CN114790488A - MNP (MNP) marker locus of staphylococcus aureus, primer composition, kit and application thereof - Google Patents

Abstract

The present invention discloses an MNP marker site, primer composition, kit and application of Staphylococcus aureus. The genomic region with multiple nucleotide polymorphisms in the interior includes marker sites of MNP-1 to MNP-15; the primers are shown in SEQ ID NO.1 to SEQ ID NO.30. The MNP marker site can specifically identify Staphylococcus aureus and finely distinguish different races; the primers do not interfere with each other, and the multiplex amplification and sequencing technology can be integrated to perform all marker sites of multiple samples at one time. Sequence analysis has the advantages of high-throughput, multi-target, high-sensitivity, high-precision, and culture-free detection. It can be applied to the identification and genetic variation detection of large-scale samples of Staphylococcus aureus. Health monitoring is of great significance.

Description

A kind of MNP marker site of staphylococcus aureus, primer composition, test kit and application thereof

technical field

The embodiments of the present invention relate to the field of biotechnology, and in particular, to an MNP marker site of Staphylococcus aureus, a primer composition, a kit and applications thereof.

Background technique

Staphylococcus aureus belongs to the genus Staphylococcus and is a representative of Gram-positive bacteria. , purulent sores, air, sewage and other environments are also ubiquitous. Under the right conditions, Staphylococcus aureus can produce enterotoxins, which can cause food poisoning. In recent years, reports of food poisoning caused by Staphylococcus aureus have emerged in an endless stream. Food poisoning caused by Staphylococcus aureus accounts for about 25% of foodborne microbial food poisoning incidents. Staphylococcus aureus has become second only to Salmonella and parahaemolyticus. The third largest microbial pathogen. In addition, Staphylococcus aureus is also a commonly used model pathogenic microorganism for laboratory research. As a group organism, in the interaction with the host and the environment, individuals in the group will mutate, resulting in the failure of detection methods or treatment methods; for laboratory research, this kind of undetectable mutation will lead to different experiments. The same named strains at different times in the same laboratory or in the same laboratory are actually not the same, resulting in non-reproducible and incomparable experimental results. The inter-laboratory heterogeneity of human Hella cells has led to substantial incomparability of experimental results and data waste. Therefore, it is of great significance to develop a rapid, accurate, and variable-monitoring detection and analysis method for Staphylococcus aureus for the scientific research and application of Staphylococcus aureus.

Classical detection methods for Staphylococcus aureus, including isolation and culture, PCR technology, whole genome and metagenomic sequencing, etc., exist one or more in terms of time length, operational complexity, detection throughput, accuracy and sensitivity of detection variation, and cost. limited. The targeted molecular marker detection technology integrating ultra-multiplex PCR amplification and high-throughput sequencing can target and enrich target microorganisms in samples with low microbial content, avoiding a large amount of data waste and background caused by whole genome and metagenomic sequencing. Noise has the advantages of less sample required, accurate diagnosis results, saving data volume, and detecting low-frequency variation. 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 in small numbers in microorganisms; SNP markers are huge in number and densely distributed, and are dimorphic markers, and the polymorphism of a single SNP marker is not enough to capture potential alleles in a microbial population genetic diversity.

Therefore, it is an urgent technical problem to develop novel molecular markers of high polymorphism of Staphylococcus aureus and detection technology for culture-free detection.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Staphylococcus aureus-specific MNP marker site, a primer composition, a kit and its application, which can qualitatively identify and detect the variation of Staphylococcus aureus, and have multi-target, high-throughput, High sensitivity and fine typing effect.

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

In a first aspect of the present invention, an MNP marker site of Staphylococcus aureus is provided, and the MNP marker site refers to the MNP marker locus screened on the Staphylococcus aureus genome that is distinguished from other species and has multiple species within the species. The genomic region of the nucleotide polymorphism, including the marker sites of MNP-1 to MNP-15 on the reference sequence of Staphylococcus aureus.

In the above technical solution, the marker sites of MNP-1 to MNP-15 are specifically shown in Table 1 of the specification, and the start and end positions of the MNP markers marked in Table 1 are based on the reference corresponding to the same row of MNP in Table 1. sequence is determined.

In the second aspect of the present invention, a multiplex PCR primer composition for detecting the MNP marker site is provided, the multiplex PCR primer composition includes 15 pairs of primers, and the specific primer sequence is as SEQ ID NO.1 -shown in SEQ ID NO.30.

In the above technical solution, the primers for each MNP marker site include an upper primer and a lower primer, as shown in Table 1 of the specification.

In a third aspect of the present invention, there is provided a detection kit for detecting the MNP marker site of Staphylococcus aureus, the kit comprising the primer composition.

Further, the kit also includes a multiplex PCR master mix.

In the fourth aspect of the present invention, there is provided the application of the MNP marker site of Staphylococcus aureus or the multiplex PCR primer composition or the detection kit in the identification of Staphylococcus aureus.

In the fifth aspect of the present invention, the MNP marker site of the Staphylococcus aureus or the multiplex PCR primer composition or the detection kit is provided to detect the inheritance of the Staphylococcus aureus within and between strains Variation applications.

In the sixth aspect of the present invention, there is provided the application of the MNP marker site of Staphylococcus aureus or the multiplex PCR primer composition or the detection kit in constructing a Staphylococcus aureus database.

In the seventh aspect of the present invention, there is provided the application of the MNP marker site of Staphylococcus aureus or the multiplex PCR primer composition or the detection kit in the fine typing detection of Staphylococcus aureus .

In the above-mentioned application, the specific operation steps are:

First, obtain the total bacterial DNA of the sample to be tested; use the kit of the present invention to perform the first round of multiplex PCR amplification on the total DNA and blank control, and the number of cycles is not more than 25; after the amplification product is purified, Add sample tags and next-generation sequencing adapters based on the second round of PCR amplification; quantify the second-round amplification products after purification; when detecting multiple strains, mix the second-round amplification products in equal amounts and perform high-throughput Sequencing; the sequencing results are compared with the reference sequence of Staphylococcus aureus, and the number of detected sequences and genotype data in the total DNA are obtained. According to the number of sequencing sequences of Staphylococcus aureus and the number of detected MNP sites obtained from the total DNA and the blank control, data quality control and data analysis are performed on the sequencing data of the total DNA to obtain the number of detected MNP sites order, the number of sequencing sequences covering each of the MNP loci and the genotype data of the MNP locus.

When used for the identification of Staphylococcus aureus, according to the number of sequencing sequences of Staphylococcus aureus detected in the sample to be tested and the blank control and the number of detected MNP sites, after quality control, it is determined whether the sample to be tested contains Nucleic acids of Staphylococcus aureus. Wherein, the quality control scheme and determination method use DNA of Staphylococcus aureus with a known copy number as the detection sample, and evaluate the sensitivity, accuracy and specificity of the kit for detecting Staphylococcus aureus, and formulate the The quality control scheme and determination method of the kit for the detection of Staphylococcus aureus.

When used for the detection of genetic variation in Staphylococcus aureus, the detection of genetic variation between strains and within strains is included. The detection of genetic variation among strains includes using the described kit and method to obtain the genotype data of each of the 15 MNP sites of the strains to be compared. Through genotype comparison, analyze whether there are differences in the main genotypes of the strains to be compared at the 15 MNP sites. If there is variation in the major genotype of at least one MNP site of the strains to be compared, it is determined that there is genetic variation between the two strains. As an alternative, the 15 loci of the strain to be compared can also be amplified by single-plex PCR, and then Sanger sequencing of the amplified product can be performed. After the sequence is obtained, the genotype of each MNP locus of the strain to be compared can be determined. Compare. If there are MNP sites with inconsistent major genotypes, there is variation between the strains to be compared. When detecting the genetic variation within the strain, the statistical model is used to determine whether the minor genotype other than the major genotype is detected at the MNP site of the strain to be tested. If the strain to be tested has a subgenotype at at least one MNP site, it is determined that there is a genetic variation within the strain to be tested.

When used to construct a Staphylococcus aureus DNA fingerprint database, the genotype data of the MNP site of Staphylococcus aureus identified from the sample is entered into a database file to form a Staphylococcus aureus DNA fingerprint database; each time When identifying different samples, by comparing with the DNA fingerprint database of the Staphylococcus aureus, identify whether the Staphylococcus aureus in the sample and the strain in the database have a major genotype at the MNP site (at one MNP site). S. aureus with major genotype differences in at least 1 MNP site is a new variant type, which is included in the DNA fingerprint database.

When it is used for Staphylococcus aureus typing, it is to identify the Staphylococcus aureus in the sample to be tested, and obtain the genotype of each MNP site; The Staphylococcus aureus DNA fingerprint database constitutes a Staphylococcus aureus reference sequence library; the genotype of Staphylococcus aureus in the sample to be tested is compared with the reference sequence library of the Staphylococcus aureus, and the genetically consistent or the most Close strains to obtain the typing of Staphylococcus aureus in the sample to be tested. According to the comparison result with the reference sequence library, identify whether the Staphylococcus aureus in the sample is an existing type or a new variant, and realize the fine typing of the Staphylococcus aureus. The invention is the first in the field of Staphylococcus aureus, and there is no relevant literature report; MNP markers are mainly developed based on reference sequences, and according to the reported resequencing data of representative races of Staphylococcus aureus, it can be mined to distinguish it from other species on a large scale , MNP sites with polymorphism and conserved sequences on both sides of Staphylococcus aureus species; MNP site detection primers suitable for multiplex PCR amplification can be designed through the conserved sequences on both sides of the MNP site; According to the test results, a set of MNP sites with the largest polymorphism and high specificity, the most compatible primer combination and the detection kit can be screened out.

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

The invention provides an MNP marker site of Staphylococcus aureus, a primer composition, a kit and applications thereof. The provided 15 MNP sites of Staphylococcus aureus and their primer combinations can be used for multiple PCR amplification, and the next-generation sequencing platform can be used to sequence the amplification products, which can meet the requirements of high-throughput and high-efficiency detection of Staphylococcus aureus. , high-accuracy and high-sensitivity detection requirements, to meet the requirements of Staphylococcus aureus standard, shareable fingerprint data construction; accurate detection of genetic variation among Staphylococcus aureus strains; identification of Staphylococcus aureus homozygous and heterozygous To meet the needs of the research and to provide technical support for the scientific research and scientific monitoring of Staphylococcus aureus.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments. Obviously, the drawings in the following description are some implementations of the embodiments of the present invention. For example, for those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

Fig. 1 is the schematic diagram of MNP marker polymorphism;

Fig. 2 is the screening and primer design flow chart of Staphylococcus aureus MNP marker site;

Figure 3 is a flow chart of the detection of MNP marker sites.

Detailed ways

Hereinafter, the embodiments of the present invention will be described in detail with reference to specific implementation manners and examples, and the advantages and various effects of the embodiments of the present invention will be more clearly presented thereby. Those skilled in the art should understand that these specific implementation manners and examples are used to illustrate the embodiments of the present invention, but not to limit the embodiments of the present invention.

Throughout the specification, unless specifically stated otherwise, terms used herein are to be understood as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention belong. In case of conflict, the present specification takes precedence.

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

The technical solutions of the embodiments of the present application are to solve the above-mentioned technical problems, and the general idea is as follows:

The present invention develops a new species-specific molecular marker-MNP marker suitable for detecting population organisms. MNP markers refer to polymorphism markers caused by multiple nucleotides in a region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) Alleles are abundant, and there are 2 ⁿ alleles on a single MNP locus, which is higher than SSR and SNP; (2) The ability to distinguish species is strong, Species identification can be achieved with only a small amount of MNP labeling, reducing detection error rates. The MNP labeling method based on ultra-multiplex PCR combined with second-generation high-throughput sequencing technology to detect MNP labeling has the following advantages: (1) The output is the base sequence, and a standardized database can be constructed for sharing and sharing without parallel experiments; (2) High Efficiency, using sample DNA barcodes to break through the limitation of the number of sequencing samples, it can type tens of thousands of MNP loci in hundreds of samples at one time; (3) High sensitivity, using multiplex PCR to detect multiple targets at one time, avoid The failure to amplify a single target leads to high false negatives and low sensitivity; (4) high accuracy, using the second-generation high-throughput sequencer to sequence the amplified product hundreds of times.

In view of the above advantages and characteristics, MNP marker and its detection technology MNP marker method can realize the classification and traceability of multi-allelic genotypes of population organisms, and has application potential in the identification of pathogenic microorganisms, the construction of fingerprint database, and the detection of genetic variation. At present, in microorganisms, there is no report on MNP markers, and corresponding technologies are also lacking. Therefore, the present invention develops an MNP marker site of Staphylococcus aureus, which is screened on the Staphylococcus aureus genome to distinguish it from other species and has multiple nucleotide polymorphisms within the species. The genomic region includes marker sites of MNP-1 to MNP-15 with CP000253 as the reference genome.

Next, the present invention develops a multiplex PCR primer composition for detecting the MNP marker site of Staphylococcus aureus, the multiplex PCR primer composition includes 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are as shown in SEQ ID NO.1 to SEQ ID NO.30. The primers do not conflict with each other and can be amplified efficiently by multiplex PCR.

The multiplex PCR primer composition can be used as a detection kit for detecting the MNP marker site of the Staphylococcus aureus.

The kit of the present invention can accurately and sensitively detect Staphylococcus aureus as low as 10 copies/reaction.

The MNP marker of the present invention and the kit have high specificity in detecting Staphylococcus aureus in a complex template.

The MNP marker site, primer composition, kit and application of a Staphylococcus aureus of the present application will be described in detail below with reference to the examples, comparative examples and experimental data.

Example 1. Screening of Staphylococcus aureus MNP marker sites and design of multiple PCR amplification primers

S1. Screening of MNP marker sites of Staphylococcus aureus

Based on the complete or partial genome sequences of 10,864 different isolates of Staphylococcus aureus published on the Internet, 15 MNP marker sites were obtained through sequence alignment. For species for which there is no genome data online, the genome sequence information of the representative race of the microbial species to be detected can also be obtained by high-throughput sequencing, where the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure the polymorphism of the selected marker, the genome sequences of at least 10 genetically representative isolates are generally used as a reference. The 15 MNP marker sites screened are shown in Table 1:

Table 1 - The MNP marker site and the starting position of the detection primer on the reference sequence

The step S1 specifically includes:

Selecting the genome sequence of a representative strain of the Staphylococcus aureus as the reference genome, and aligning the genome sequence with the reference genome to obtain the single nucleic acid polymorphism sites of each strain of the Staphylococcus aureus ;

On the reference genome, a window of 100-300 bp is used as a window and a step size of 1 bp is used to perform window translation, and a plurality of candidate MNP site regions are obtained by screening, wherein the candidate MNP site region contains ≥ 2 of the single nuclei A nucleotide variation site, and the single nucleic acid polymorphism site does not exist on the 30bp sequence at both ends;

In the candidate polynucleotide polymorphism site region, the region with the discrimination degree DP≥0.2 is selected as the MNP marker site; wherein, DP=d/t, t is in the candidate polynucleotide polymorphism site The comparison log of all races in the region when pairwise compared, d is the sample log of the difference of at least two single nucleic acid polymorphisms in the candidate polynucleotide polymorphism site region.

As an optional embodiment, other step sizes can also be selected when screening with a window of 100-300 bp on the reference genome. In this embodiment, a step size of 1 bp is used, which is conducive to comprehensive screening.

S2. Design of multiplex PCR amplification primers

The multiplex PCR amplification primers of the MNP site are designed by primer design software. The primer design follows that the primers do not interfere with each other. All primers can be combined into a primer pool for multiplex PCR amplification, that is, all designed primers can be used in one amplification reaction. normal amplification.

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

S3. Evaluation of detection efficiency of primer combinations

The MNP marker detection method is to amplify all MNP sites at one time by multiple PCR, sequence the amplified products by second-generation high-throughput sequencing, analyze the sequencing data, and evaluate the detected sites according to the detected sites. Compatibility of primer combinations described.

Use Staphylococcus aureus DNA with a known copy number, add it to human genomic DNA, prepare a template of 1000 copies/reaction, use the described primer combination, and screen according to the detection of MNP sites in the 4 libraries Amplify the primer combination with uniform and optimal compatibility, and finally screen out the primer combination of the 15 MNP sites described in Table 1 of the present invention.

Threshold setting and performance evaluation of MNP sites and primers described in Example 2 to identify Staphylococcus aureus

1. Detection of MNP markers

In this example, a Staphylococcus aureus nucleic acid standard with a known copy number was added to human genomic DNA to prepare 1 copy/reaction, 10 copies/reaction and 100 copies/reaction mock samples of Staphylococcus aureus. At the same time, an equal volume of sterile water was set as a blank control. A total of 4 samples were collected, and 3 replicate libraries were constructed for each sample every day, and were continuously detected for 4 days, that is, 12 sets of sequencing data were obtained for each sample, as shown in Table 2. The reproducibility, accuracy and sensitivity of the detection method were evaluated according to the number of sequencing fragments and loci of S. aureus MNP loci detected in the blank control and S. aureus nucleic acid standard in 12 replicate experiments , to formulate the thresholds for contamination of the quality control system and detection of target pathogens. The detection flow of MNP labeling is shown in Figure 3.

Table 2-Detection sensitivity and stability analysis of MNP labeling method of Staphylococcus aureus

As shown in Table 2, the kit can stably detect more than 8 MNP sites in samples with 10 copies/reaction, while at most 1 MNP site can be detected in samples with 0 copies/reaction. The kit can clearly distinguish samples with 10 copies/reaction and 0 copies/reaction, with technical stability and detection sensitivity as low as 10 copies/reaction.

2. Evaluation of the reproducibility and accuracy of the MNP labeling detection kit for the detection of Staphylococcus aureus

Based on whether the genotypes of the co-detected loci were reproducible in the two replicates, the reproducibility and accuracy of the MNP marker detection method in the detection of S. aureus were evaluated. Specifically, 12 sets of data of 100-copy samples were compared in pairs, and the results are shown in Table 3.

Table 3 - Evaluation of reproducibility and accuracy of S. aureus MNP marker detection method

It can be seen from Table 3 that the number of MNP loci with differences in main genotypes is 0; according to the principle of reproducible genotypes between two repeated experiments, the accuracy rate a=1-(1-r)/2 =0.5+0.5r, r represents the reproducibility, that is, the ratio of the number of reproducible loci of the main genotype to the number of common loci. In the reproducibility test of this project, the logarithm of the difference of the major genotypes of MNP markers between different libraries and different building batches of each sample is 0, the reproducibility rate is r=100%, and the accuracy rate is a=100%.

3. Threshold determination of Staphylococcus aureus detected by MNP labeling detection kit

As shown in Table 2, the sequence aligned to Staphylococcus aureus can be detected in 1 copy/reaction sample, covering at least 1 MNP site. The sequences of Staphylococcus aureus were also detected in some blank controls. Due to the extreme sensitivity of the MNP marker detection method, data contamination during detection can easily lead to false positives. Therefore, a quality control plan is formulated in this example, as follows:

1) The amount of sequencing data is greater than 4.5 million bases. The calculation is based on the fact that the number of MNP sites detected in 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 be guaranteed to cover each position in one experiment. The number of sequencing fragments of the site reaches 1000 times, ensuring the accurate analysis of the base sequence of each MNP site.

2) Determine whether the pollution is acceptable according to the signal index S of Staphylococcus aureus in the test sample and the noise index P of Staphylococcus aureus in the blank control, wherein:

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

The signal index of the test sample is S=nt/Nt, where nt and Nt represent the number of sequencing fragments and the total number of sequencing fragments of Staphylococcus aureus in the test sample, respectively.

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

Table 4 - Signal-to-Noise Ratio of Staphylococcus aureus in Test Samples

The results are shown in Table 4. The average noise index of Staphylococcus aureus in the blank control was 0.04%, while the average signal index in the 1-copy sample was 0.19%. The signal of the 1-copy sample and the blank control The average value of the noise ratio is 5.2, therefore, the present invention stipulates that when the signal-to-noise ratio is greater than 10 times, it can be determined that the pollution in the detection system is acceptable. The average signal-to-noise ratio between the 10-copy sample and the blank control was 81.6. In the 12 sets of data with 10 copies/reaction, at least 8 MNP sites were stably detected, accounting for 53.3% of the total sites. Therefore, under the condition of ensuring the accuracy, this standard stipulates that the signal-to-noise ratio judgment threshold of Staphylococcus aureus is 40, that is, when the signal-to-noise ratio of Staphylococcus aureus in the sample is greater than 40, and the locus detection rate is greater than or equal to 30% , it was determined that the nucleic acid of Staphylococcus aureus was detected in the sample.

Therefore, the kit provided by the present invention can accurately and sensitively detect as low as 10 copies/reaction of Staphylococcus aureus.

4. Evaluation of the specificity of MNP marker detection method for the detection of Staphylococcus aureus

Staphylococcus aureus and Mycobacterium tuberculosis, Acinetobacter strains, Bordetella pertussis, Bordetella hordeii, 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, Streptococcus pneumoniae, Streptococcus pyogenes The DNAs were mixed together in equimolar amounts to prepare a mixed template, and the blank template was used as a control to detect Staphylococcus aureus in the mixed template by the method provided by the present invention. Three repeated experiments were carried out, and after analysis according to the described quality control scheme and the judgment threshold, only 15 MNP sites of the Staphylococcus aureus could be specifically detected in the three repeated experiments, and the signal-to-noise ratios were sequentially 673.4, 752.6 and 634.8 were reached, indicating the high specificity of MNP labeling and the kit to detect target microorganisms in complex templates.

Example 3. Detection of genetic variation among Staphylococcus aureus strains

Six progeny strains of a Staphylococcus aureus strain kept in the same laboratory were detected using the kit and the MNP marker site detection method. The samples were named S1-S6 in turn, and the average coverage of each sample was 1416 times, all 15 MNP markers could be detected in each strain (Table 5). The fingerprints of the 6 strains were compared in pairs, and the results are shown in Table 5. There are 1 (S-2) and 5 Staphylococcus aureus tested in the same batch. There are major genotypes of some sites. Differences (Table 5), there is inter-strain variation.

Table 5-6 Detection and Analysis of Staphylococcus aureus

As can be seen from Table 5, the test kit of the present invention can be used to ensure the genetic consistency of the same named Staphylococcus aureus strains in different laboratories by detecting the application of the MNP marker to identify the genetic variation between strains, thereby ensuring the comparability of the research results. It is of great significance for the scientific research of Staphylococcus aureus. In clinical practice, the diagnostic scheme can be considered according to whether the differential loci affects drug resistance.

Example 4. Detection of genetic variation within Staphylococcus aureus strains

As a colony organism, some individuals in the Staphylococcus aureus colony mutate, so that the colony is no longer homozygous, forming a heterogeneous heterozygous colony, which affects especially the stability and consistency of the phenotype of the microorganisms used in the test. This variant is shown as an allelic genotype other than the main genotype of the locus when molecular marker detection is performed on the population. When the variant individuals have not accumulated yet, they only account for a very small part of the population, showing low-frequency alleles. Low-frequency alleles are often mixed with technical errors, making them indistinguishable with existing techniques. What the present invention detects is the MNP marker with high polymorphism. The technical error rate of MNP markers is significantly lower than that of SNP markers, based on the fact that the probability of multiple errors occurring simultaneously is lower than the probability of one error occurring.

The authenticity assessment of the sub-alleles in this example is carried out as follows: First, the alleles with strand preference (the ratio of the number of sequenced sequences covered on the DNA duplex) are excluded according to the following rules: the strand preference is greater than 10 fold, or the difference in strand preference from the major allele is greater than 5-fold.

Genotypes without strand bias were judged for authenticity based on the number and proportion of sequenced sequences in Table 6. Table 6 lists the calculation based on the _BINOM.INV function under the probability guarantee of α=99.9999%, when emax (n=1) and emax (n≥2) are 1.03% and _0.0994 %, respectively, in each site The critical value of the number of sequencing sequences of the sub-allelic type, only when the number of sequencing sequences of the sub-allelic type exceeds the critical value is determined as the true sub-allelic type. When there are multiple candidate suballeles, multiple corrections are performed on the P values of each candidate allele, and candidate alleles with FDR<0.5% are judged to be true suballeles.

The parameters e _max (n=1) and e _max (n≧2) involved in Table 6 refer to the highest proportion of the number of sequenced sequences carrying the wrong alleles of n SNPs to the total number of sequenced sequences at the site. The _emax (n=1) and emax (n≧2) of 1.03% and _0.0994 %, respectively, were obtained from the frequencies of all minor genotypes detected at 930 homozygous MNP loci.

Table 6 - Critical values for determining minor alleles at partial sequencing depths

According to the above parameters, the nucleic acids of the two strains with different genotypes shown in Table 5 were divided into the following eight ratios: 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/ 100, 7/100 mixed, artificial heterozygous samples were prepared, each sample was tested 3 times, and a total of 24 sequencing data were obtained. Through accurate comparison with the genotypes of the MNP loci of the two strains, loci with heterozygous genotypes were detected in 24 artificial heterozygous samples, indicating the developed MNP marker detection of Mycoplasma pneumoniae. Applicability of the method in detecting genetic variation within strain populations.

Example 5. Construction of Staphylococcus aureus DNA fingerprint database

The DNA of all strains or samples used to construct the Staphylococcus aureus DNA fingerprint database was extracted by conventional CTAB method, commercial kits and other methods, and the quality of DNA was detected by agarose gel and UV spectrophotometer. If the ratio of the absorbance values at 260 nm and 230 nm of the extracted DNA is greater than 2.0, and the ratio of the absorbance values at 260 nm and 280 nm is between 1.6 and 1.8, the main band of DNA electrophoresis is obvious, and there is no obvious degradation and RNA residues. Related quality requirements, follow-up experiments can be carried out.

After the sequence data of the above 6 strains were compared with the reference genotype, the main genotype of each locus of each strain was obtained, and the MNP fingerprint of each strain was formed. The obtained MNP fingerprint of each strain was entered into the database file to form a Staphylococcus aureus DNA fingerprint database.

The constructed MNP fingerprint database is based on the gene sequences of the detected strains, so it is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared, and can be updated at any time. Compare the MNP fingerprints of the strains obtained by each detection with the MNP fingerprint database constructed based on the existing genomic data, and enter the MNP fingerprints of the strains with major genotype differences into the constructed MNP fingerprint database to achieve the real-time database. Update and co-build and share.

Example 6. Application in fine typing of Staphylococcus aureus

The above-mentioned 6 Staphylococcus aureus strains were detected by using the primer combination and the MNP marker site detection method, and the MNP fingerprint of each strain was obtained. The DNA fingerprints of each strain are compared pairwise and compared with the constructed fingerprint database. The same as the existing fingerprint database is defined as the existing variant, and there is a major genotype difference at at least one MNP site. , defined as a new variant to achieve fine typing of Staphylococcus aureus. The detection of 6 Staphylococcus aureus samples is shown in Table 5. Among the 6 Staphylococcus aureus samples tested, 1 and the other 5 samples have differences in the main genotypes at 2 MNP loci, which may be different variants . Therefore, the resolution of the described method for Staphylococcus aureus reaches the level of single base, which can realize the fine typing of Staphylococcus aureus in the sample.

Finally, it should also be noted that the terms "comprising", "comprising" or any other variation thereof are intended to encompass a non-exclusive inclusion such that a process, method, article or device comprising a series of elements includes not only those elements, but also Also included are other elements not expressly listed or inherent to such a process, method, article or apparatus.

While preferred embodiments of the embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as 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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<212> DNA

<213> Artificial Sequence

<400> 13

cagatcgatg aattaaagta gggtgt 26

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 14

tagaagacac tgatgctatc gatca 25

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 15

agaagcaatt aagtatagtg acattcca 28

<210> 16

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 16

gcttgtattt caatcgacat taagca 26

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 17

ttaatggctt taaccacttc gtctg 25

<210> 18

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 18

agcttcagat aaagtattaa atcagca 27

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 19

caacgcagca gtatttgata aattt 25

<210> 20

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 20

tcaaatcatg acaaagattt ttacttca 28

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 21

gatacttttt cggcgtctgt ctt 23

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 22

aggtgaagaa gaattagaag aagca 25

<210> 23

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 23

aacaccttga tatgtattgc aaaattt 27

<210> 24

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 24

gttgcacccc caaatatata aagaca 26

<210> 25

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 25

tcaagattga taggggcacc tc 22

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 26

aatacattaa agacgtcagt atgcg 25

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 27

atataaagtc gatatcgtag ctggg 25

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 28

ttcatgctct cttaacatag gaact 25

<210> 29

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 29

tgtagagtct aggacattga tttctgt 27

<210> 30

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 30

accatttatt ccatactaaa caaaagaggt 30

Claims (8)

1. a MNP marker site of staphylococcus aureus, it is characterised in that the MNP marker site is to be screened on the staphylococcus aureus genome to be distinguished from other species and have multiple nucleotide polymorphisms in species Sexual genomic regions, including the marker sites of MNP-1 to MNP-15 with CP000253 as the reference genome. 2. a multiplex PCR primer composition for detecting the described Staphylococcus aureus MNP marker site of claim 1, it is characterized in that, described multiplex PCR primer composition comprises 15 pairs of primers, the core of described 15 pairs of primers The nucleotide sequences are shown in SEQ ID NO.1 to SEQ ID NO.30. 3 . A detection kit for detecting the MNP marker site of Staphylococcus aureus according to claim 1 , wherein the kit comprises the primer composition according to claim 2 . 4 . 4. The detection kit according to claim 3, wherein the kit further comprises a multiplex PCR premix. 5. the identification of the MNP marker site of a staphylococcus aureus of claim 1 or the primer composition described in claim 2 or the arbitrary described detection kit of claim 3-4 in staphylococcus aureus and application in the preparation of identification products of Staphylococcus aureus. 6. a MNP marker site of staphylococcus aureus according to claim 1 or primer composition according to claim 2 or the detection kit described in any one of claims 3-4 is detecting staphylococcus aureus bacterial strain Applications of intra- and inter-strain genetic variation. 7. a MNP marker site of staphylococcus aureus according to claim 1 or the primer composition according to claim 2 or the detection kit described in any one of claims 3-4 is building staphylococcus aureus database applications in . 8. a MNP marker site of staphylococcus aureus according to claim 1 or primer composition according to claim 2 or the detection kit described in any one of claims 3-4 in staphylococcus aureus fine fraction. application in type detection.

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