CN115029453B

CN115029453B - MNP (MNP) marking site of streptococcus pyogenes, primer composition, kit and application of MNP marking site

Info

Publication number: CN115029453B
Application number: CN202111354807.1A
Authority: CN
Inventors: 陈利红; 李论; 高利芬; 方治伟; 李甜甜; 肖华锋; 周俊飞; 彭海; 万人静
Original assignee: Jianghan University
Current assignee: Jianghan University
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2023-06-16
Anticipated expiration: 2041-11-16
Also published as: CN115029453A

Abstract

The invention discloses a streptococcus pyogenes specific MNP (MNP) marker locus, a primer composition, a kit and application thereof, wherein the MNP marker locus refers to a genome region which is screened on a streptococcus pyogenes genome and is separated from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker loci of MNP-1-MNP-15; the primer is shown as SEQ ID NO.1-SEQ ID NO. 30. The MNP marker locus can specifically identify streptococcus pyogenes and finely distinguish different species; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that the sequence analysis can be carried out on all the marker loci of multiple samples at one time, the method has the advantages of high throughput, multiple targets, high sensitivity and culture-free detection, can be applied to the identification and genetic variation detection of streptococcus pyogenes of large-scale samples, and has important significance on scientific research and monitoring of the streptococcus pyogenes.

Description

MNP (MNP) marking site of streptococcus pyogenes, primer composition, kit and application of MNP marking site

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marking site of streptococcus pyogenes, a primer composition, a kit and application thereof.

Background

Streptococcus pyogenes (Streptococcus pyogenes) is one of important pathogenic bacteria of human body, can cause a series of infections such as acute pharyngitis, acute tonsillitis, scarlet fever, impetigo and acute pharyngitis of children, can be transmitted through food, water source, air and other ways, and is helpful for the occurrence of Streptococcus pyogenes infection due to poor living, poor sanitary conditions, crowded living, close contact and the like. Therefore, the rapid and accurate detection of streptococcus pyogenes has important significance for timely preventing and controlling the epidemic and transmission of pathogenic bacteria. In addition, streptococcus pyogenes is also a common model of pathogenic microorganisms for laboratory research. As a group organism, individuals in the group can be mutated in interaction with hosts and environments. For laboratory studies, such undetectable variations can result in strains of the same name in different laboratories or different times in the same laboratory being virtually different, resulting in irreproducible and incomparable experimental results. Heterogeneity between human hela cell laboratories has resulted in a significant amount of incomparable experimental results and wasted data. Therefore, developing a rapid, accurate and monitorable mutation detection and analysis method for streptococcus pyogenes has important significance for scientific research and application of streptococcus pyogenes.

Laboratory diagnostic methods for streptococcus pyogenes mainly include bacterial culture and identification, nucleic acid detection and serological tests, among which the most common are nucleic acid detection based on the specific polymerase chain reaction (polymerase chain reaction, PCR). With the development of sequencing technology, whole genome and metagenome sequencing are also used in different sample assays. These existing techniques have one or more limitations in terms of duration, complexity of operation, throughput of detection, accuracy and sensitivity of detection variation, cost, and the like. The targeted molecular marker detection technology integrating the ultra-multiplex PCR amplification and the high-throughput sequencing can enrich target microorganisms in a sample with low microorganism content in a targeted manner, avoids the limitation of massive data waste and background noise caused by the separation culture of the whole genome depending on pathogenic bacteria and metagenome sequencing, and has the advantages of small sample requirement, accurate diagnosis result, data quantity conservation, low-frequency variation detection and culture-free. The molecular markers detected by the existing targeted detection technology mainly comprise SNP and SSR markers. SSR markers are the most well-accepted markers for polymorphism, but are small in number in microorganisms; the number of SNP markers is huge, the distribution is dense, and the polymorphism of single SNP marker is insufficient to capture the potential allelic diversity in microorganism population.

Therefore, developing a novel molecular marker with high polymorphism of streptococcus pyogenes and a detection technology thereof becomes a technical problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a specific MNP (MNP) marking site of streptococcus pyogenes, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on the streptococcus pyogenes and have the effects of multiple targets, high flux, high sensitivity and fine typing.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect of the invention there is provided a streptococcus pyogenes specific MNP marker locus, which is a genomic region screened on the streptococcus pyogenes genome that is distinct from other species and has a plurality of nucleotide polymorphisms within the species, comprising the marker locus of MNP-1 to MNP-15 on a streptococcus pyogenes reference sequence.

In the above technical scheme, the marking sites of MNP-1 to MNP-15 are specifically shown in the specification table 1, and the starting and ending positions of the MNP marks marked in the table 1 are determined based on the reference sequences corresponding to the same row of MNPs in the table 1.

In a second aspect of the present invention, there is provided a multiplex PCR primer composition for detecting the MNP marker loci, the multiplex PCR primer composition comprising 15 pairs of primers, the specific primer sequences being shown in SEQ ID NO.1-SEQ ID NO. 30.

In the above technical solution, the primers of each MNP marker locus include an upper primer and a lower primer, and are specifically shown in table 1 of the specification.

In a third aspect of the invention, there is provided a detection kit for detecting the MNP marker locus of streptococcus pyogenes, the kit comprising the primer composition.

Further, the kit further comprises a multiplex PCR premix.

In a fourth aspect of the invention, there is provided the use of said MNP-labeling site of Streptococcus pyogenes or said multiplex PCR primer composition or said detection kit for the qualitative detection of Streptococcus pyogenes for non-diagnostic purposes and for the preparation of a product for the qualitative detection of Streptococcus pyogenes.

In a fifth aspect of the invention, there is provided the use of the MNP marker locus of streptococcus pyogenes or the multiplex PCR primer composition or the detection kit for detecting genetic variation within and between streptococcus pyogenes strains.

In a sixth aspect of the invention, there is provided the use of the MNP marker locus of streptococcus pyogenes or the multiplex PCR primer composition or the detection kit in the construction of a streptococcus pyogenes database.

In a seventh aspect of the invention, there is provided the use of the MNP marker locus of streptococcus pyogenes or the multiplex PCR primer composition or the detection kit in the detection of the fine type of streptococcus pyogenes.

In the application, the specific operation steps are as follows: firstly, obtaining total bacterial DNA of a sample to be detected; performing a first round of multiplex PCR amplification on the total DNA and the blank control by using the kit, wherein the number of cycles is not higher than 25; purifying the amplified product, and then adding a sample tag and a second generation sequencing joint based on the second-round PCR amplification; quantifying after purifying the second round of amplification products; detecting a plurality of strains by mixing the amplification products of the second round in equal amounts and then performing high throughput sequencing; and comparing the sequencing result with the reference sequence of the streptococcus pyogenes to obtain the number of detection sequences and genotype data of the total DNA. And carrying out data quality control and data analysis on the sequencing data of the total DNA according to the number of the sequencing sequences of streptococcus pyogenes and the number of the detected MNP sites obtained in the total DNA and the blank control, and obtaining the number of the detected MNP sites, the number of the sequencing sequences covering each MNP site and the genotype data of the MNP sites.

When the method is used for identifying streptococcus pyogenes, whether the sample to be detected contains nucleic acid of streptococcus pyogenes is judged after quality control according to the number of sequencing sequences of the streptococcus pyogenes detected in the sample to be detected and a blank control and the number of MNP sites detected. The quality control scheme and the judging method are characterized in that DNA of streptococcus pyogenes with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the streptococcus pyogenes are evaluated, and the quality control scheme and the judging method for detecting the streptococcus pyogenes by the kit are formulated.

When used in the detection of genetic variation in Streptococcus pyogenes, it includes the detection of genetic variation between strains and within strains. The detection of genetic variation among strains comprises the steps of obtaining genotype data of 15 MNP sites of each strain to be compared by using the kit and the method. By genotype comparison, the strains to be compared are analyzed for differences in major genotypes at the 15 MNP sites. If the strain to be compared has a variation in the main genotype of at least one MNP site, it is determined that there is a genetic variation in both. Alternatively, 15 loci of the strain to be compared may be amplified by single PCR, and then Sanger sequencing is performed on the amplified products to obtain sequences, and then the genotypes of each MNP locus of the strain to be compared are aligned. If there are MNP sites of inconsistent main genotypes, there are variations between the strains to be compared. When detecting genetic variation inside the strain, determining whether the secondary genotype other than the primary genotype is detected at the MNP locus of the strain to be detected through a statistical model. If the strain to be tested has the subgenotype at least one MNP site, judging that the strain to be tested has genetic variation.

When used for constructing a streptococcus pyogenes DNA fingerprint database, genotype data of the MNP locus of streptococcus pyogenes identified from a sample is recorded into a database file to form the streptococcus pyogenes DNA fingerprint database; and (3) each time different samples are identified, comparing the samples with a DNA fingerprint database of the streptococcus pyogenes, and identifying whether the streptococcus pyogenes in the samples has a difference of a main genotype (a genotype supported by more than 50% of sequencing fragments at one MNP site) with strains in the database, wherein the streptococcus pyogenes with the main genotype difference at least 1 MNP site is a new mutation type, and recording the new mutation type in the DNA fingerprint database.

When the method is used for typing streptococcus pyogenes, the streptococcus pyogenes in the sample to be tested is identified, and the genotype of each MNP locus is obtained; collecting genome sequences of streptococcus pyogenes disclosed on the net and constructing a streptococcus pyogenes DNA fingerprint database to form a streptococcus pyogenes reference sequence library; comparing the genotype of the streptococcus pyogenes in the sample to be detected with a reference sequence library of the streptococcus pyogenes, and screening strains which are genetically identical or closest to each other to obtain the typing of the streptococcus pyogenes in the sample to be detected. And identifying whether the streptococcus pyogenes in the sample is an existing type or a new type according to the comparison result with the reference sequence library, and realizing the fine typing of the streptococcus pyogenes.

The invention belongs to the field of streptococcus pyogenes and is not reported in related documents; MNP markers are developed mainly based on reference sequences, and MNP sites which are large-scale and are distinguished from other species, polymorphic in the streptococcus pyogenes species and conserved in sequences at two sides can be mined according to reported resequencing data of the streptococcus pyogenes representative race; MNP site detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of the MNP site; and then a set of MNP locus with the largest polymorphism and high specificity and a primer combination with the best compatibility can be screened out according to the test result of the standard substance.

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

the invention provides an MNP (MNP) marking site of streptococcus pyogenes, a primer composition, a kit and application thereof. The provided 15 MNP loci of streptococcus pyogenes and the primer combination thereof can be used for multiplex PCR amplification, and the amplification products are sequenced by fusing a second generation sequencing platform, so that the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection of the combined mycobacterium are met, and the requirements of accurate detection of genetic variation among and in streptococcus pyogenes strains and accurate subdivision are met; meets the requirements of streptococcus pyogenes standard and sharable fingerprint data construction, and provides technical support for scientific research and medical monitoring of streptococcus pyogenes.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of MNP marker polymorphism;

FIG. 2 is a flow chart of screening and primer design for MNP marker loci of Streptococcus pyogenes;

FIG. 3 is a flow chart of detection of MNP marker loci.

Detailed Description

The advantages and various effects of the embodiments of the present invention will be more clearly apparent from the following detailed description and examples. Those skilled in the art will appreciate that these specific implementations and examples are provided to illustrate, but not limit, examples of the present invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. In case of conflict, the present specification will control.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or may be prepared by existing methods.

The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:

the invention develops a novel molecular marker-MNP marker which is suitable for detecting group organisms and is specific to species. MNP markers are indicated inPolymorphic markers caused by multiple nucleotides in a region of the genome. MNP markers have the following advantages over SSR markers and SNP markers: (1) The alleles are abundant, and 2 are arranged on single MNP locus ⁿ Species alleles higher than SSR and SNP; (2) The species distinguishing capability is strong, the species identification can be realized by only a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting MNP labels based on the combination of super multiplex PCR and a second generation high throughput sequencing technology has the following advantages: (1) The output is a base sequence, a standardized database can be constructed for sharing without parallel experiments; (2) The method has high efficiency, breaks through the limitation of the number of sequencing samples by using the sample DNA bar code, and can type tens of thousands of MNP sites of hundreds of samples at one time; (3) High sensitivity, multiple targets are detected at one time by using multiple PCR, and high false negative and low sensitivity caused by single target amplification failure are avoided; (4) High accuracy, and sequencing the amplified product hundreds of times by using a second-generation high-throughput sequencer.

In view of the advantages and the characteristics, the MNP marking and the detection technology thereof can realize classification and tracing of the multi-allele types of the group organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, genetic variation detection and the like. At present, no report about MNP labeling exists in microorganisms, and corresponding technology is lacking. Thus, the present invention developed MNP marker loci for genomic regions screened on the Streptococcus pyogenes genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including marker loci for MNP-1-MNP-15 of the AE004092 reference genome.

Next, the present invention has developed a multiplex PCR primer composition for detecting the MNP marker loci of Streptococcus pyogenes, characterized in that the multiplex PCR primer composition comprises 15 pairs of primers, the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO.1 to SEQ ID NO. 30. The primers do not collide with each other, and efficient amplification can be performed by multiplex PCR.

The multiplex PCR primer composition can be used as a detection kit for detecting the MNP marker locus of streptococcus pyogenes.

The kit of the invention can accurately and sensitively detect streptococcus pyogenes with the concentration as low as 10 copies/reaction.

The MNP markers and the kits of the invention detect the high specificity of Streptococcus pyogenes in complex templates.

The MNP marker loci, primer compositions, kits and uses thereof of one of the streptococcus pyogenes of the present application will be described in detail below in conjunction with examples, comparative examples and experimental data.

Example 1 screening of MNP marker loci of Streptococcus pyogenes and design of multiplex PCR amplification primers

S1, screening MNP (MNP) marker locus of streptococcus pyogenes

Based on the complete or partial sequences of the genome of 499 different isolates of Streptococcus pyogenes disclosed on the net, 15 MNP marker loci were obtained by sequence alignment. For species on which no genomic data is present on the net, genomic sequence information representing a minispecies of the microorganism species to be detected may also be obtained by high throughput sequencing, which may be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected markers, genomic sequences of at least 10 genetically representative isolates are generally used as reference. The 15 MNP marker loci screened are shown in table 1:

TABLE 1 MNP marker loci and detection primers starting position on the reference sequence

The step S1 specifically includes:

selecting a genome sequence of a representative strain of streptococcus pyogenes as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphism sites of each strain of streptococcus pyogenes;

on the reference genome, carrying out window translation by taking 100-300 bp as a window and taking 1bp as a step length, and screening to obtain a plurality of candidate MNP site areas, wherein the candidate MNP site areas contain more than or equal to 2 single nucleotide variation sites, and the single nucleotide polymorphism sites do not exist on sequences of 30bp at both ends;

screening a region with the discrimination DP more than or equal to 0.2 from the candidate polynucleotide polymorphism site region as an MNP marking site; wherein dp=d/t, t is the log of comparisons when all the minor species are compared pairwise in the candidate polynucleotide polymorphic site region, and d is the log of samples of differences in at least two single nucleic acid polymorphisms in the candidate polynucleotide polymorphic site region.

As an optional implementation mode, when screening is performed on the reference genome by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, so that the comprehensive screening is facilitated.

S2, design of multiplex PCR amplification primer

The multiplex PCR amplification primers of the MNP locus are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.

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

S3, evaluating detection efficiency of primer combination

The detection method of the MNP marker is that all MNP loci are amplified at one time through multiplex PCR, amplification products are sequenced through second-generation high-throughput sequencing, sequencing data are analyzed, and compatibility of the primer combination is evaluated according to the detected loci.

DNA of a purchased Streptococcus pyogenes standard strain ATCC12344 (goods No.: VIP (B) 10223) was used, and after quantitative analysis by digital PCR, the DNA was added to human genomic DNA to prepare 1000 copies/reaction templates, and the primer combinations were used to screen for a primer combination of optimal compatibility with uniform amplification according to detection of MNP sites in 4 libraries, and finally, the primer combinations of 15 MNP sites according to Table 1 of the present invention were selected.

Example 2 MNP site and primer identification threshold settings and Performance assessment of Streptococcus pyogenes

1. Detection of MNP markers

In this example, 1 copy/reaction, 10 copy/reaction and 100 copy/reaction of Streptococcus pyogenes simulated samples were prepared using purchased DNA of Streptococcus pyogenes standard strain ATCC12344 (cargo number: VIP (B) 10223), quantified by digital PCR, and added to human genomic DNA. An equal volume of sterile water was set at the same time as a blank. A total of 4 samples, each of which was constructed as 3 replicate libraries per day, were tested continuously for 4 days, i.e., 12 sets of sequencing data were obtained per sample, as shown in table 2. And (3) evaluating the reproducibility, accuracy and sensitivity of the detection method according to the number of sequencing fragments and the number of sites of the MNP site of the streptococcus pyogenes detected in the blank control and the streptococcus pyogenes nucleic acid standard substance in 12 repeated experiments, and formulating a threshold value for pollution of a quality control system and detection of a target pathogen. The detection flow of MNP markers is shown in fig. 3.

TABLE 2 detection sensitivity and stability analysis of MNP labeling method of Streptococcus pyogenes

As shown in Table 2, the kit can stably detect more than 10 MNP sites in a 10-copy/reaction sample, and can detect at most 1 MNP site in a 0-copy/reaction sample, and the kit can clearly distinguish between the 10-copy/reaction sample and the 0-copy/reaction sample, and has technical stability and detection sensitivity as low as 10-copy/reaction.

2. Reproducibility and accuracy assessment of MNP (MNP) marker detection kit for detecting streptococcus pyogenes

Based on whether the genotype of the co-detection site is reproducible in the two replicates, the reproducibility and accuracy of the MNP marker detection method for detecting streptococcus pyogenes is assessed. Specifically, the data of 12 sets of 100 copies/reaction samples were compared in pairs, respectively, and the results are shown in Table 3.

TABLE 3 reproducibility and accuracy assessment of methods for detection of MNP markers in Streptococcus pyogenes

As can be seen from Table 3, the number of MNP sites having a difference in the main genotypes was 0; according to the principle that the reproducible genotypes are considered to be accurate between 2 repeated experiments, the accuracy a=1- (1-r)/2=0.5+0.5r, and r represents the reproducibility, namely the ratio of the reproducible site number of the main genotype to the common site number. In the project reproducibility test, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.

3. Threshold judgment for detecting streptococcus pyogenes by MNP (MNP) marker detection kit

As shown in Table 2, the sequences aligned to Streptococcus pyogenes could be detected in 1 copy/reaction sample, covering at least 1 MNP site. The sequence of Streptococcus pyogenes was also detected in part of the blank. Because of the extreme sensitivity of MNP marker detection methods, contamination of the data in the detection is prone to false positives. Therefore, the quality control scheme is formulated in this example, and is specifically as follows:

1) The amount of sequencing data is greater than 4.5 megabases. The measurement and calculation basis is that the number of MNP loci detected by each sample is 15, and the length of one sequencing fragment is 300 bases, so that when the data size is more than 4.5 megabases, most samples can ensure that the number of sequencing fragments covering each locus reaches 1000 times by one experiment, and ensure the accurate analysis of the base sequence of each MNP locus.

2) Determining whether the contamination is acceptable based on the signal index S of streptococcus pyogenes in the test sample and the noise index P of streptococcus pyogenes in the blank, wherein:

the noise figure p=nc/Nc for the control, where Nc and Nc represent the number of sequenced fragments and total sequenced fragment number of streptococcus pyogenes, respectively, in the control.

The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments and total sequenced fragment number, respectively, of streptococcus pyogenes in the test sample.

3) Calculating the detection rate of MNP marking sites in a test sample, wherein the detection rate refers to the ratio of the number of detected sites to the number of total designed sites.

TABLE 4 SNR of Streptococcus pyogenes in samples to be tested

As shown in Table 4, the average noise figure of Streptococcus pyogenes in the control was 0.02%, the average signal figure in the 1-copy sample was 0.28%, and the average signal-to-noise ratio of the 1-copy sample and the control was 12.5, so that the present invention provides that when the signal-to-noise ratio was more than 15-fold, it was judged that the contamination in the detection system was acceptable.

The average signal to noise ratio of the 10 copies of the sample and the blank was 129.48, and at least 10 MNP sites were stably detected in the 10 copies/reaction 15 sets of data, accounting for 66.7% of the total sites. Thus, under the condition of ensuring accuracy, the standard prescribes that the judgment standard for the positive of the streptococcus pyogenes is that the detection of the nucleic acid of the streptococcus pyogenes in the sample is judged when the signal to noise ratio of the streptococcus pyogenes in the sample is more than 30 and the site detection rate is more than or equal to 30%.

Therefore, the kit provided by the invention can accurately and sensitively detect streptococcus pyogenes with the copy/reaction as low as 10.

4. Specific evaluation of MNP marker detection method for detecting streptococcus pyogenes

The method comprises the steps of artificially mixing streptococcus pyogenes with nucleic acid of mycobacterium tuberculosis, acinetobacter strain, pertussis bauhinia, huo Shibao termyces, chlamydia pneumoniae, mycoplasma pneumoniae, EB virus, haemophilus influenzae, varicella zoster virus, cytomegalovirus, herpes simplex virus, human bocavirus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus and streptococcus pneumoniae to prepare a mixed template, detecting streptococcus pyogenes in the mixed template by adopting the method provided by the invention by using a blank template as a reference, and performing 3 repeated experiments. After sequence comparison and analysis according to the quality control scheme and the judgment threshold, 15 MNP sites of streptococcus pyogenes can be specifically detected in 3 repeated experiments, which shows that the MNP markers and the kit detect the high specificity of target microorganisms in complex templates.

Example 3 detection of genetic variation between Streptococcus pyogenes strains

The kit and MNP labeling site detection method are utilized to detect 6 sub-generation strains of 1 strain of streptococcus pyogenes provided by disease control of Hubei province, samples are sequentially named as S1-S6, the average coverage of sequencing of each sample is 1416 times, and all 15 MNP labels can be detected by each strain (Table 5). The fingerprints of 6 strains were aligned pairwise, and the results are shown in Table 5, where 1 part (S-2) and 5 parts of Streptococcus pyogenes detected together in the same batch all have a major genotype difference at part of the sites (Table 5), indicating that there is a variation between strains.

TABLE 5 detection assay for 6 Streptococcus pyogenes

As can be seen from Table 5, the application of the kit of the invention in identifying genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of streptococcus pyogenes strains named identically in different laboratories, thereby ensuring the comparability of research results, which has great significance for scientific research of streptococcus pyogenes. In clinical terms, one can take into account the diagnostic regimen as to whether the site of the difference affects resistance.

Example 4 detection of genetic variation inside Streptococcus pyogenes Strain

As a group organism, the individuals in the streptococcus pyogenes group are mutated, so that the group is no longer homozygous to form a heterogeneous heterozygous group, and the stability and consistency of the phenotype of the microorganism for the test are influenced. Such variants, when detected by molecular marker detection on the population, appear as alleles outside the major genotype of the locus. When variant individuals have not accumulated, they occupy a very small proportion of the population and exhibit a low frequency of allelic forms. Low frequency alleles tend to mix with technical errors, making the prior art indistinguishable. The present invention detects MNP markers with high polymorphism. Based on the fact that the probability of occurrence of a plurality of errors is lower than that of one error, the technical error rate of MNP markers is significantly lower than that of SNP markers.

The authenticity assessment of the secondary isogenotypes of this example was performed as follows: the allelotype with strand preference (ratio of the number of sequencing sequences covered on the DNA duplex) is first excluded according to the following rule: the strand preference is greater than 10-fold, or the difference from the strand preference of the major allele is greater than 5-fold.

Genotypes without strand preference were judged for authenticity based on the number and proportion of sequenced sequences in table 6. Table 6 lists e calculated based on binom. Inv function under the probability guarantee of α=99.9999% _max (n=1) and e _max (n.gtoreq.2) is 1.03% and 0.0994%, respectively, and the true hypogenotype is judged only when the number of sequences of the hypogenotype exceeds the critical value. When a plurality of candidate minor alleles exist, multiple correction is carried out on the P value of each candidate allele type, and FDR is carried out<0.5% of candidate alleles are judged to be true minor genotypes.

Parameter e related to Table 6 _max (n=1) and e _max (n.gtoreq.2) refers to the highest proportion of the total sequence of the locus of the sequence of the wrong allele carrying n SNPs. e, e _max (n=1) and e _max (n.gtoreq.2) 1.03% and 0.0994%, respectively, are obtained from the frequency of all minor genotypes detected at 930 homozygous MNP sites.

TABLE 6-threshold for determining the hypo-isogenotypes at partial sequencing depth

According to the above parameters, nucleic acids of two strains having a difference in genotype were mixed in the following 8 ratios of 1/1000,3/1000,5/1000,7/1000,1/100,3/100,5/100,7/100 to prepare artificial heterozygous samples, each sample was tested 3 times for repetition, and 24 sequencing data were obtained in total. Through the accurate comparison with the genotypes of MNP loci of the two strains, loci with heterozygous genotypes are detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marker detection method for streptococcus pyogenes in detecting genetic variation inside a strain population is demonstrated.

Example 5 construction of DNA fingerprint database of Streptococcus pyogenes

All strains or DNA of samples used for constructing a streptococcus pyogenes DNA fingerprint database are extracted by using a conventional CTAB method, a commercial kit and other methods, and the quality of the DNA is detected by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm and 230nm is more than 2.0, the ratio of the absorbance values of 260nm and 280nm is between 1.6 and 1.8, the DNA electrophoresis main band is obvious, no obvious degradation and RNA residues exist, the genome DNA reaches the relevant quality requirements, and the subsequent experiments can be carried out.

And (3) comparing the sequencing data of the 6 strains with the reference genotype, and obtaining the main genotype of each site of each strain to form the MNP fingerprint of each strain. And recording the obtained MNP fingerprint of each strain into a database file to form a streptococcus pyogenes DNA fingerprint database.

The constructed MNP fingerprint database is based on the gene sequence of the detected strain, is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared and being updated at any time. And comparing the MNP fingerprint of the strain obtained by each detection with an MNP fingerprint database constructed based on the existing genome data, and inputting the MNP fingerprint of the strain with the main genotype difference into the constructed MNP fingerprint database to achieve real-time updating and co-construction sharing of the database.

Example 6 use in the fine subdivision of Streptococcus pyogenes

Firstly, constructing a reference sequence library of streptococcus pyogenes, which consists of a published genome sequence of the streptococcus pyogenes and a constructed fingerprint database of the streptococcus pyogenes; obtaining MNP fingerprints of streptococcus pyogenes in each sample to be tested by using the primer combination and the MNP marker locus detection method described in example 2; comparing the DNA fingerprint of each strain with a constructed reference sequence library, and screening to obtain the strain with the closest genetic distance in the sequence library; 100% identical to the genotype of the existing strain, the existing variant, the new variant with major genotype difference at least one MNP site, realizes the fine typing of streptococcus pyogenes.

The typing results of 6 parts of Streptococcus pyogenes in this example are shown in Table 5, and 6 parts of strains can be classified into 2 types. Wherein 5 strains with consistent genotypes are consistent with the ATCC12344 strain in the reference sequence library, which is considered as a very similar strain of ATCC12344 strain, and the other variant strain and ATCC12344 have a main genotype difference of 1 MNP site, which is judged as a new variant strain, so that the fine typing of streptococcus pyogenes is realized.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiment and all such alterations and modifications as fall within the scope of the embodiments of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, the embodiments of the present invention are intended to include such modifications and alterations insofar as they come within the scope of the embodiments of the invention as claimed and the equivalents thereof.

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<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

aatcagtacg atttgggagt tcaaa 25

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

agcaatacct aattctgctg ctaca 25

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tagaaacacc tgagacagtt atggc 25

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ccgttaaaag aatgccaagt ctagg 25

<210> 9

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

accaaccttg atgcaaacct ttatt 25

<210> 10

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

agtaaactca aatgagggaa tctttt 26

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tgaaactact agaggactct ttttcct 27

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

taatgcgaat tttctcatcg gcatt 25

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cttcagctgc aatctgacga ttatc 25

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

aaaatgatag tcaagaagtg aagcg 25

<210> 15

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

agaccttatt aaactttttg ccttttt 27

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gttttagggg ctgtaagaca aacaa 25

<210> 17

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gtaattgcat tattcaagta cctcgc 26

<210> 18

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

aaagcagcca agcataataa tctgg 25

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

cagttcgtgc tctgccag 18

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

gcatttggcc tgattgcatc a 21

<210> 21

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

caccacttcc tcagattcag ctc 23

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

gcggttatgt ttcgcttcta gttta 25

<210> 23

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

cttcatcgtc aacttgttga acagt 25

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

agctatttgg aacactcgtt tagaa 25

<210> 25

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

tggtttatgg gttttgattt ttcagg 26

<210> 26

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

agtcattttc cttttatctt cccttct 27

<210> 27

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

gccgtctgag accaaagcg 19

<210> 28

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

cagaagtagc aatcagataa tcgtct 26

<210> 29

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

catattagta ggttggtaaa aataagtatg atg 33

<210> 30

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

gactaccgtg catatagtgg aatc 24

Claims

1. A multiplex PCR primer composition for detecting streptococcus pyogenes, which is characterized by comprising 15 pairs of primers, wherein the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO.1-SEQ ID NO. 30.

2. A test kit for detecting streptococcus pyogenes, comprising the primer composition of claim 1.

3. The test kit of claim 2, wherein the kit further comprises a multiplex PCR premix.

4. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for the qualitative detection of streptococcus pyogenes for non-diagnostic purposes and for the preparation of a product for the qualitative detection of streptococcus pyogenes.

5. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for detecting genetic variation within and among streptococcus pyogenes strains.

6. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for constructing a streptococcus pyogenes database.

7. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 in the detection of streptococcus pyogenes fine-particle types.