CN114790488A - MNP (MNP) marker locus of staphylococcus aureus, primer composition, kit and application thereof - Google Patents
MNP (MNP) marker locus of staphylococcus aureus, primer composition, kit and application thereof Download PDFInfo
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Abstract
The invention discloses an MNP marker locus of staphylococcus aureus, a primer composition, a kit and application thereof, wherein the MNP marker locus refers to a genome region which is screened on a staphylococcus aureus genome, is distinguished 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 staphylococcus aureus and finely distinguish different races; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that sequence analysis can be performed on all marked sites of multiple samples at one time, the method has the detection advantages of high throughput, multiple targets, high sensitivity, high accuracy and no culture, 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
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to an MNP (protein) marker locus of staphylococcus aureus, a primer composition, a kit and application thereof.
Background
Staphylococcus aureus (Staphylococcus aureus) belongs to the genus Staphylococcus, is a gram-positive bacterium representative, is a common food-borne pathogenic microorganism, and is commonly parasitic on skin, nasal cavity, throat, intestines and stomach, carbuncle, suppurative sore mouth of human and animals, and is ubiquitous in air, sewage and other environments. Under appropriate conditions, staphylococcus aureus can produce enterotoxin, causing food poisoning. In recent years, food poisoning caused by staphylococcus aureus is reported to be endless, the food poisoning caused by staphylococcus aureus accounts for about 25% of food-borne microbial food poisoning events, and staphylococcus aureus becomes the third microbial pathogenic bacteria second only to salmonella and parachromobacter. In addition, staphylococcus aureus is also a common model pathogenic microorganism for laboratories to study. When the gene is used as a group organism, individuals in the group can generate variation in the interaction with a host and the environment, so that the detection method or the treatment method is invalid; for laboratory studies, such inconspicuous variations can result in different laboratories or the same laboratory differing in the fact that the same named strains are not identical at different times, leading to non-reproducible and non-comparable experimental results. Heterogeneity between human Hella cell laboratories has resulted in a large number of incomparable experimental results and wasted data. Therefore, the development of a rapid and accurate staphylococcus aureus detection and analysis method capable of monitoring variation is of great significance to scientific research and application of staphylococcus aureus.
The classical staphylococcus aureus detection method comprises isolation culture, a PCR technology, whole genome and metagenome sequencing and the like, and has one or more limitations in the aspects of time length, operation complexity, detection flux, detection variation accuracy and sensitivity, cost and the like. The targeted molecular marker detection technology combining the ultra-multiplex PCR amplification and the high-throughput sequencing can be used for enriching target microorganisms in a sample with low microorganism content in a targeted manner, avoids a large amount of data waste and background noise caused by whole genome and metagenome sequencing, and has the advantages of less sample requirement, accurate diagnosis result, data quantity saving and low-frequency variation detection. The molecular markers detected by the existing targeted detection technology mainly comprise SNP (single nucleotide polymorphism) markers and SSR (simple sequence repeat) markers. SSR markers are generally accepted as the most polymorphic markers, but are few in microorganisms; the SNP markers are large in number, densely distributed and are binary markers, and the polymorphism of a single SNP marker is insufficient to capture the potential allelic diversity in a microbial population.
Therefore, the development of a novel molecular marker for high polymorphism of staphylococcus aureus and a culture-free detection technique has become an urgent technical problem to be solved.
Disclosure of Invention
The invention aims to provide a specific MNP marker locus of staphylococcus aureus, a primer composition, a kit and application thereof, which can be used for qualitatively identifying and detecting variation of staphylococcus aureus and have the effects of multiple targets, high flux, high sensitivity and fine typing.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, the MNP marker site of staphylococcus aureus is provided, and refers to a genome region which is screened on the staphylococcus aureus genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and the MNP marker site comprises the marker sites of MNP-1-MNP-15 on a 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 specification, and the starting and ending positions of the MNP label marked in table 1 are determined based on the reference sequence corresponding to the same row of MNP in table 1.
In a second aspect of the invention, a multiplex PCR primer composition for detecting said MNP marker site is provided, said multiplex PCR primer composition comprising 15 pairs of primers, the specific primer sequence being shown in SEQ ID No.1-SEQ ID No. 30.
In the above technical scheme, the primer of each MNP marker site includes an upper primer and a lower primer, which are specifically shown in table 1 of the specification.
In a third aspect of the invention, a detection kit for detecting the MNP marker site of the staphylococcus aureus is provided, and the kit comprises the primer composition.
Further, the kit also comprises a multiplex PCR premix.
In the fourth aspect of the invention, the application of the MNP marker locus of staphylococcus aureus, the multiplex PCR primer composition or the detection kit in the identification of staphylococcus aureus is provided.
In the fifth aspect of the invention, the MNP marker locus of the staphylococcus aureus, the multiplex PCR primer composition or the detection kit is applied to the detection of the genetic variation inside and among strains of the staphylococcus aureus.
In a sixth aspect of the 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 a seventh aspect of the invention, 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 is provided.
In the above application, the specific operation steps are as follows:
firstly, obtaining the total DNA of bacteria of a sample to be detected; carrying out first round of multiplex PCR amplification on the total DNA and a blank control by using the kit disclosed by the invention, wherein the cycle number is not higher than 25; after purifying the amplification product, adding a sample label based on the second round of PCR amplification and a second-generation sequencing adaptor; purifying and quantifying the second round amplification product; when a plurality of strains are detected, performing high-throughput sequencing by equivalently mixing second round amplification products; and comparing the sequencing result with the reference sequence of the staphylococcus aureus to obtain the number and genotype data of the detection sequences in the total DNA. And performing data quality control and data analysis on the sequencing data of the total DNA according to the quantity of the staphylococcus aureus sequencing sequences obtained from the total DNA and the blank control and the number of detected MNP loci to obtain the number of the detected MNP loci, the number of the sequencing sequences covering each MNP locus and the MNP locus genotype data.
When the method is used for identifying the staphylococcus aureus, whether the nucleic acid of the staphylococcus aureus exists in the sample to be detected or not is judged after quality control is carried out according to the sequencing sequence quantity of the staphylococcus aureus detected in the sample to be detected and the blank control and the number of the detected MNP sites. The quality control scheme and the judgment method are characterized in that the DNA of the staphylococcus aureus with known copy number is used as a detection sample, the sensitivity, the accuracy and the specificity of the kit for detecting the staphylococcus aureus are evaluated, and the quality control scheme and the judgment method for the kit for detecting the staphylococcus aureus are formulated.
When used for detecting the genetic variation of staphylococcus aureus, the method comprises the detection of the genetic variation among strains and in the strains. The detection of genetic variation among strains comprises the steps of obtaining genotype data of each strain to be compared at 15 MNP sites by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared on the 15 MNP sites have difference or not through genotype comparison. If the strains to be compared have variation in the major genotype of at least one MNP site, the strains are judged to have genetic variation. Alternatively, 15 sites of the strains to be compared can be amplified respectively by single PCR, and then the amplified products are subjected to Sanger sequencing, and after the sequences are obtained, the genotypes of each MNP site of the strains to be compared are compared. If there are MNP sites with inconsistent master genotypes, there are variations between the strains to be compared. When the genetic variation in the strain is detected, whether a minor genotype other than the major genotype is detected at the MNP site of the strain to be detected is determined by a statistical model. If the to-be-detected strain has a minor genotype at least one MNP site, determining that genetic variation exists in the to-be-detected strain.
When the method is used for constructing a staphylococcus aureus DNA fingerprint database, the genotype data of the MNP locus of the staphylococcus aureus identified from a sample is recorded into a database file to form the staphylococcus aureus DNA fingerprint database; when different samples are identified each time, the DNA fingerprint database of the staphylococcus aureus is compared, whether the staphylococcus aureus in the samples has the difference of the major genotypes (more than 50 percent of genotypes supported by sequencing fragments at one MNP site) at the MNP sites with the strains in the database is identified, the staphylococcus aureus with the major genotype difference at least 1 MNP site is the new variation type, and the new variation type is collected into the DNA fingerprint database.
When the method is used for typing the staphylococcus aureus, the staphylococcus aureus in a sample to be tested is identified to obtain the genotype of each MNP site; collecting the genome sequence of staphylococcus aureus disclosed on the net and the constructed staphylococcus aureus DNA fingerprint database to form a staphylococcus aureus reference sequence database; and comparing the genotype of the staphylococcus aureus in the sample to be detected with the reference sequence library of the staphylococcus aureus, and screening genetically identical or closest strains to obtain the type of the staphylococcus aureus in the sample to be detected. And identifying whether the staphylococcus aureus in the sample is an existing type or a new variant according to the comparison result with the reference sequence library, thereby realizing the fine typing of the staphylococcus aureus. The invention belongs to the initiative in the field of staphylococcus aureus, and is not reported in related documents; the MNP marker is mainly developed based on a reference sequence, and MNP sites which are largely distinguished from other species, are polymorphic in staphylococcus aureus species and have conserved sequences on two sides can be mined according to reported re-sequencing data of staphylococcus aureus representing small species; MNP locus detection primers suitable for multiplex PCR amplification can be designed through conserved sequences on two sides of the MNP locus; and then according to the test result of the standard product, a set of MNP sites with the largest polymorphism and high specificity and a primer combination with the best compatibility and a 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 (MNP) marker locus of staphylococcus aureus, a primer composition, a kit and application thereof. The provided 15 MNP sites of the staphylococcus aureus and the primer combination thereof can carry out multiple PCR amplification, fuse a second-generation sequencing platform to carry out sequencing on an amplification product, meet the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on the staphylococcus aureus and meet the requirements of standard and sharable fingerprint data construction of the staphylococcus aureus; the need to accurately detect genetic variation among staphylococcus aureus strains; the method has the advantages that the requirements of homozygosis and heterozygosis of the staphylococcus aureus are identified, and the technical support is provided for scientific research and scientific monitoring of the staphylococcus aureus.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of the screening and primer design of the MNP marker sites of Staphylococcus aureus;
FIG. 3 is a flow chart of detection of MNP marker sites.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the embodiments of the present invention will be more clearly apparent therefrom. It should be understood by those skilled in the art that the detailed description and examples are intended to illustrate, but not limit, the embodiments of the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
the invention develops a novel species-specific molecular marker-MNP marker which is suitable for detecting group organisms. MNP markers refer to polymorphic markers arising from multiple nucleotides within a region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) abundant alleles, 2 at a single MNP locus n Species alleles, higher than SSR and SNP; (2) the species distinguishing capability is strong, species identification can be realized only by a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting the MNP label based on the combination of the super-multiplex PCR and the second-generation high-throughput sequencing technology has the following advantages: (1) the output is a base sequence, and a standardized database can be constructed for sharing without parallel experiments; (2) the method has high efficiency, breaks through the limitation of the quantity of sequencing samples by using the DNA barcodes of the samples, and can type tens of thousands of MNP sites of hundreds of samples at one time; (3) the sensitivity is high, multiple targets are detected at one time by utilizing multiple PCR, and high false negative and low sensitivity caused by amplification failure of a single target are avoided; (4) high accuracy, using the second generation high throughput sequencer to sequence the amplification product hundreds of times.
In view of the advantages and characteristics, the MNP marker and the detection technology thereof, namely the MNP marking method, can realize the classification and tracing of the multi-allelic genotypes of the population organisms, and have application potential in the aspects of identification of pathogenic microorganisms, fingerprint database construction, genetic variation detection and the like. At present, no report on MNP labeling exists in microorganisms, and corresponding technologies are lacked. Therefore, the invention develops the MNP marker sites of the staphylococcus aureus, which are the genome regions which are screened on the staphylococcus aureus genome, are distinguished from other species and have a plurality of nucleotide polymorphisms in the species, including the marker sites of MNP-1-MNP-15 taking CP000253 as a reference genome.
Then, the invention develops a multiplex PCR primer composition for detecting the MNP marker locus of the staphylococcus aureus, wherein the multiplex PCR primer composition comprises 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO.1-SEQ ID NO. 30. The primers do not conflict with each other, and efficient amplification can be performed through multiplex PCR.
The multiplex PCR primer composition can be used as a detection kit for detecting the MNP marker locus of staphylococcus aureus.
The kit can accurately and sensitively detect staphylococcus aureus with the copy/reaction as low as 10.
The MNP marker and the kit have high specificity in detecting staphylococcus aureus in a complex template.
The MNP marker site, primer composition, kit and use thereof of staphylococcus aureus of the present application will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1 screening of the MNP marker sites of Staphylococcus aureus and design of multiplex PCR amplification primers
S1 screening of staphylococcus aureus MNP marker locus
Based on the complete or partial genome sequence of 10864 different staphylococcus aureus isolates disclosed in the network, 15 MNP marker loci are obtained through sequence alignment. For species without genomic data on the net, the genomic sequence information of the representative microspecies of the microbial species to be detected can also be obtained by high-throughput sequencing, wherein the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure polymorphism of the marker screened, the genomic sequence of at least 10 genetically representative isolates is generally used as a reference. The 15 MNP marker sites screened are shown in table 1:
TABLE 1-the MNP marker sites and the starting positions of the detection primers on the reference sequence
The step S1 specifically includes:
selecting a genome sequence of a representative strain of the staphylococcus aureus as a reference genome, and performing sequence comparison on the genome sequence and the reference genome to obtain single nucleic acid polymorphic sites of each strain of the staphylococcus aureus;
on the reference genome, window translation is carried out by taking 100-300 bp as a window and 1bp as a step length, and a plurality of candidate MNP site regions are obtained by screening, wherein the candidate MNP site regions 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 two ends;
screening a region with the division DP of more than or equal to 0.2 in the candidate polynucleotide polymorphic site region as an MNP marker site; wherein DP is d/t, t is the log of comparisons for all the minor species in the candidate polynucleotide polymorphic site region when compared two by two, d is the log of samples for which at least two single nucleic acid polymorphisms in the candidate polynucleotide polymorphic site region are different.
As an optional implementation mode, when the reference genome is screened by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, which is beneficial to comprehensive screening.
S2 design of multiplex PCR amplification primers
And designing the multiplex PCR amplification primers of the MNP sites through primer design software, wherein 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 normally amplified in one amplification reaction.
In this embodiment, the primers used to identify the MNP marker sites are shown in table 1.
S3 evaluation of detection efficiency of primer combination
The detection method of the MNP marker comprises the steps of amplifying all MNP sites at one time through multiple PCR, sequencing amplification products through second-generation high-throughput sequencing, analyzing sequencing data, and evaluating the compatibility of the primer combination according to the detected sites.
The method comprises the steps of adding staphylococcus aureus DNA with a known copy number into human genome DNA to prepare a 1000-copy/reaction template, screening primer combinations with uniform amplification and optimal compatibility according to detection conditions of MNP sites in 4 libraries by using the primer combinations, and finally screening out primer compositions of 15 MNP sites disclosed in the table 1.
Threshold setting and Performance evaluation of the MNP sites and primers described in example 2 for the identification of Staphylococcus aureus
1. Detection of MNP markers
In this example, 1, 10 and 100 copies/reaction of staphylococcus aureus mimetics were prepared by adding known copy numbers of a staphylococcus aureus nucleic acid standard to human genomic DNA. An equal volume of sterile water was also set as a blank. A total of 4 samples were obtained, each sample was constructed into 3 duplicate libraries each day, and the assay was continued for 4 days, i.e. 12 sets of sequencing data were obtained for each sample, as shown in table 2. According to the sequencing fragment number and the site number of the MNP site of the staphylococcus aureus detected in a blank control and a staphylococcus aureus nucleic acid standard substance in 12 repeated experiments, the repeatability, the accuracy and the sensitivity of the detection method are evaluated, and the threshold value for detecting the pollution of a quality control system and the target pathogen is set. The detection procedure for MNP markers is shown in FIG. 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 1 MNP site at most in a sample of 0 copies/reaction, the kit can obviously distinguish the samples of 10 copies/reaction and 0 copies/reaction, and has technical stability and detection sensitivity as low as 10 copies/reaction.
2. Evaluation of reproducibility and accuracy of MNP (MNP) marker detection kit for detecting staphylococcus aureus
And evaluating the reproducibility and accuracy of the MNP marker detection method for detecting the staphylococcus aureus based on whether the genotype of the co-detected site can be reproduced in the two repetitions. Specifically, 12 sets of data of 100 copies of the sample were each subjected to a pair-wise comparison, and the results are shown in table 3.
TABLE 3 reproducibility and accuracy assessment of Staphylococcus aureus MNP marker detection methods
As can be seen from Table 3, the number of MNP sites with differences in major genotypes is 0; according to the principle that the genotype reproducible among 2 repeated experiments is considered to be accurate, the accuracy rate a is 1- (1-r)/2 is 0.5+0.5r, and r represents the reproducibility rate, namely, the ratio of the number of reproducible loci of the main genotype to the number of common loci. In the reproducibility test of the project, the logarithm of difference of the MNP labeling main genotypes among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100%, and the accuracy a is 100%.
3. Threshold judgment method for detecting staphylococcus aureus by MNP (MNP) marker detection kit
As shown in Table 2, sequences aligned to Staphylococcus aureus were detected in 1 copy/reaction sample, covering at least 1 MNP site. While in the partial blank control, the sequence of Staphylococcus aureus was also detected. Due to the extreme sensitivity of MNP marker detection methods, contamination of the data during detection is likely to result in the generation of false positives. Therefore, in this example, a quality control scheme is established, specifically as follows:
1) the amount of sequencing data was greater than 4.5 megabases. The measuring and calculating basis is that the number of MNP sites detected by each sample is 15, and the length of a sequencing fragment is 300 bases, so that when the data volume is more than 4.5 million bases, the number of the sequencing fragments covering each site can be ensured to reach 1000 times by one-time experiment of most samples, and the accurate analysis of the base sequence of each MNP site is ensured.
2) And judging whether the pollution is acceptable according to the signal index S of the staphylococcus aureus in the test sample and the noise index P of the staphylococcus aureus in the blank control, wherein:
and the blank control noise index P is Nc/Nc, wherein Nc and Nc respectively represent the number of sequencing fragments of staphylococcus aureus and the number of total sequencing fragments in the blank control.
And the signal index S of the test sample is Nt/Nt, wherein Nt and Nt respectively represent the number of the sequencing fragments of staphylococcus aureus and the number of the total sequencing fragments in the test sample.
3) And (3) calculating the detection rate of the MNP marker loci in the test sample, wherein the detection rate refers to the ratio of the number of the detected loci to the number of the total design loci.
TABLE 4 SNR of Staphylococcus aureus in samples tested
As shown in Table 4, the average value of the noise index of Staphylococcus aureus in the blank was 0.04%, while the average value of the signal index in the sample of 1 copy was 0.19%, and the average value of the signal-to-noise ratio of the sample of 1 copy and the blank was 5.2, and thus, the present invention provides that when the signal-to-noise ratio is more than 10 times, contamination in the detection system can be judged to be acceptable. The average of the signal-to-noise ratios of the 10 copies of the sample and the blank was 81.6, and at least 8 MNP sites were stably detected in 12 sets of data of 10 copies/reaction, accounting for 53.3% of the total sites. Therefore, the standard provides that the signal-to-noise ratio determination threshold of the staphylococcus aureus is 40 under the condition of ensuring the accuracy, namely, when the signal-to-noise ratio of the staphylococcus aureus in the sample is more than 40 and the site detection rate is more than or equal to 30%, the nucleic acid of the staphylococcus aureus in the sample is determined to be detected.
Therefore, the kit provided by the invention can accurately and sensitively detect staphylococcus aureus with the copy/response as low as 10.
4. Specificity evaluation of MNP (MNP) marker detection method for detecting staphylococcus aureus
Artificially mixing DNAs of staphylococcus aureus, mycobacterium tuberculosis, acinetobacter strains, bordetella pertussis, bordetella hollisae, 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, streptococcus pneumoniae and streptococcus pyogenes together according to equimolar amount to prepare a mixed template, and detecting the staphylococcus aureus in the mixed template by using a blank template as a control by adopting the method provided by the invention. After 3 repeated experiments are carried out and analyzed according to the quality control scheme and the judgment threshold value, only 15 MNP sites of the staphylococcus aureus can be specifically detected in the 3 repeated experiments, the signal-to-noise ratio reaches 673.4, 752.6 and 634.8 in sequence, and the MNP marker and the kit are indicated to detect the high specificity of the target microorganism in a complex template.
Example 3 detection of genetic variation among Staphylococcus aureus strains
6 progeny strains of a staphylococcus aureus strain stored in the same laboratory are detected by using the kit and the MNP marker locus detection method, samples are named as S1-S6 in sequence, the sequencing average coverage multiple of each sample reaches 1416 times, and all 15 MNP markers can be detected from each strain (Table 5). The fingerprints of 6 strains are compared in pairs, and the results are shown in table 5, wherein 1 part (S-2) of staphylococcus aureus and 5 parts (S-2) of staphylococcus aureus detected in the same batch have main genotype difference of partial sites (table 5) and variation among strains.
TABLE 5-6 detection assay for 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 the MNP marker to identify the genetic variation among the strains, thereby ensuring the comparability of the research results, which is of great significance to the scientific research of Staphylococcus aureus. In clinical settings, diagnostic protocols can be considered as to whether differential sites affect drug resistance.
Example 4 detection of genetic variation within Staphylococcus aureus strains
As a colony organism, the individual variation of a part of the staphylococcus aureus colony can make the colony not homozygous any more, and a heterogeneous heterozygous colony is formed, so that the stability and the consistency of the phenotype of the microorganism for testing are influenced. Such variants exhibit an allelic profile outside the major genotype of the locus when tested for molecular markers in a population. When the variant individuals have not accumulated, they represent a very small fraction of the population and are characterized by a low frequency of alleles. Low frequency alleles tend to be confused with technical errors, making prior art techniques difficult to distinguish. The present invention detects highly polymorphic MNP markers. The technical error rate of MNP markers is significantly lower than that of SNP markers, based on the fact that the probability of multiple errors occurring simultaneously is lower than that of one error.
The authenticity assessment of the sub-allelic genotypes of this example was performed as follows: allelic types with strand bias (ratio of the number of sequencing sequences overlaid on a DNA double strand) were first excluded according to the following rule: the strand preference is greater than 10-fold, or the difference from the strand preference of the dominant allele is greater than 5-fold.
Genotypes without strand preference were judged for authenticity based on the number and proportion of sequences sequenced in table 6. Inv function calculation under a 99.9999% probability guarantee, e max (n-1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, the number of sequenced sequences of the sub-allelic gene in each locus is a critical value, and only when the number of sequenced sequences of the sub-allelic gene exceeds the critical value, the true sub-allelic gene is determined. When multiple candidate sub-alleles are present, multiple corrections are made to the P-value for each candidate allele, FDR<0.5% of the candidate alleles were judged to be true sub-allelic genotypes.
Table 6 relates to the parameter e max (n-1) and e max (n.gtoreq.2) means that the highest proportion of the number of sequencing sequences carrying the wrong allele of n SNPs to the total number of sequencing sequences at that site. e.g. of a cylinder max (n-1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, were obtained from the frequency of all minor alleles detected at 930 homozygous MNP sites.
TABLE 6-determination of sub-allelic genotype at partial sequencing depth of cut-off values
Nucleic acids of two strains differing in genotype as shown in Table 5 were mixed in the following 8 ratios 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100, 7/100 according to the above parameters to prepare artificial heterozygous samples, and each sample was examined for 3 replicates to obtain a total of 24 sequencing data. The loci with heterozygous genotypes are detected in 24 artificial heterozygous samples by accurately comparing the genotypes of the MNP loci of the two strains, thereby demonstrating the applicability of the developed MNP marker detection method for the mycoplasma pneumoniae in detecting the genetic variation in the strain population.
Example 5 construction of Staphylococcus aureus DNA fingerprint database
Extracting DNA of all strains or samples for constructing a DNA fingerprint database of staphylococcus aureus by using a conventional CTAB method, a commercial kit and other methods, and detecting the quality of the DNA by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm to 230nm is more than 2.0, the ratio of the absorbance values of 260nm to 280nm is between 1.6 and 1.8, the main band of the DNA electrophoresis is obvious, and obvious degradation and RNA residue do not exist, the genome DNA reaches the relevant quality requirement, and subsequent experiments can be carried out.
And (3) comparing the sequencing data of the 6 strains with the reference genotype to obtain the major genotype of each site of each strain and form the MNP fingerprint of each strain. And inputting the obtained MNP fingerprint of each strain into a database file to form a staphylococcus aureus DNA fingerprint database.
The constructed MNP fingerprint database is based on the gene sequences of detected strains, is compatible with all high-throughput sequencing data, and has the characteristics of complete co-construction and sharing and update at any time. The MNP fingerprint of the strains obtained by each detection is compared with an MNP fingerprint database constructed based on the existing genome data, and the MNP fingerprint of the strains with the differences in main genotypes is recorded into the constructed MNP fingerprint database, so that the real-time updating and co-construction sharing of the database are achieved.
Example 6 application in Fine typing of Staphylococcus aureus
The primer combination and the MNP marker locus detection method are utilized to detect the 6 staphylococcus aureus strains, and the MNP fingerprint of each strain is obtained. And comparing the DNA fingerprints of each strain pairwise and the constructed fingerprint database, defining the DNA fingerprints as existing variants which are the same as those of the existing fingerprint database, and defining the DNA fingerprints as new variants with main genotype difference at least one MNP site, so as to realize the fine typing of the staphylococcus aureus. Detection of 6 s.aureus samples as shown in table 5, the 6 s.aureus detected were 1 and 5 others differing in the major genotype at the 2 MNP sites, possibly in different variants. Therefore, the method achieves the resolution of the staphylococcus aureus to the level of a single base, and can realize the fine typing of the staphylococcus aureus in the sample.
Finally, it should be further 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. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments 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 in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, provided that such 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 intended to include such modifications and variations as well.
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Claims (8)
1. The MNP marker site of staphylococcus aureus is a genome region which is screened from staphylococcus aureus genomes and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and the MNP marker site comprises marker sites of MNP-1-MNP-15 taking CP000253 as a reference genome.
2. The multiplex PCR primer composition for detecting the MNP marker locus of the staphylococcus aureus in claim 1, which comprises 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.
3. A test kit for detecting the MNP marker site of staphylococcus aureus of claim 1, comprising the primer composition of claim 2.
4. The test kit of claim 3, wherein the kit further comprises a multiplex PCR premix.
5. Use of the MNP marker site of staphylococcus aureus according to claim 1, or the primer composition according to claim 2, or the test kit according to any one of claims 3-4 for the identification of staphylococcus aureus and for the preparation of products for the identification of staphylococcus aureus.
6. Use of the MNP marker site of staphylococcus aureus of claim 1, or the primer composition of claim 2, or the test kit of any one of claims 3-4, for detecting genetic variations in and among staphylococcus aureus strains.
7. Use of the MNP marker site of staphylococcus aureus of claim 1 or the primer composition of claim 2 or the test kit of any one of claims 3-4 for constructing a database of staphylococcus aureus.
8. Use of the MNP marker site of staphylococcus aureus according to claim 1 or the primer composition according to claim 2 or the test kit according to any one of claims 3-4 for the fine typing of staphylococcus aureus.
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Citations (3)
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|---|---|---|---|---|
| US20030175716A1 (en) * | 2002-03-13 | 2003-09-18 | Lu-Yieng Liu | Method for detecting staphylococcus aureus |
| CN102816844A (en) * | 2012-08-08 | 2012-12-12 | 许龙岩 | Primer, kit and method for detecting staphylococcus aureus through PCR(Polymerase Chain Reaction)-pyrophosphate method |
| WO2018200887A1 (en) * | 2017-04-26 | 2018-11-01 | The Translational Genomics Research Institute | Methods and assays for subtyping staphylococcus aureus clonal complex 8 strains |
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| US20030175716A1 (en) * | 2002-03-13 | 2003-09-18 | Lu-Yieng Liu | Method for detecting staphylococcus aureus |
| CN102816844A (en) * | 2012-08-08 | 2012-12-12 | 许龙岩 | Primer, kit and method for detecting staphylococcus aureus through PCR(Polymerase Chain Reaction)-pyrophosphate method |
| WO2018200887A1 (en) * | 2017-04-26 | 2018-11-01 | The Translational Genomics Research Institute | Methods and assays for subtyping staphylococcus aureus clonal complex 8 strains |
Non-Patent Citations (1)
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| 窦宇红;梁鸿;何;刘和录;刘琼;马彩凤;李玉霞;: "qPCR快速检测金黄色葡萄球菌及MRSA方法的建立及评价", 中国感染控制杂志, no. 09 * |
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