CN114790486A - MNP (protein-binding protein) marker site of bacillus anthracis, primer composition, kit and application of kit - Google Patents
MNP (protein-binding protein) marker site of bacillus anthracis, primer composition, kit and application of kit Download PDFInfo
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Abstract
The invention discloses an MNP marker site, a primer composition, a kit and application of bacillus anthracis, wherein the MNP marker site refers to a genome region which is screened on a bacillus anthracis genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker sites of MNP-1-MNP-11; the primer is shown in SEQ ID NO. 1-SEQ ID NO. 22. The MNP marker site can specifically identify the bacillus anthracis and finely distinguish different subtypes; 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 marked sites of multiple samples at one time, the advantages of high throughput, multiple targets, high sensitivity and no culture are achieved, the method can be applied to identification and genetic variation detection of the bacillus anthracis of large-scale samples, and the method has important significance for epidemic prevention monitoring and scientific research of the bacillus anthracis.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marker site of bacillus anthracis, a primer composition, a kit and application thereof.
Background
Bacillus anthracis (Bacillus anthracaci) belongs to the aerobic Bacillus genus, and is a pathogenic bacterium that causes anthrax in livestock, wild animals and humans (zoonosis). Herdsmen, farmers, fur and butcherers are susceptible to infections, which people often eat or come into contact with dead animals, and have been regarded by the empire as one of the lethal agents. Clinically, it is manifested mainly by local skin necrosis and specific dark scab, or by acute infection of lung, intestinal tract and meninges, sometimes accompanied by septicemia. Anthrax caused by this bacterium occurs almost all over the world, all the year round, and thus, the harm of anthrax to public health and economic development of society has hitherto been a considerable proportion.
The classical bacillus anthracis detection method comprises separation 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, accuracy and sensitivity of detection variation, 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; SNP markers are large in number, densely distributed, and polymorphic, single SNP markers are insufficient to capture the potential allelic diversity in a microbial population.
Therefore, the development of novel molecular markers with high polymorphism of pathogenic microorganism bacillus anthracis and a detection technology thereof become technical problems to be solved urgently.
Disclosure of Invention
The invention aims to provide an MNP (MNP) marker site of bacillus anthracis, a primer composition, a kit and application thereof, which can be used for qualitative identification and variation detection of bacillus anthracis 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 loci of the bacillus anthracis are provided, and the MNP marker loci are genome regions which are specific to species screened on the bacillus anthracis genome and have a plurality of nucleotide polymorphisms in the species, including the marker loci of MNP-1-MNP-11 on AE016879 genome.
In the above technical scheme, the labeling sites of MNP-1 to MNP-11 are specifically shown in Table 1 in the specification, and the starting and ending positions of the MNP label marked in Table 1 are determined based on the AE016879 sequence.
In a second aspect of the invention, a multiplex PCR primer composition for detecting said MNP marker locus is provided, said multiplex PCR primer composition comprising 11 pairs of primers, the nucleotide sequences of said 11 pairs of primers are shown in SEQ ID No.1 to SEQ ID No. 22.
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 bacillus anthracis 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 the bacillus anthracis, the multiplex PCR primer composition or the detection kit in the detection of the bacillus anthracis is provided.
In the fifth aspect of the invention, the application of the MNP marker locus of the bacillus anthracis, the multiple PCR primer composition or the detection kit in detecting the genetic variation inside and among strains of the bacillus anthracis is provided.
In the sixth aspect of the invention, the application of the MNP marker locus of the bacillus anthracis, the multiplex PCR primer composition or the detection kit in the construction of a bacillus anthracis database is provided.
In the seventh aspect of the invention, the application of the MNP marker site of the bacillus anthracis, the multiplex PCR primer composition or the detection kit in the fine typing detection of the bacillus anthracis is provided.
In the applications of the bacillus anthracis detection, the detection of genetic variation in bacillus anthracis strains and among strains, the construction of a bacillus anthracis database and the fine typing detection of bacillus anthracis, firstly, the total bacterial DNA of a sample to be detected is obtained; 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 the amplification product is purified, adding a label and a second-generation sequencing adaptor of the sample through a second round of PCR amplification; purifying and quantifying the second round amplification product; when a plurality of strains are detected, performing high-throughput sequencing by equivalently mixing the second round amplification products; and comparing the sequencing result with the reference sequence of the bacillus anthracis to obtain the number and genotype data of the detected sequences in the total DNA. And performing data quality control and data analysis on the sequencing data of the total DNA according to the number of the sequencing sequences of the bacillus anthracis obtained from the total DNA and the blank control and the number of the detected MNP sites to obtain the number of the detected MNP sites, the number of the sequencing sequences covering each MNP site and the genotype data of the MNP sites.
When the method is used for identifying the bacillus anthracis, whether the nucleic acid of the bacillus anthracis exists in a sample to be detected or not is judged after quality control is carried out according to the number of sequencing sequences of the bacillus anthracis detected in the sample to be detected and a blank control and the number of MNP (detected protein) sites. The quality control scheme and the determination method are characterized in that the DNA of the Bacillus anthracis strain with known copy number is used as a detection sample, the sensitivity, the accuracy and the specificity of the kit for detecting the Bacillus anthracis are evaluated, and the quality control scheme and the determination method for detecting the Bacillus anthracis by the kit are formulated.
When used for detecting genetic variation of the bacillus anthracis, 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 the strains to be compared at 11 MNP sites by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared on the 11 MNP sites are different or not by genotype comparison. If the strains to be compared have a variation in the major genotype of at least one MNP site, the strains are judged to have genetic variation. As an alternative, 11 sites of the strains to be compared can be amplified respectively through single PCR, and then the amplification products are subjected to Sanger sequencing, and after the sequences are obtained, the genotypes of all MNP sites 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 DNA fingerprint database of the bacillus anthracis, the genotype data of the MNP site of the bacillus anthracis identified from a sample is recorded into a database file to form the DNA fingerprint database of the bacillus anthracis; when different samples are identified each time, whether the bacillus anthracis in the samples has the difference of the major genotype (more than 50 percent of the genotype supported by the sequencing fragment at one MNP site) with the strains in the database at the MNP site is identified by comparing with the DNA fingerprint database of the bacillus anthracis, and the bacillus anthracis with the difference of the major genotype at least 1 MNP site is a new variant type and is included in the DNA fingerprint database. Therefore, the DNA fingerprint database can be continuously enriched by utilizing the primer combination.
When the method is used for typing the bacillus anthracis, the bacillus anthracis in a sample to be tested is identified to obtain the genotype of each MNP locus; collecting the genome sequence of the bacillus anthracis disclosed on the network and the constructed bacillus anthracis DNA fingerprint database to form a bacillus anthracis reference sequence database; and comparing the genotype of the bacillus anthracis in the sample to be detected with the reference sequence library of the bacillus anthracis, and screening out strains which are genetically identical or closest to each other to obtain the type of the bacillus anthracis in the sample to be detected. And identifying whether the bacillus anthracis in the sample is an existing type or a new type by consistent comparison with the bacillus anthracis reference sequence library, and completing the typing of the bacillus anthracis in the sample. The invention belongs to the initiative in the field of bacillus anthracis, and is not reported in related documents; the MNP marker is mainly mined based on the reported re-sequencing data of the anthrax bacillus representative microspecies, and MNP marker sites which are specific to the anthrax bacillus and have high discrimination on various microspecies of the anthrax bacillus are searched; sequences on two sides of MNP markers are highly conserved among all small strains of the Haliotis holtzia hollandii, and a conserved area is used for designing a multiplex PCR amplification primer; and then according to the test result of the standard product, a set of MNP sites with the largest polymorphism and high specificity, a primer combination with the best compatibility and a detection kit are screened.
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 site of bacillus anthracis, a primer composition, a kit and application thereof. The provided 11 MNP sites of the bacillus anthracis and the primer combination thereof can carry out multiple PCR amplification, and are fused with a second-generation sequencing platform to carry out sequencing on an amplification product, thereby meeting the requirements of carrying out high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on the bacillus anthracis and the requirement of accurately detecting genetic variation among bacillus anthracis strains; the requirement for identifying the degradation of the bacillus anthracis population is met; the method meets the requirements of shareable fingerprint data construction of the bacillus anthracis standard, and provides technical support for scientific research and epidemic prevention monitoring of the bacillus anthracis.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of screening and primer design for the MNP marker sites of Bacillus anthracis;
FIG. 3 is a flow chart of detection of MNP marker sites.
Detailed Description
The embodiments of the present invention will be specifically explained 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 presented thereby. 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:
screening MNP markers suitable for detecting the population organisms as detection targets. 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, singleAt MNP site 2 n Species alleles, higher than SSR and SNP, are suitable for detection of microorganisms, which are typical group organisms; (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 MNP labels is combined with the super-multiplex PCR and the second-generation high-throughput sequencing technology, and 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 the characteristics, the MNP marker and the detection technology MNP marking method thereof can realize the classification and the tracing of the multi-allelic genotypes of the population organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, detection of genetic variation and the like. At present, no report on MNP labeling exists in microorganisms, and corresponding technologies are lacked. The development, screening and application of the MNP marking method have better application basis in plants.
Therefore, the invention develops the MNP marker sites of the bacillus anthracis, which are screened genome regions which are distinguished from other species and have a plurality of nucleotide polymorphisms in the species on the genome of the bacillus anthracis, and comprise the marker sites of MNP-1-MNP-11 on the AE016879 genome.
Then, the invention develops a multiplex PCR primer composition for detecting the MNP marker locus of the bacillus anthracis, wherein the multiplex PCR primer composition comprises 11 pairs of primers, and the nucleotide sequences of the 11 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 22. The primers are not conflicted with each other, and can be efficiently amplified through multiple PCR;
the multiplex PCR primer composition can be used for a detection kit for detecting the MNP marker locus of the bacillus anthracis.
The kit provided by the invention can sensitively detect 1 copy/reaction of bacillus anthracis.
In the reproducibility test of the invention, the logarithm of difference of the MNP marked main genotype among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100%, and the accuracy a is 100%.
The MNP marker and the kit of the invention have high specificity in detecting target microorganisms in a complex template.
The MNP marker site, primer composition, kit and application of Bacillus anthracis of the present application will be described in detail with reference to examples, comparative examples and experimental data.
Example 1 screening of MNP marker loci of Bacillus anthracis and design of multiplex PCR amplification primers
S1 screening of MNP marker loci of bacillus anthracis
Based on the complete or partial genome sequence of 259 different anthrax bacillus isolates published in the network, 11 MNP marker sites are obtained through sequence comparison. For species without genome data on the network, the genome sequence information of the representative microspecies of the microbial species to be detected can also be obtained through high-throughput sequencing, wherein the high-throughput sequencing can be whole genome sequencing or simplified genome sequencing. In order to ensure polymorphism of the selected marker, the genomic sequence of at least 10 representative strains having diversity is generally used as a reference. The 11 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 subtype of the bacillus anthracis 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 bacillus anthracis;
on the reference genome, performing window translation by taking 100-300 bp as a window and 1bp as a step length, and screening to obtain a plurality of candidate MNP site regions, 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 ═ d/t, t is the comparison logarithm of all the minor species in the region of the candidate polynucleotide polymorphic site when compared pairwise, d is the sample logarithm of at least two single nucleic acid polymorphisms that differ in the region of the candidate polynucleotide polymorphic site.
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 step size adopted in the implementation mode is 1bp, so that comprehensive screening is facilitated.
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.
S3 evaluation of detection efficiency of primer combination
Adding a bacillus anthracis counting standard with a known copy number into human genome DNA to prepare a 1000-copy/reaction simulation template, detecting by the MNP marker detection method to construct 4 repeated sequencing libraries, screening primer combinations with uniform amplification and optimal compatibility according to the detection condition of MNP sites in the 4 libraries, and finally screening the primer compositions of 11 MNP sites in the table 1.
Example 2 detection of Bacillus anthracis by MNP sites and primers
1. Detection of MNP markers
Bacillus anthracis mock samples were prepared for 1 copy/reaction, 10 copies/reaction, and 100 copies/reaction using known copy number bacillus anthracis counting standards, added 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. The detection procedure for MNP markers is shown in FIG. 3. According to the sequencing fragment number and the site number of the MNP site of the bacillus anthracis detected in the blank control and the bacillus anthracis nucleic acid standard 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.
TABLE 2 detection sensitivity and stability analysis of MNP labeling method for Bacillus anthracis
As shown in Table 2, the kit can stably detect 3 MNP sites in a sample of 1 copy/reaction and detect 1 MNP site at most in a sample of 0 copy/reaction, can obviously distinguish samples of 1 copy/reaction and 0 copy/reaction, and has technical stability and detection sensitivity as low as 1 copy/reaction.
2. Reproducibility and accuracy evaluation of detection of bacillus anthracis by MNP (MNP) marker detection kit
And evaluating the repeatability and accuracy of the MNP marker detection method for detecting the bacillus anthracis based on whether the genotype of the co-detected site can be reproduced in the two times of repetition. Specifically, two-by-two comparisons were made for each of 12 sets of data for 100 copies of the sample, and the results are shown in table 3.
TABLE 3 reproducibility and accuracy assessment of Bacillus anthracis MNP marker detection method
As can be seen from Table 3, the number of MNP sites differing in major genotypes was 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 invention, the logarithm of difference of the MNP marked main genotype 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 for detecting bacillus anthracis by MNP (MNP) marker detection kit
The sequence of B.anthracis was detected in a partial blank. 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 as follows:
1) the amount of sequencing data was greater than 3.3 megabases. The measuring and calculating basis is that the number of MNP sites detected by each sample is 11, the length of a sequencing fragment is 300 bases, so that when the data volume is more than 3.3 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 bacillus anthracis in the test sample and the noise index P of the bacillus anthracis in the blank control, wherein:
the blank noise index P is Nc/Nc, where Nc and Nc represent the number of sequencing fragments of B.anthracis and the total number of sequencing fragments in the blank, respectively.
And the signal index S of the test sample is Nt/Nt, wherein Nt and Nt respectively represent the number of sequencing fragments of the bacillus anthracis and the total number of 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. The results are shown in Table 4;
TABLE 4 Signal to noise ratio of Bacillus anthracis in samples to be tested
As can be seen from Table 4, the average noise index of B.anthracis in the blank was 0.03%, while the average signal index in the 1 copy sample was 0.39%, and the average signal-to-noise ratio of the 1 copy sample and blank was 11.5, which resulted in stable detection of at least 3 MNP sites, accounting for 27.3% of the total sites. Therefore, the kit provided by the invention can sensitively detect 1 copy/response of bacillus anthracis.
The present invention provides that contamination in the detection system can be judged to be acceptable when the signal-to-noise ratio is greater than 11 times. And when the signal-to-noise ratio of the bacillus anthracis in the sample is more than 11 and the site detection rate is more than or equal to 27.3 percent, judging that the nucleic acid of the bacillus anthracis is detected in the sample.
Therefore, the kit provided by the invention can sensitively detect the copy/reaction bacillus anthracis.
4. Specificity evaluation of MNP (MNP) marker detection kit for detecting bacillus anthracis
Artificially mixing DNA equimolar amounts of bacillus anthracis, acinetobacter, adenovirus, Bordetella holtziae, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, EB virus, haemophilus influenzae, varicella zoster virus, cytomegalovirus, herpes simplex virus, human bocavirus, Klebsiella pneumoniae, Legionella, Moraxella catarrhalis, Pseudomonas aeruginosa, Rickettsia, staphylococcus aureus, streptococcus pneumoniae and streptococcus pyogenes together to prepare a mixed template, and using sterile water as a blank control to detect the bacillus anthracis in the mixed template 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, only 11 MNP sites of the bacillus anthracis in the mixed template can be specifically detected in the 3 repeated experiments, the signal-to-noise ratio is 3578.3, 3974.7 and 3616.4 in sequence, and the MNP markers and the kit are indicated to detect the high specificity of the target microorganism in the complex template.
Example 3 detection of genetic variation between strains of Bacillus anthracis
6 sub-generations of collected one strain of bacillus anthracis were tested by the kit and the MNP marker locus test method, the samples were named S1-S6 in sequence, the average coverage factor of each MNP locus in sequencing was 1103 times, and all 11 MNP markers could be detected in each strain (Table 5). The fingerprints of the 6 strains are compared pairwise, and the results are shown in table 5, wherein 1 part (S-2) of bacillus anthracis and 5 parts of bacillus anthracis detected together in the same batch have main genotype differences of partial sites (table 5), and variation exists among the strains.
TABLE 5-6 detection assay for Bacillus anthracis
As can be seen from Table 5, the kit can be used for ensuring the genetic consistency of the same named Bacillus anthracis strains in different laboratories by detecting the application of MNP markers to identify genetic variation among the strains, thereby ensuring the comparability of research results, which has important significance for scientific research of Bacillus anthracis. Clinically, diagnostic protocols can be weighed against whether differential sites affect drug resistance.
Example 4 detection of genetic variation in Bacillus anthracis
The detection of genetic variation of Bacillus anthracis includes variation between strains and variation within strains. Because the bacillus anthracis is parasitic in the host, the genetic variation of the bacillus anthracis between hosts and inside the hosts is detected. The variation among hosts is detected by comparing the main genotypes, the obtained fingerprint spectrums of the bacillus anthracis are compared pairwise, the main genotypes are identified with 100% reproducibility and accuracy based on an MNP marking method, and the main genotype difference of one site of two strains can be detected.
But difficult to detect are variations within the host of B.anthracis. Bacillus anthracis, a population organism, has a mutation in the host or within the population and, when the population is subjected to molecular marker detection, exhibits an allelic form outside the major genotype of the locus. 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 invention distinguishes real sub-allelic genotypes and wrong genotypes caused by technical errors through a statistical model. Specifically, the method comprises the following steps:
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 ratio of sequenced sequences in table 6. Inv function calculation with 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 suballelic genotype at each locus is a critical value, and only when the number of sequenced sequences of the suballelic genotype exceeds the critical value, the true suballelic genotype 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 refers to the parameter e max (n-1) and e max (n.gtoreq.2) means carrying n SThe highest ratio of the number of sequenced sequences of the wrong allele of NP to the total number of sequenced sequences at that site. e.g. of the type max (n-1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, were obtained from the frequency of all the minor alleles detected at 930 homozygous MNP sites.
TABLE 6-determination of sub-allelic genotype at partial sequencing depth of cut-off values
The DNA of Bacillus anthracis in different variants was mixed according to the above parameters in the following 8 ratios 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100, 7/100 to prepare artificial heterozygous samples, each sample tested for 3 replicates to obtain a total of 24 sequencing data. By accurately comparing the gene types of the MNP loci of the two variants of the bacillus anthracis, the heterozygous gene type loci can be detected in 24 artificial heterozygous samples, thereby demonstrating the applicability of the developed MNP marker detection method of the bacillus anthracis in detecting the genetic variation of the strains.
Example 5 construction of Bacillus anthracis DNA fingerprint database
The DNA of all strains or samples for constructing the DNA fingerprint database of the bacillus anthracis is extracted by methods such as a conventional CTAB method, a commercial kit and the like, and the quality of the DNA is detected by 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 no obvious degradation or RNA residue exists, the genome DNA reaches the relevant quality requirement, and subsequent experiments can be carried out.
And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain the main genotype of each site of each strain, forming the MNP fingerprint of each strain, and recording the MNP fingerprint into a database file to form a DNA fingerprint database of the bacillus anthracis. The constructed MNP fingerprint database is based on the gene sequences of the detected strains and is therefore compatible with all high throughput sequencing data. The MNP fingerprint spectrum of the strain obtained by each detection is compared with the established MNP fingerprint database, and the MNP fingerprint database established by the MNP fingerprint spectrum of the strain with the difference of the main genotypes realizes the co-establishment sharing and the random updating of the database.
Example 6 application in Fine typing of Bacillus anthracis
The primer combination and the MNP marker locus detection method described in example 2 are utilized to obtain the MNP fingerprint of each strain. The DNA fingerprints of each strain are compared pairwise and are compared with a constructed fingerprint database, the DNA fingerprints are identical to the existing fingerprint database and are defined as existing variants, and the DNA fingerprints with main genotype difference at least one MNP site are defined as new variants, so that the bacillus anthracis can be finely classified.
The results of 6 anthrax bacillus tests are shown in table 5, and are consistent with the expectations, 1 part of the 6 anthrax bacillus tests and 5 other parts of the 6 anthrax bacillus tests have differences in major genotypes at 2 MNP sites, and the results of the genotype analyses differentiate the 6 strains into 2 types. Therefore, the resolution of the method for the bacillus anthracis reaches the level of a single base, and the bacillus anthracis in a sample can be finely classified.
Finally, it should also be 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 the preferred embodiment and all changes and modifications that fall within the true scope of the embodiments of the present 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 locus of the bacillus anthracis is a genome region which is screened from a bacillus anthracis genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and the MNP marker locus comprises marker loci of MNP-1-MNP-11 on an AE016879 genome.
2. The multiplex PCR primer composition for detecting the MNP marker locus of the bacillus anthracis as set forth in claim 1, which is characterized in that the multiplex PCR primer composition comprises 11 pairs of primers, and the nucleotide sequences of the 11 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 22.
3. A test kit for detecting the MNP marker site of Bacillus anthracis as set forth in claim 1, wherein said kit includes the primer composition as set forth in 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 Bacillus anthracis as set forth in claim 1, or the primer composition as set forth in claim 2, or the detection kit as set forth in any one of claims 3-4 for qualitative detection of Bacillus anthracis and for preparation of products for detection of Bacillus anthracis.
6. Use of the MNP marker site of Bacillus anthracis as set forth in claim 1 or the primer composition as set forth in claim 2 or the detection kit as set forth in any one of claims 3-4 for detecting genetic variation within and among strains of Bacillus anthracis.
7. Use of the MNP marker site of Bacillus anthracis as set forth in claim 1, or the primer composition as set forth in claim 2, or the detection kit as set forth in any one of claims 3-4 for constructing a Bacillus anthracis database.
8. Use of the MNP marker site of Bacillus anthracis as set forth in claim 1, or the primer composition as set forth in claim 2, or the detection kit as set forth in any one of claims 3-4 for the fine typing detection of Bacillus anthracis.
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