The strains in S-4 and the reference sequence library were inconsistent in the major genotypes of 2 MNP markers and were determined to be new variants. It can be seen that the resolution of the method described above for Streptococcus pneumoniae has reached the level of single bases, and the fine typing of Streptococcus pneumoniae in the sample can be achieved.

Example 5: Detection of genetic variation among strains of Streptococcus pneumoniae

Genetic variation detection of Streptococcus pneumoniae includes variation between strains and within strains. Since Streptococcus pneumoniae parasitizes in the host, the genetic variation of Streptococcus pneumoniae between hosts and within hosts is detected. Variation between hosts is detected by comparing the main genotype. The fingerprints of Streptococcus pneumoniae obtained are compared pairwise. Based on the 100% reproducibility and accuracy of the main genotype identification by the MNP labeling method, a difference in the main genotype of one marker between two strains can be detected.

As shown in Table 5, 5 of the 6 progeny strains had the same genotype and no genetic variation among the strains; however, there was genetic variation among the strains numbered S-4 and other strains.

It can be seen that the application of the kit for identifying genetic variation between 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 of great significance for scientific research on Streptococcus pneumoniae.

Example 6: Detection of genetic variation within strains of Streptococcus pneumoniae

As a group organism, Streptococcus pneumoniae mutates in the host or in the group. When the group is tested for molecular markers, it manifests as an allele type other than the main genotype of the marker. When the mutant individuals have not yet accumulated, they only account for a very small part of the group and manifest as a low-frequency allele type, which is difficult to detect with the existing technology. Low-frequency allele types are often mixed with technical errors, making it difficult to distinguish with the existing technology. The present invention detects highly polymorphic MNP markers. Based on the fact that the probability of multiple errors occurring simultaneously is lower than the probability of one error occurring, the technical error rate of MNP markers is significantly lower than that of SNP markers. The present invention distinguishes between true secondary allele types and erroneous genotypes caused by technical errors through statistical models. Specifically:

The authenticity evaluation of the secondary allele type in this embodiment is carried out as follows: first, the allele type with chain preference (the ratio of the number of sequencing sequences covered on the DNA double strands) is excluded according to the following rules: the chain preference is greater than 10 times, or the difference in chain preference with the major allele type is greater than 5 times.

Genotypes without strand bias are re-used to verify their authenticity using statistical models. The principle of the statistical model is to assume that the candidate minor allele is the product of the major allele due to PCR or sequencing errors. Under this assumption, the number of sequencing sequences (c) supported by the candidate minor allele conforms to the binomial distribution based on the marker sequencing depth and the amplicon sequencing error rate e(n), where n refers to the number of SNPs that differ between the candidate minor allele and the major genotype. If the number of sequencing sequences supporting the candidate allele exceeds the critical value, the assumption that the allele is the product of the major allele due to PCR or sequencing errors is overturned and it is determined to be the true minor allele. When there are multiple candidate minor alleles, the P value of each candidate allele is multi-corrected, and the candidate allele with FDR<0.5% is considered to be the true minor allele.

The parameter involved in the statistical model, the error rate of amplicon sequencing, e(n), refers to the highest ratio of the number of sequencing sequences carrying the wrong allele of n SNPs to the total number of sequencing sequences of the marker. In this embodiment, the frequency of the minor allele genotypes detected in the expected homozygous MNP marker, that is, the error rate of these genotypes, is calculated to obtain e _max (n=1) and e _max (n≥2) in the statistical model.

The calculation is as follows:

The number of bases that differ between the ith minor allele and the major genotype is ni, the number of sequencing sequences it supports is ci, and the error rate ei is the ratio of the number of sequencing sequences ci of the minor allele to the total number of sequencing sequences N of the marker, that is, ei = ci/N;

Calculate e _max (n) at different sequencing depths. This example summarizes the error rates of all minor alleles of all 930 homozygous MNP markers. When the sequencing depth is >= 1000X, e _max (n=1) and e _max (n≥2) are 1.03% and 0.0994%, respectively.

In order to detect low-frequency variations in microorganisms in a population, the sequencing depth needs to reach more than 1000X. Then, based on the BINOM.INV function, the critical value of the number of sequencing sequences of the sub-allele type in each marker is calculated under the probability guarantee of α=99.9999%, when e _max (n=1) and e _max (n≥2) are 1.03% and 0.0994% respectively. Only when the number of sequencing sequences of the sub-allele type exceeds the critical value is it determined to be a true sub-allele type (Table 4). When there are multiple candidate sub-alleles, the P value of each candidate allele type is multi-corrected, and the candidate allele with FDR<0.5% is determined to be a true sub-allele type.

Table 4 Critical values for determining minor allele types at certain sequencing depths

According to the above parameters, DNA of S-1 and S-4 strains with inter-strain variation in Table 5 were mixed according to the following 8 ratios: 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100, 7/100, to prepare artificial heterozygous samples, and each sample was tested 3 times to obtain a total of 24 sequencing data. By accurately comparing the genotypes of the MNP markers of the two variants of Streptococcus pneumoniae, the heterozygous genotype markers were detected in all 24 artificial heterozygous samples, indicating the applicability of the developed MNP marker detection method for Streptococcus pneumoniae in detecting genetic variation of strains.

Finally, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

Although the preferred embodiments of the present invention have been described, other changes and modifications may be made to these embodiments once those skilled in the art have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

Obviously, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if these modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

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tgttagcaga tggtattttg caatcaagat attttgaaaa tttgaaaaat agttggaaag 60

atttagatat agctgtagtc ggaattggtg attttagcaa taaaggaaag catcaatggt 120

tagacatgct tacagaggat gacttt 146

<210> 5

<211> 150

<212> DNA

<213> Artificial Sequence

<400> 5

acttttgtca aggttctgtc gcgtgtcata ggtaaaaaac tagatcagca aggtctggaa 60

aatccagcta tcccaaccag tccaagcagc aagaccttag ccaaggacac cttgcaagct 120

ctctatcctg ccaaacagga gttttacctg 150

<210> 6

<211> 141

<212> DNA

<213> Artificial Sequence

<400> 6

cctgcttcat cgtgtccatg tacttgttga tgattttaac ccaagtgtct gtatcacttg 60

gagctctctt gctcttattt acatattccc agttctcaac atcataatag atagggtaag 120

acaggttcat attgtatttc t 141

<210> 7

<211> 150

<212> DNA

<213> Artificial Sequence

<400> 7

aaacaagctc cgaatatctc tccccttggt agctgcaccg atagacagac gcagattttc 60

atcttctgag tgaatagtca gctctaagcc actatccaaa gccttgtcat tgagcagggt 120

gacatatttg gttagggttg cttttgaaaa 150

<210> 8

<211> 149

<212> DNA

<213> Artificial Sequence

<400> 8

ttcacctcgt taatcaatcc tttgatgtca atctttccat ttggagttag tggcaaactg 60

tctcggtaaa ggaatttaga tggcatcata taggacatca tgatgtctgt caggtcttcc 120

ttgatggcct tggtaatatc gatatctcg 149

<210> 9

<211> 150

<212> DNA

<213> Artificial Sequence

<400> 9

agaattccaa gaattttgca aggatacggt cctagttgcc cacaatgcta cctttgacgt 60

tggctttatg aatgctaatt atgagcgtca tgatcttcca aagattagtc agccagttat 120

tgatacgctg gagtttgcta gaaacctcta 150

<210> 10

<211> 150

<212> DNA

<213> Artificial Sequence

<400> 10

caaagtggaa gccttctgga ttttcaacca tggttccagc taccaaagct cgagtatctg 60

catctgtgat ttcttacttc ttatcggcca gtgccttgaa cttagcaaag agtggtttga 120

tatcctcttc tgtaaaatct agggccaatt 150

<210> 11

<211> 150

<212> DNA

<213> Artificial Sequence

<400> 11

cactagggct ccgatgacaa tacttgcgat aaatagaagg acagttccag ggtttggagc 60

gaccatgata cggtcgatat attcttggga ttttcctctt gccagaagag tagccatata 120

ggctttgggc gcaatccaca taagcaagat 150

<210> 12

<211> 146

<212> DNA

<213> Artificial Sequence

<400> 12

aggttctgaa tatgcaaata ctgtcactca aggtatcgta tccagtctca atagaaatgt 60

atccttaaaa tcggaagatg gacaagctat ttctacaaaa gccatccaaa ctgatactgc 120

tattaaccca ggtaactctg gcggcc 146

<210> 13

<211> 150

<212> DNA

<213> Artificial Sequence

<400> 13

tgattcctca tcagcagtag caaagtgggt aaagattcct tcaacacaaa caccgtgttg 60

ttggagcaaa tcttgagcct gctcaacctc acttgcctct ctaaaaccaa tccgtcccat 120

ccctgaatca atcttgaggt ggactgtcaa 150

<210> 14

<211> 143

<212> DNA

<213> Artificial Sequence

<400> 14

tcgctaatac catattagta tcctttcttt tatctacaca aagaataaca cacttatgtt 60

aaccctatat gaactttaat aaaaaactaa tctgtctaca agctaatctt aaattccata 120

ccgtgttcat aagggagaaa act 143

<210> 15

<211> 150

<212> DNA

<213> Artificial Sequence

<400> 15

agtaccagta attcctttgg tttgagatca atttcttcat ttttataatg tgctttgtaa 60

gaggtaaaat ccactgttac atcctgatat cgccaaatat cctctatgac gtagtaacgt 120

ctgataagcg ccttaatacg cttgacaagc 150

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 16

cgtttctaca aagactggaa cctat 25

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 17

tcgctttgtt gctggtagc 19

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 18

ttggtctgaa atagccatgg c 21

<210> 19

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 19

tgttagcaga tggtattttg caatca 26

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 20

acttttgtca aggttctgtc gc 22

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 21

cctgcttcat cgtgtccatg ta 22

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 22

aaacaagctc cgaatatctc tccc 24

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 23

ttcacctcgt taatcaatcc tttga 25

<210> 24

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 24

agaattccaa gaattttgca aggat 25

<210> 25

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 25

caaagtggaa gccttctgga ttttc 25

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 26

cactagggct ccgatgacaa tac 23

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 27

aggttctgaa tatgcaaata ctgtc 25

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 28

tgattcctca tcagcagtag caa 23

<210> 29

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 29

tcgctaatac catattagta tcctttct 28

<210> 30

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 30

agtaccagta attcctttgg tttga 25

<210> 31

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 31

ggcaacaatg accgtattac aa 22

<210> 32

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 32

cagttgttgg caatcaaaga gc 22

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 33

gttctcctca tggacgagcc 20

<210> 34

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 34

aaagtcatcc tctgtaagca tgtct 25

<210> 35

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 35

caggtaaaac tcctgtttgg cag 23

<210> 36

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 36

agaaatacaa tatgaacctg tcttaccc 28

<210> 37

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 37

ttttcaaaag caaccctaac caaat 25

<210> 38

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 38

cgagatatcg atattaccaa ggcca 25

<210> 39

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 39

tagaggtttc tagcaaactc cagc 24

<210> 40

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 40

aattggccct agattttaca gaaga 25

<210> 41

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 41

atcttgctta tgtggattgc gc 22

<210> 42

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 42

ggccgccaga gttacctg 18

<210> 43

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 43

ttgacagtcc acctcaagat tgatt 25

<210> 44

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 44

agttttctcc cttatgaaca cggta 25

<210> 45

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 45

gcttgtcaag cgtattaagg cg 22

Claims (9)

1. The core MNP marker combination of streptococcus pneumoniae is characterized by comprising 15 markers, and the specific nucleotide sequences of the core MNP marker combination are shown as SEQ ID NO.1-SEQ ID NO. 15.

2. A multiplex PCR primer pair combination for detecting the streptococcus pneumoniae core MNP marker combination according to claim 1, wherein the multiplex PCR primer pair combination comprises 15 pairs of primers, the specific primer nucleotide sequences are shown in SEQ ID No.16-SEQ ID No. 45.

3. A test kit for detecting the streptococcus pneumoniae core MNP marker combination according to claim 1, wherein the kit comprises the primer pair combination according to claim 2.

4. The test kit of claim 3, wherein the kit further comprises a multiplex PCR premix.

5. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 for qualitative detection of streptococcus pneumoniae of non-diagnostic interest.

6. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 in the preparation of a qualitative detection product of streptococcus pneumoniae.

7. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 for the non-diagnostic detection of streptococcus pneumoniae micro-types.

8. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 for constructing a streptococcus pneumoniae database.

9. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 for the detection of genetic variation within and between streptococcus pneumoniae strains for non-diagnostic purposes.