CN114790488B - MNP (MNP) marking site of staphylococcus aureus, primer composition, kit and application of MNP marking site - Google Patents

Abstract

The invention discloses an MNP (MNP) marking site, a primer composition, a kit and application thereof, wherein the MNP marking site refers to a genome region which is screened on a staphylococcus aureus genome and is separated from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marking sites of MNP-1-MNP-15; the primer is shown as SEQ ID NO.1-SEQ ID NO. 30. The MNP marker loci can specifically identify staphylococcus aureus 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 performed on all the marker loci of multiple samples at one time, the method has the detection advantages of high flux, multiple targets, high sensitivity, high precision and culture free, can be applied to identification and genetic variation detection of staphylococcus aureus of large-scale samples, and has important significance on scientific research and sanitary monitoring of staphylococcus aureus.

Description

MNP marking site, primer composition, test kit and method for Staphylococcus aureus its application

Technical field

The embodiments of the present invention relate to the field of biotechnology, and in particular to an MNP labeling site, primer composition, kit and application of Staphylococcus aureus.

Background technique

Staphylococcus aureus belongs to the genus Staphylococcus and is a representative of Gram-positive bacteria. It is a common food-borne pathogenic microorganism that often parasitizes the skin, nasal cavity, throat, intestines and stomach, and carbuncles of humans and animals. , purulent sores, air, sewage and other environments are also everywhere. Under appropriate conditions, Staphylococcus aureus can produce enterotoxin and cause food poisoning. In recent years, reports of food poisoning caused by Staphylococcus aureus have emerged one after another. Food poisoning caused by Staphylococcus aureus accounts for about 25% of food-borne microbial food poisoning incidents. Staphylococcus aureus has become second only to Salmonella and Bacillus parahaemolyticus. The third most common microbial pathogenic bacteria. In addition, Staphylococcus aureus is also a commonly used model pathogenic microorganism in laboratory research. As a group organism, in its interaction with the host and the environment, individuals within the group will mutate, leading to the failure of detection methods or treatment methods; for laboratory research, this kind of imperceptible mutation will lead to different experiments. The strains with the same name in different laboratories or in the same laboratory are actually not the same, resulting in irreproducible and incomparable experimental results. The heterogeneity among human Hella cell laboratories has led to a large number of incomparable experimental results and data waste. Therefore, the development of a fast, accurate, and variation-monitorable Staphylococcus aureus detection and analysis method is of great significance for the scientific research and application of Staphylococcus aureus.

Classic Staphylococcus aureus detection methods, including isolation and culture, PCR technology, whole genome and metagenomic sequencing, etc., have one or more limitations in terms of time duration, operational complexity, detection throughput, accuracy and sensitivity of detection variation, and cost. limitations. Targeted molecular marker detection technology that combines 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, it has the advantages of requiring less samples, accurate diagnostic results, saving data volume, and detecting low-frequency variations. 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, making them dimorphic markers. The polymorphism of a single SNP marker is not enough to capture potential alleles in microbial populations. Genetic diversity.

Therefore, the development of new highly polymorphic molecular markers and culture-free detection technology for Staphylococcus aureus has become an urgent technical issue to be solved.

Contents of the invention

The purpose of the present invention is to provide a Staphylococcus aureus-specific MNP labeling site, primer composition, kit and application thereof, which can carry out qualitative identification and mutation detection of Staphylococcus aureus, and has the characteristics of multi-target, high-throughput, Highly sensitive and fine typing effect.

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

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

In the above technical solution, the labeling sites of MNP-1 to MNP-15 are specifically shown in Table 1 of the instruction manual. The starting and ending positions of the MNP labeling marked in Table 1 are based on the reference corresponding to the same row of MNP in Table 1. The 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 such as SEQ ID NO.1 -SEQ ID NO.30.

In the above technical solution, the primers for each MNP labeling site include upper primers and lower primers, as shown in Table 1 of the instruction manual.

In a third aspect of the present invention, a detection kit for detecting the Staphylococcus aureus MNP labeling site is provided, and the kit includes the primer composition.

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

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

In the fifth aspect of the present invention, the MNP marker site of Staphylococcus aureus or the multiplex PCR primer composition or the detection kit is provided for detecting genetics within and between strains of Staphylococcus aureus. Application in variation.

In the sixth aspect of the present invention, 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 is provided.

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 fine typing detection of Staphylococcus aureus. .

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

The first step is to 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, with the number of cycles not higher than 25; after purifying the amplification product, Add sample tags and second-generation sequencing adapters based on the second round of PCR amplification; purify and quantify the second-round amplification products; when detecting multiple strains, mix the second-round amplification products in equal amounts and perform high-throughput Sequencing; the sequencing results are compared to the reference sequence of Staphylococcus aureus to obtain the number of detected sequences and genotype data in the total DNA. According to the number of Staphylococcus aureus sequencing sequences and the number of detected MNP sites obtained in the total DNA and the blank control, perform data quality control and data analysis on the sequencing data of the total DNA to obtain the number of detected MNP sites. The purpose is to include the number of sequencing sequences covering each MNP site and the genotype data of the MNP site.

When used for the identification of Staphylococcus aureus, based on 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, quality control is performed to determine whether the sample to be tested contains Nucleic acid of Staphylococcus aureus. Among them, the quality control plan and the determination method are based on the DNA of Staphylococcus aureus with a known copy number as the detection sample, and the sensitivity, accuracy and specificity of the kit for detecting Staphylococcus aureus are evaluated, and the described Quality control plan and determination method for the detection of Staphylococcus aureus by the kit.

When used for the detection of genetic variation in Staphylococcus aureus, it includes detection of genetic variation between strains and within strains. The detection of genetic variation between strains includes using the described kit and method to obtain genotype data of each strain to be compared at 15 MNP sites. Through genotype comparison, analyze whether there are differences in the major 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, you can also use single-plex PCR to amplify the 15 sites of the strain to be compared, and then perform Sanger sequencing on the amplified products. After obtaining the sequence, the genotype of each MNP site of the strain to be compared can be Make a comparison. If there are MNP sites with inconsistent major genotypes, there is variation between the strains to be compared. When detecting genetic variation within a strain, a statistical model is used to determine whether a 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 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 the database file to form a DNA fingerprint database of Staphylococcus aureus; each time When identifying different samples, compare it with the DNA fingerprint database of Staphylococcus aureus to identify whether the Staphylococcus aureus in the sample has a major genotype at the MNP site (at one MNP site) with the strains in the database. Staphylococcus aureus with a genotype difference supported by more than 50% of the sequencing fragments, and Staphylococcus aureus with a major genotype difference at at least 1 MNP site is a new variant type and is included in the DNA fingerprint database.

When used for Staphylococcus aureus typing, the Staphylococcus aureus in the sample to be tested is identified to obtain the genotype of each MNP site; the genome sequences of Staphylococcus aureus published online and the constructed ones are collected 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 Staphylococcus aureus, and the genetically consistent or most optimal Staphylococcus aureus reference sequence library is screened. Close strains to obtain the typing of Staphylococcus aureus in the sample to be tested. According to the comparison results with the reference sequence library, it is identified whether the Staphylococcus aureus in the sample is an existing type or a new variant, thereby achieving precise typing of Staphylococcus aureus. This invention is the first of its kind in the field of Staphylococcus aureus and has not been reported in relevant literature; MNP markers are mainly developed based on reference sequences. Based on the reported resequencing data of representative strains of Staphylococcus aureus, large-scale differentiation from other species can be mined , a polymorphic MNP site with 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; and then based on the standard Based on the test results, a set of MNP sites with the largest polymorphism and high specificity, the most compatible primer combination and 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 labeling site of Staphylococcus aureus, a primer composition, a test kit and an application thereof. The provided 15 MNP sites of Staphylococcus aureus and their primer combinations can be used for multiplex PCR amplification and integrated with the second-generation sequencing platform to sequence the amplified products to meet the needs of high-throughput and high-efficiency analysis of Staphylococcus aureus. , the need for high-accuracy and high-sensitivity detection to meet the requirements for standard and shareable fingerprint data construction of Staphylococcus aureus; the need for accurate detection of genetic variation among Staphylococcus aureus strains; identification of homozygous and heterozygous Staphylococcus aureus meet the needs and provide technical support for scientific research and scientific monitoring of Staphylococcus aureus.

Description of the drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, a brief introduction will be made below to the drawings needed 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 be obtained based on these drawings without exerting creative efforts.

Figure 1 is a schematic diagram of MNP marker polymorphism;

Figure 2 is a flow chart for screening and primer design of Staphylococcus aureus MNP marker sites;

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

Detailed ways

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

Throughout this specification, unless otherwise specifically stated, the terms used herein are to be understood as having the meaning 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 embodiments of the invention belong. If there is any conflict, this manual takes precedence.

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

The technical solutions of the embodiments of this application are to solve the above technical problems. The general idea is as follows:

The present invention develops a new species-specific molecular marker - MNP marker, which is suitable for detecting group organisms. MNP markers refer to polymorphic markers caused by multiple nucleotides within a region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) Alleles are abundant, with 2 ⁿ alleles on a single MNP site, which is higher than SSR and SNP; (2) Species discrimination ability is strong, Only a small amount of MNP markers are needed to achieve species identification, reducing detection error rates. The MNP labeling method based on ultra-multiplex PCR combined with second-generation high-throughput sequencing technology to detect MNP markers has the following advantages: (1) The output is the base sequence, without the need for parallel experiments, and a standardized database can be constructed for sharing; (2) High Efficiency, using sample DNA barcodes to break through the limitation of the number of sequencing samples, and can type tens of thousands of MNP sites in hundreds or thousands of samples at one time; (3) High sensitivity, using multiplex PCR to detect multiple targets at one time, avoiding Failure in amplification of a single target results in high false negatives and low sensitivity; (4) High accuracy, using second-generation high-throughput sequencers to sequence the amplification products hundreds of times.

In view of the above advantages and characteristics, MNP markers and their detection technology, MNP marker method, can realize the classification and traceability of multi-allelic types of population organisms, and have application potential in the identification of pathogenic microorganisms, construction of fingerprint databases, and detection of genetic variation. Currently, there are no reports on MNP labeling in microorganisms, and there is a lack of corresponding technology. Therefore, the present invention develops MNP marker sites for Staphylococcus aureus, which are screened on the Staphylococcus aureus genome and are distinguished from other species and have multiple nucleotide polymorphisms within the species. The genomic region includes the marker sites of MNP-1 to MNP-15 using CP000253 as the reference genome.

Next, the present invention developed a multiplex PCR primer composition for detecting the Staphylococcus aureus MNP marker site. The multiplex PCR primer composition included 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers were such as SEQ. ID NO.1～SEQ ID NO.30 are shown. The primers do not conflict with each other and can perform efficient amplification through multiplex PCR.

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

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

The MNP label and the kit of the present invention have high specificity in detecting Staphylococcus aureus in complex templates.

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

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

S1. Screening of Staphylococcus aureus MNP marker sites

Based on the complete or partial genome sequences of 10,864 different Staphylococcus aureus isolates published online, 15 MNP marker sites were obtained through sequence alignment. For species for which genome data do not exist online, the genome sequence information of representative races of the microbial species to be detected can also be obtained through high-throughput sequencing, where high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure the polymorphism of the screened markers, the genome sequences of at least 10 genetically representative isolates are generally used as references. The 15 screened MNP marker sites are shown in Table 1:

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

The step S1 specifically includes:

The genome sequence of a representative strain of Staphylococcus aureus is selected as a reference genome, and the genome sequence and the reference genome are compared to obtain single nucleic acid polymorphism sites of each strain of Staphylococcus aureus. ;

On the reference genome, use 100 to 300 bp as a window and 1 bp as a step to perform window translation and screen to obtain multiple candidate MNP site regions, wherein the candidate MNP site region contains ≥ 2 of the single nuclei The nucleotide variation site, and the single nucleic acid polymorphism site does not exist in the sequence of 30 bp at each end;

In the candidate polynucleotide polymorphism site region, a region with a discrimination DP≥0.2 is selected as an MNP marker site; where, DP=d/t, t is at the candidate polynucleotide polymorphism site The number of comparison logs when comparing all races in the region pairwise, d is the number of sample logs with differences in at least two single nucleic acid polymorphisms in the candidate polynucleotide polymorphism site region.

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

S2. Design of multiplex PCR amplification primers

Use primer design software to design multiplex PCR amplification primers for the MNP site. Primer design follows the principle that 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. All of them amplified normally.

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

S3. Evaluation of detection efficiency of primer combinations

The detection method of MNP markers is to amplify all MNP sites at once through multiplex PCR, sequence the amplification products through second-generation high-throughput sequencing, analyze the sequencing data, and evaluate the results based on the detected sites. Compatibility of the primer combinations described.

Use Staphylococcus aureus DNA with a known copy number and add it to human genomic DNA to prepare a template of 1000 copies/reaction. Use the primer combination to screen based on the detection of MNP sites in 4 libraries. A primer combination with uniform amplification and optimal compatibility was finally screened out for the 15 MNP site primer combinations described in Table 1 of the present invention.

Threshold setting and performance evaluation of MNP sites and primers for identification of Staphylococcus aureus described in Example 2

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 Staphylococcus aureus simulated samples of 1 copy/reaction, 10 copies/reaction and 100 copies/reaction. An equal volume of sterile water was set at the same time as a blank control. There were a total of 4 samples, and 3 duplicate libraries were constructed for each sample every day and tested continuously for 4 days, that is, 12 sets of sequencing data were obtained for each sample, as shown in Table 2. Based on the number of sequencing fragments and sites of Staphylococcus aureus MNP sites detected in the blank control and Staphylococcus aureus nucleic acid standards in 12 repeated experiments, the reproducibility, accuracy, and sensitivity of the detection method were evaluated. , establish thresholds for quality control system contamination and target pathogen detection. The detection process of MNP labeling is shown in Figure 3.

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

As shown in Table 2, the kit can stably detect more than 8 MNP sites in a sample of 10 copies/reaction, and can detect up to 1 MNP site in a sample of 0 copies/reaction. The kit can clearly differentiate between samples with 10 copies/reaction and 0 copies/reaction, with technical stability and detection sensitivity as low as 10 copies/reaction.

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

Based on whether the genotypes of the commonly detected sites are reproducible in two repetitions, the reproducibility and accuracy of the MNP marker detection method in detecting Staphylococcus aureus were evaluated. Specifically, pairwise comparisons were performed on 12 sets of data of 100 copies of samples, and the results are shown in Table 3.

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

As can be seen from Table 3, the number of MNP sites with differences in major genotypes is all 0; based on the principle that reproducible genotypes between two repeated experiments are considered accurate, the accuracy rate a=1-(1-r)/2 =0.5+0.5r, r represents the recurrence rate, that is, the ratio of the number of reproducible sites of the major genotype to the number of shared sites. In the reproducibility test of this project, the logarithm of the difference in MNP marker major genotypes between different libraries and different library construction batches for each sample was 0, the reproducibility rate r=100%, and the accuracy rate a=100%.

3. Determination of threshold value for detecting Staphylococcus aureus by MNP labeling detection kit

As shown in Table 2, sequences aligned to Staphylococcus aureus can be detected in 1 copy/reaction sample, covering at least 1 MNP site. Staphylococcus aureus sequences were also detected in some blank controls. Because the MNP labeling detection method is extremely sensitive, data contamination during detection can easily lead to false positives. Therefore, a quality control plan is developed 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 detected MNP sites for each sample is 15, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 4.5 million bases, most samples can be guaranteed to cover every site in one experiment. The number of sequencing fragments at each point reaches 1,000 times, ensuring accurate analysis of the base sequence of each MNP site.

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

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

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

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

Table 4 - Signal-to-noise ratio of Staphylococcus aureus in samples to be tested

The results are shown in Table 4. The average noise index of Staphylococcus aureus in the blank control is 0.04%, while the average signal index in the 1-copy sample is 0.19%. The signal between 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 of 10 copies of the sample and blank control was 81.6. In 12 sets of data of 10 copies/reaction, at least 8 MNP sites could be stably detected, accounting for 53.3% of the total sites. Therefore, while ensuring accuracy, this standard stipulates that the signal-to-noise ratio judgment threshold for Staphylococcus aureus is 40, that is, when the signal-to-noise ratio of Staphylococcus aureus in the sample is greater than 40, and the site 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 Staphylococcus aureus as low as 10 copies/reaction.

4. Specificity evaluation of MNP labeling detection method for detecting Staphylococcus aureus

Artificially combine Staphylococcus aureus and Mycobacterium tuberculosis, Acinetobacter strains, Bordetella pertussis, Bordetella hossenii, Chlamydia pneumoniae, Mycoplasma pneumoniae, Epstein-Barr virus, Haemophilus influenzae, varicella zoster virus, Cytomegalovirus, herpes simplex virus, human bocavirus, Klebsiella pneumoniae, Legionella spp., Moraxella catarrhalis, Pseudomonas aeruginosa, Rickettsia spp., Streptococcus pneumoniae, Streptococcus pyogenes DNA is mixed together in equimolar amounts to prepare a mixed template, and the blank template is used as a control, and the method provided by the invention is used to detect Staphylococcus aureus in the mixed template. Three repeated experiments were conducted, and after analysis according to the described quality control scheme and judgment threshold, only 15 MNP sites of Staphylococcus aureus could be specifically detected in the three repeated experiments, and the signal-to-noise ratios were in order. reached 673.4, 752.6 and 634.8, indicating the high specificity of MNP labeling and the kit in detecting target microorganisms in complex templates.

Example 3. Detection of genetic variation among Staphylococcus aureus strains

The described kit and MNP marker site detection method were used to detect 6 progeny strains of a Staphylococcus aureus strain stored in the same laboratory. The samples were named S1-S6 in sequence. The average sequencing coverage of each sample reached 1416 times, all 15 MNP markers can be detected in each strain (Table 5). The fingerprints of the 6 strains were compared in pairs. The results are shown in Table 5. One sample (S-2) and 5 samples of Staphylococcus aureus tested together with the same batch all had major genotypes at 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 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 MNP markers to identify genetic variations between strains, thereby ensuring the comparability of research results. This The scientific research on Staphylococcus aureus is of great significance. In clinical practice, diagnostic solutions can be considered based on whether different sites affect drug resistance.

Example 4. Detection of genetic variation within Staphylococcus aureus strains

As a group organism, some individuals within the Staphylococcus aureus group mutate, causing the group to no longer be homozygous and form a heterogeneous heterozygous group, which affects especially the stability and consistency of the phenotype of the experimental microorganisms. This variant appears as an allele type other than the main genotype of the locus when molecular markers are tested on the population. When mutated individuals have not yet accumulated, they only account for a very small part of the population and exhibit low frequency alleles. Low-frequency allele types are often mixed with technical errors, making them difficult to distinguish with current 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 authenticity assessment of minor allelic types in this example is performed as follows: First, allelic types with strand preference (the ratio of the number of sequencing sequences covered on the DNA double strand) are excluded according to the following rules: strand preference is greater than 10 times, or the strand preference difference from the major allele type is greater than 5 times.

The authenticity of genotypes without strand preference is determined based on the number and proportion of sequencing sequences in Table 6. Table 6 lists the calculation based on the BINOM.INV function under the probability guarantee of α=99.9999%, e _max (n=1) and e _max (n≥2) are 1.03% and 0.0994% respectively, in each site The critical value of the number of sequencing sequences of the minor allele type. Only when the number of sequencing sequences of the minor allele type exceeds the critical value, it is determined to be the true minor allele type. When there are multiple candidate minor alleles, multiple corrections are performed on the P value of each candidate allele type, and the candidate allele with FDR <0.5% is judged to be the true minor allele type.

The parameters e _max (n=1) and e _max (n≥2) involved in Table 6 refer to the highest proportion of the number of sequencing sequences carrying the wrong allele of n SNPs to the total number of sequencing sequences at the site. e _max (n=1) and e _max (n≥2) of 1.03% and 0.0994%, respectively, were obtained based on the frequencies of all minor allele types detected at 930 homozygous MNP loci.

Table 6-Critical values for determining minor allele types under partial sequencing depth

According to the above parameters, the nucleic acids of the two strains with different genotypes shown in Table 5 are divided into the following eight ratios: 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/ 100, 7/100 were mixed to prepare artificial hybrid samples. Each sample was tested three times and repeated to obtain a total of 24 sequencing data. Through accurate comparison with the genotypes of the MNP sites of the two strains, the sites with hybrid genotypes were detected in 24 artificial hybrid samples, which illustrates the developed MNP marker detection of Mycoplasma pneumoniae. Applicability of the method to detect genetic variation within a strain population.

Example 5. Construction of Staphylococcus aureus DNA fingerprint database

Use conventional CTAB methods, commercial kits and other methods to extract the DNA of all strains or samples used to construct the Staphylococcus aureus DNA fingerprint database, and use agarose gel and UV spectrophotometer to detect the quality of the DNA. If the ratio of the absorbance values of the extracted DNA at 260nm and 230nm is greater than 2.0, the ratio of the absorbance values 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 has reached Relevant quality requirements can be followed by subsequent experiments.

After comparing the sequencing data of the above six strains with the reference genotypes, the major genotype of each site of each strain was obtained, and the MNP fingerprint of each strain was formed. The obtained MNP fingerprint of each strain was entered into a database file to form a Staphylococcus aureus DNA fingerprint database.

The MNP fingerprint database constructed is based on the genetic sequence of the tested strain, so it is compatible with all high-throughput sequencing data and has the characteristics of being completely co-constructed and shared and can be updated at any time. The MNP fingerprints of the strains obtained in each test are compared with the MNP fingerprint database constructed based on existing genome data, and the MNP fingerprints of strains with different major genotypes are entered into the constructed MNP fingerprint database to achieve real-time access to the database. Update and co-build sharing.

Example 6. Application in fine typing of Staphylococcus aureus

The above-mentioned 6 Staphylococcus aureus strains were detected using the primer combination and MNP labeling 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. If they are the same as the existing fingerprint database, they are defined as existing variants and there is a major genotypic difference at at least one MNP site. , defined as a new variant, enabling fine typing of Staphylococcus aureus. The detection of 6 Staphylococcus aureus samples is shown in Table 5. One of the 6 Staphylococcus aureus samples detected and the other 5 samples have differences in the major genotypes of the two MNP sites, which may be different variants. . Therefore, the method's resolution for Staphylococcus aureus reaches the single-base level, and can achieve precise typing of Staphylococcus aureus in the sample.

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

Although preferred embodiments of the embodiments of the present invention have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once the basic inventive concepts are apparent. Therefore, it is intended that the appended claims be construed to include the preferred embodiments and all changes and modifications that fall within the scope of embodiments of the 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. In this way, 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 equivalent technologies, then the embodiments of the present invention are also intended to include these modifications and variations.

sequence list

<110> Jianghan University

<120> A MNP labeling site, primer composition, kit and application of Staphylococcus aureus

<160> 30

<170> SIPOSequenceListing 1.0

<210> 1

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 1

ggatacaact attacgccat ttcaga 26

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 2

ttatgatcac gacgaacatg ttcca 25

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 3

tcaaaattaa agttctacat tggtgatg 28

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 4

atatttgtct tcactgcctc aactg 25

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 5

cgaaactatt gctaaagtcc tggaa 25

<210> 6

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 6

tgtgttgatt ttccattata aacgtc 26

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 7

tcaagtgttt caatattttc aacagca 27

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 8

tcaaccattt gaagattgtt gtaca 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 9

cagcttctct aatttcttct gccac 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 10

tggacatgtt cttgactttg aagtg 25

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 11

tgcgtgataa aaacgataat gccat 25

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 12

aatgaaacat cacgtaacat ttgatac 27

<210> 13

<211> 26

<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 (7)

1. A multiplex PCR primer composition for detecting Staphylococcus aureus MNP marker sites, characterized in that the multiplex PCR primer composition includes 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are such as SEQ ID NO.1 ~ SEQ IDNO.30 are shown; the MNP marker site is a genomic region screened on the Staphylococcus aureus genome that is different from other species and has multiple nucleotide polymorphisms within the species, including CP000253 was used as the marker sites of MNP-1 to MNP-15 in the reference genome. 2. A detection kit for detecting Staphylococcus aureus MNP marking sites, characterized in that the kit includes the primer composition of claim 1. 3. The detection kit according to claim 2, characterized in that the kit further includes a multiplex PCR master mix. 4. Application of the primer composition according to claim 1 or the detection kit according to any one of claims 2 to 3 in the non-diagnostic identification of Staphylococcus aureus and the preparation of Staphylococcus aureus identification products. 5. Application of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 in detecting genetic variation within and between strains of Staphylococcus aureus for non-diagnostic purposes. 6. Application of the primer composition according to claim 1 or the detection kit according to any one of claims 2 to 3 in constructing a Staphylococcus aureus database. 7. Application of the primer composition according to claim 1 or the detection kit according to any one of claims 2 to 3 in the non-diagnostic fine typing detection of Staphylococcus aureus.