CN114790486B - MNP (MNP) marking site of bacillus anthracis, primer composition, kit and application of MNP marking site - Google Patents
MNP (MNP) marking site of bacillus anthracis, primer composition, kit and application of MNP marking site Download PDFInfo
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Abstract
The invention discloses an MNP (MNP) marking site of bacillus anthracis, a primer composition, a kit and application thereof, wherein the MNP marking site refers to a genome region which is screened on the genome of bacillus anthracis and is separated from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marking sites of MNP-1-MNP-11; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 22. The MNP marker locus can specifically identify bacillus anthracis and finely distinguish different subtypes; the primers are not interfered with each other, and the method synthesizes multiple amplification and sequencing technologies, can perform sequence analysis on all marking sites of multiple samples at one time, has the advantages of high flux, multiple targets, high sensitivity and culture free, can be applied to identification and genetic variation detection of bacillus anthracis of large-scale samples, and has important significance on epidemic prevention monitoring and scientific research of bacillus anthracis.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to MNP (MNP) marking sites of bacillus anthracis, a primer composition, a kit and application thereof.
Background
Bacillus anthracis (Bacillus anthraci), which belongs to the genus Bacillus aerodans, is a pathogenic bacterium that can cause anthrax (zoonosis) in domestic animals, wild animals and humans. Herders, farmers, fur and slaughter workers are susceptible to infection, and people are often infected by eating or by contact with dead animals. Clinically, it is mainly manifested as local skin necrosis and specific black scab, or acute infections of the lung, intestinal tract and meninges, sometimes accompanied by septicemia. Anthrax caused by this bacterium occurs almost worldwide throughout the four seasons, and thus bacillus anthracis is a hazard to public health and economic development and thus has heretofore been a considerable weight.
Classical bacillus anthracis detection methods, including isolation and culture, PCR techniques, whole genome and metagenome sequencing, etc., have one or more limitations in terms of duration, complexity of operation, detection throughput, accuracy and sensitivity of detection variation, cost, etc. The targeted molecular marker detection technology integrating the ultra-multiplex PCR amplification and the high-throughput sequencing can enrich target microorganisms in a sample with low microorganism content in a targeted manner, avoids a large amount of data waste and background noise caused by sequencing of a whole genome and a metagenome, and has the advantages of small 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 and SSR markers. SSR markers are the most well-accepted markers for polymorphism, but are small in number in microorganisms; the number of SNP markers is huge, the distribution is dense, and the polymorphism of single SNP marker is insufficient to capture the potential allelic diversity in microorganism population.
Therefore, the development of a novel molecular marker with high polymorphism of pathogenic microorganism bacillus anthracis and a detection technology thereof become a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide MNP (MNP) marking sites of bacillus anthracis, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on bacillus anthracis and have the effects of multiple targets, high flux, high sensitivity and fine typing.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention there is provided an MNP marker locus of Bacillus anthracis, the MNP marker locus being a species specific genomic region screened on the Bacillus anthracis genome and having a plurality of nucleotide polymorphisms within the species, including the marker locus of MNP-1 to MNP-11 on the AE016879 genome.
In the above technical scheme, the marking sites of MNP-1 to MNP-11 are specifically shown in the specification table 1, and the starting and ending positions of the MNP marks marked in the table 1 are determined based on AE016879 sequence.
In a second aspect of the present invention, there is provided a multiplex PCR primer composition for detecting the MNP marker loci, the multiplex PCR primer composition comprising 11 pairs of primers, the nucleotide sequences of the 11 pairs of primers being shown as SEQ ID NO.1 to SEQ ID NO. 22.
In the above technical solution, the primers of each MNP marker locus include an upper primer and a lower primer, and are specifically shown in table 1 of the specification.
In a third aspect of the invention, there is provided a detection kit for detecting the MNP marker locus of Bacillus anthracis, the kit comprising the primer composition.
Further, the kit further comprises a multiplex PCR premix.
In a fourth aspect of the invention, there is provided the use of said MNP marker locus of Bacillus anthracis or said multiplex PCR primer composition or said detection kit in the detection of Bacillus anthracis.
In a fifth aspect of the invention, there is provided the use of said MNP marker locus of Bacillus anthracis or said multiplex PCR primer composition or said detection kit for detecting genetic variation within and between Bacillus anthracis strains.
In a sixth aspect of the invention, there is provided the use of said MNP marker locus of Bacillus anthracis or said multiplex PCR primer composition or said detection kit for the construction of a database of Bacillus anthracis.
In a seventh aspect of the invention, there is provided the use of said MNP-labeling site of Bacillus anthracis or said multiplex PCR primer composition or said detection kit for the accurate and fine detection of Bacillus anthracis.
In the applications of the bacillus anthracis detection, the detection of genetic variation in bacillus anthracis strains and among the strains, the construction of bacillus anthracis database and the accurate subdivision detection of bacillus anthracis, firstly, the total DNA of bacteria of a sample to be detected is obtained; performing a first round of multiplex PCR amplification on the total DNA and the blank control by using the kit, wherein the number of cycles is not higher than 25; after the amplified product is purified, adding a label and a second generation sequencing joint of the sample through a second round of PCR amplification; quantifying after purifying the second round of amplification products; detecting a plurality of strains by mixing the amplification products of the second round in equal amounts and then performing high throughput sequencing; and comparing the sequencing result with the reference sequence of the bacillus anthracis to obtain the number of detection sequences and genotype data of the total DNA. And carrying out data quality control and data analysis on the sequencing data of the total DNA according to the number of the sequencing sequences of bacillus anthracis obtained in the total DNA and the blank control and the number of the detected MNP sites, and obtaining the number of the detected MNP sites, the number of the sequencing sequences covering each MNP site and the genotype data of the MNP sites.
When the method is used for identifying bacillus anthracis, whether the sample to be detected contains bacillus anthracis nucleic acid or not is judged after quality control according to the number of sequencing sequences of bacillus anthracis detected in the sample to be detected and a blank control and the number of MNP loci detected. The quality control scheme and the judging method are characterized in that DNA of bacillus anthracis strains with known copy numbers is used as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting bacillus anthracis are evaluated, and the quality control scheme and the judging method when the kit detects bacillus anthracis are formulated.
When used for bacillus anthracis genetic variation detection, it includes inter-strain and intra-strain genetic variation detection. The detection of genetic variation among strains comprises the steps of obtaining genotype data of 11 MNP loci of each strain to be compared by using the kit and the method. By genotype comparison, the strains to be compared are analyzed for differences in major genotypes at the 11 MNP sites. If the strain to be compared has a variation in the main genotype of at least one MNP site, it is determined that there is a genetic variation in both. Alternatively, 11 loci of the strains to be compared may be amplified by single PCR, and then Sanger sequencing is performed on the amplified products to obtain sequences, and then the genotypes of each MNP locus of the strains to be compared are aligned. If there are MNP sites of inconsistent main genotypes, there are variations between the strains to be compared. When detecting genetic variation inside the strain, determining whether the secondary genotype other than the primary genotype is detected at the MNP locus of the strain to be detected through a statistical model. If the strain to be tested has the subgenotype at least one MNP site, judging that the strain to be tested has genetic variation.
When the method is used for constructing a bacillus anthracis DNA fingerprint database, genotype data of the MNP locus of bacillus anthracis identified from a sample is recorded into a database file to form the bacillus anthracis DNA fingerprint database; and (3) each time different samples are identified, comparing the samples with a DNA fingerprint database of bacillus anthracis, identifying whether the bacillus anthracis in the samples has a difference of main genotypes (with more than 50% of genotypes supported by sequencing fragments at one MNP site) with strains in the database, wherein the bacillus anthracis with the main genotypes difference at least 1 MNP site is a new mutation type, and recording the new mutation type into 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 bacillus anthracis, bacillus anthracis in a sample to be tested is identified, and the genotype of each MNP locus is obtained; collecting genome sequences of bacillus anthracis disclosed on the net and constructing a bacillus anthracis DNA fingerprint database to form a bacillus anthracis reference sequence library; and comparing the genotype of bacillus anthracis in the sample to be detected with a reference sequence library of the bacillus anthracis, and screening strains which are genetically consistent 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 of an existing type or a new type by comparison consistent with the bacillus anthracis reference sequence library, and completing the typing of the bacillus anthracis in the sample. The invention is initiated in the bacillus anthracis field, and is not reported in related documents; the MNP mark is mainly mined based on reported resequencing data of bacillus anthracis representative species, and MNP mark sites which are specific to the bacillus anthracis and have high distinction degree on various species of bacillus anthracis are searched; the sequences at two sides of MNP mark are highly conserved among Huo Shibao tertbacterium species, and the conserved region is used for designing multiplex PCR amplification primers; and then according to the test result of the standard substance, a set of primer combination with the largest polymorphism, high specificity MNP locus and the best compatibility and a detection kit are screened.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
the invention provides MNP (MNP) marking sites of bacillus anthracis, a primer composition, a kit and application thereof. The provided 11 MNP loci of bacillus anthracis and the primer combination thereof can be used for multiplex PCR amplification, and the amplification products are sequenced by fusing a second generation sequencing platform, so that the requirements of high throughput, high efficiency, high accuracy and high sensitivity detection of bacillus anthracis are met, and the requirement of accurately detecting genetic variation among bacillus anthracis strains is met; meets the requirement of identifying the degradation of bacillus anthracis population; meets the requirements of the bacillus anthracis standard and the sharable fingerprint data construction, and provides technical support for the scientific research and epidemic prevention monitoring of bacillus anthracis.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of screening and primer design of MNP marker loci of Bacillus anthracis;
FIG. 3 is a flow chart of detection of MNP marker loci.
Detailed Description
The advantages and various effects of the embodiments of the present invention will be more clearly apparent from the following detailed description and examples. Those skilled in the art will appreciate that these specific implementations and examples are provided to illustrate, but not limit, examples of the present invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or may be prepared by existing methods.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
MNP markers suitable for detection of the population organisms are screened as detection targets. MNP markers refer to polymorphic markers caused by multiple nucleotides in a region of the genome. MNP markers have the following advantages over SSR markers and SNP markers: (1) The alleles are abundant, and 2 are arranged on single MNP locus n Species alleles, higher than SSR and SNP, are suitable for detection of microorganisms, a typical population organism; (2) The species distinguishing capability is strong, the species identification can be realized by only a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting MNP labeling fuses the ultra-multiplex PCR and the second-generation high-throughput sequencing technology, and has the following advantages: (1) The output is a base sequence, which does not need to be parallelVerifying, a standardized database can be constructed for sharing; (2) The method has high efficiency, breaks through the limitation of the number of sequencing samples by using the sample DNA bar code, and can type tens of thousands of MNP sites of hundreds of samples at one time; (3) High sensitivity, multiple targets are detected at one time by using multiple PCR, and high false negative and low sensitivity caused by single target amplification failure are avoided; (4) High accuracy, and sequencing the amplified product hundreds of times by using a second-generation high-throughput sequencer.
In view of the advantages and the characteristics, the MNP marking and the detection technology thereof can realize classification and tracing of the multi-allele types of the group organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, genetic variation detection and the like. At present, no report about MNP labeling exists in microorganisms, and corresponding technology is lacking. The development, screening and application of MNP labeling method has better application foundation in plants.
Thus, the present invention developed MNP marker loci for Bacillus anthracis that of a genomic region screened on the Bacillus anthracis genome that is distinct from other species and has multiple nucleotide polymorphisms within the species, including the marker loci of MNP-1-MNP-11 on the AE016879 genome.
Next, the present invention developed a multiplex PCR primer composition for detecting the MNP marker loci of Bacillus anthracis, comprising 11 pairs of primers, the nucleotide sequences of the 11 pairs of primers being shown as SEQ ID NO.1 to SEQ ID NO. 22. The primers do not collide with each other, and efficient amplification can be performed through multiplex PCR;
the multiplex PCR primer composition can be used for a detection kit for detecting the MNP labeling site of bacillus anthracis.
The kit provided by the invention can sensitively detect 1 copy/reaction bacillus anthracis.
In the reproducibility test of the invention, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.
The MNP markers and the kits of the invention have high specificity in detecting target microorganisms in complex templates.
The MNP-labeling site, primer composition, kit and use thereof of one Bacillus anthracis of the present application will be described in detail below in conjunction with examples, comparative examples and experimental data.
Example 1 screening of MNP marker loci of Bacillus anthracis and design of multiplex PCR amplification primers
S1, screening MNP (MNP) marker locus of bacillus anthracis
Based on complete or partial sequences of genomes of 259 different isolates of bacillus anthracis disclosed on the net, 11 MNP marking sites are obtained through sequence alignment. For species on which no genomic data is present on the net, genomic sequence information representing a minispecies of the microorganism species to be detected may also be obtained by high throughput sequencing, which may be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected markers, typically at least 10 genomic sequences of representative strains with diversity are used as reference. The 11 MNP marker loci screened are shown in table 1:
TABLE 1 MNP marker loci and detection primers starting position on the reference sequence
The step S1 specifically includes:
selecting a genome sequence representing subtype of bacillus anthracis as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphism sites of each strain of bacillus anthracis;
on the reference genome, carrying out window translation by taking 100-300 bp as a window and taking 1bp as a step length, and screening to obtain a plurality of candidate MNP site areas, wherein the candidate MNP site areas contain more than or equal to 2 single nucleotide variation sites, and the single nucleotide polymorphism sites do not exist on sequences of 30bp at both ends;
screening a region with the discrimination DP more than or equal to 0.2 from the candidate polynucleotide polymorphism site region as an MNP marking site; wherein dp=d/t, t is the log of comparisons when all the minor species are compared pairwise in the candidate polynucleotide polymorphic site region, and d is the log of samples of differences in at least two single nucleic acid polymorphisms in the candidate polynucleotide polymorphic site region.
As an optional implementation mode, when screening is performed on the reference genome by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, so that the comprehensive screening is facilitated.
S2, design of multiplex PCR amplification primer
The multiplex PCR amplification primers of the MNP locus are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.
S3, evaluating detection efficiency of primer combination
The method comprises the steps of adding a bacillus anthracis counting standard with known copy number into human genome DNA to prepare a 1000-copy/reaction simulated template, detecting by the MNP mark detection method, constructing 4 repeated sequencing libraries, screening primer combinations with uniform amplification and optimal compatibility according to detection conditions of MNP sites in the 4 libraries, and finally screening the primer combinations of 11 MNP sites in the table 1.
Example 2 detection of Bacillus anthracis by MNP loci and primers
1. Detection of MNP markers
Bacillus anthracis simulated samples of 1 copy/reaction, 10 copy/reaction and 100 copy/reaction were prepared using a known copy number of Bacillus anthracis count standard added to human genomic DNA. An equal volume of sterile water was set at the same time as a blank. A total of 4 samples, each of which was constructed as 3 replicate libraries per day, were tested continuously for 4 days, i.e., 12 sets of sequencing data were obtained per sample, as shown in table 2. The detection flow of MNP markers is shown in fig. 3. And according to the number of sequencing fragments and the number of sites of MNP sites of bacillus anthracis detected in a blank control and bacillus anthracis nucleic acid standard substance in 12 repeated experiments, evaluating the reproducibility, accuracy and sensitivity of the detection method, and formulating a quality control system pollution and a threshold value for detecting a target pathogen.
TABLE 2 detection sensitivity and stability analysis of MNP labeling method of Bacillus anthracis
As shown in Table 2, the kit can stably detect 3 MNP sites in a 1-copy/reaction sample, and at most 1 MNP site in a 0-copy/reaction sample, and the kit can clearly distinguish between a 1-copy/reaction sample and a 0-copy/reaction sample, and has technical stability and detection sensitivity as low as 1-copy/reaction.
2. Reproducibility and accuracy assessment of MNP (mammalian nucleic acid) marker detection kit for detecting bacillus anthracis
Based on whether the genotype of the co-detected site is reproducible in the two replicates, the reproducibility and accuracy of detection of bacillus anthracis by the MNP marker detection method is assessed. Specifically, the data of 12 sets of 100 copies of the sample were compared in pairs, and the results are shown in Table 3.
TABLE 3 reproducibility and accuracy assessment of MNP marker detection method of Bacillus anthracis
As can be seen from Table 3, the number of MNP sites having a difference in the main genotypes was 0; according to the principle that the reproducible genotypes are considered to be accurate between 2 repeated experiments, the accuracy a=1- (1-r)/2=0.5+0.5r, and r represents the reproducibility, namely the ratio of the reproducible site number of the main genotype to the common site number. In the reproducibility test of the invention, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.
3. Threshold value judgment for detecting bacillus anthracis by MNP (MNP) mark detection kit
The sequences of bacillus anthracis were detected in a partial blank. Because of the extreme sensitivity of MNP marker detection methods, contamination of the data in the detection is prone to false positives. Therefore, the quality control scheme is formulated in this example, and is specifically as follows:
1) The amount of sequencing data is greater than 3.3 megabases. The measurement and calculation basis is that the number of MNP loci detected by each sample is 11, and the length of one sequencing fragment is 300 bases, so that when the data size is more than 3.3 megabases, most samples can ensure that the number of sequencing fragments covering each locus reaches 1000 times by one experiment, and ensure the accurate analysis of the base sequence of each MNP locus.
2) Determining whether contamination is acceptable based on the signal index S of Bacillus anthracis in the test sample and the noise index P of Bacillus anthracis in the blank, wherein:
the noise figure p=nc/Nc for the control, where Nc and Nc represent the number of sequenced fragments and total sequenced fragment number of bacillus anthracis, respectively, in the control.
The signal index of the test sample s=nt/Nt, where Nt and Nt represent the number of sequenced fragments and total sequenced fragment number of bacillus anthracis, respectively, in the test sample.
3) Calculating the detection rate of MNP marking sites in a test sample, wherein the detection rate refers to the ratio of the number of detected sites to the number of total designed sites. The results are shown in Table 4;
TABLE 4 SNR of Bacillus anthracis in samples to be tested
As can be seen from Table 4, the average noise index of Bacillus anthracis in the blank was 0.03%, 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 the blank was 11.5, so that at least 3 MNP sites were stably detected, and they were 27.3% of the total sites. Therefore, the kit provided by the invention can sensitively detect 1 copy/reaction bacillus anthracis.
The invention provides that when the signal to noise ratio is greater than 11 times, it can be determined that the contamination in the detection system is acceptable. When the signal to noise ratio of bacillus anthracis in the sample is more than 11 and the site detection rate is more than or equal to 27.3%, determining that the bacillus anthracis nucleic acid is detected in the sample.
Therefore, the kit provided by the invention can sensitively detect copy/reaction bacillus anthracis.
4. Specific evaluation of MNP marker detection kit for detecting bacillus anthracis
The method comprises the steps of artificially mixing DNA (deoxyribonucleic acid) of bacillus anthracis, acinetobacter, adenovirus, huo Shibao termyces, 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 detecting bacillus anthracis in the mixed template by using sterile water as a blank control by adopting the method provided by the invention. After 3 repeated experiments are carried out and analysis is carried out according to the quality control scheme and the judgment threshold, only 11 MNP sites of bacillus anthracis in the mixed template can be specifically detected in the 3 repeated experiments, and the signal to noise ratios are 3578.3, 3974.7 and 3616.4 in sequence, so that the high specificity of MNP labels and the detection target microorganisms in the complex template by the kit is shown.
Example 3 detection of genetic variation between Bacillus anthracis strains
The kit and MNP marker locus detection method are used for detecting the collected 6 sub-generation strains of one bacillus anthracis strain, samples are sequentially named as S1-S6, the average coverage multiple of sequencing of each MNP locus is 1103 times, and all 11 MNP markers can be detected by each strain (table 5). The fingerprints of 6 strains were aligned pairwise, and the results are shown in Table 5, in which 1 part (S-2) of the strain and 5 parts of Bacillus anthracis detected in the same batch all have a major genotype difference of partial sites (Table 5), and strain-to-strain variation exists.
TABLE 5-6 detection assay for Bacillus anthracis
As can be seen from Table 5, the application of the kit for identifying the genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of the same named bacillus anthracis strains in different laboratories, so that the comparability of research results is ensured, and the kit has great significance for the scientific research of bacillus anthracis. In clinical terms, one can take into account the diagnostic regimen as to whether the site of the difference affects resistance.
Example 4 genetic variation detection of Bacillus anthracis
Genetic variation detection of bacillus anthracis, including inter-and intra-strain variation. Since bacillus anthracis is parasitized in the host, that is, genetic variation of bacillus anthracis is detected between and within the host. The variation among hosts is detected by comparing the main genotypes, and the obtained fingerprint of bacillus anthracis is compared pairwise, so that 100% reproduction rate and accuracy of the main genotypes are identified based on MNP labeling, and the main genotype difference of two strains with one site can be detected.
And it is the variation inside the bacillus anthracis host that is difficult to detect. As a group organism, bacillus anthracis is mutated in a host or in a group, and when the group is detected by molecular marker, it is represented as an allele outside the main genotype of the site. When variant individuals have not accumulated, they occupy a very small proportion of the population and exhibit a low frequency of allelic forms. Low frequency alleles tend to mix with technical errors, making the prior art indistinguishable. The present invention detects MNP markers with high polymorphism. Based on the fact that the probability of occurrence of a plurality of errors is lower than that of one error, the technical error rate of MNP markers is significantly lower than that of SNP markers. The invention distinguishes true minor genotypes from error genotypes caused by technical errors through a statistical model. Specifically:
the authenticity assessment of the secondary isogenotypes of this example was performed as follows: the allelotype with strand preference (ratio of the number of sequencing sequences covered on the DNA duplex) is first excluded according to the following rule: the strand preference is greater than 10-fold, or the difference from the strand preference of the major allele is greater than 5-fold.
Genotypes without strand preference were judged for authenticity based on the number and proportion of sequenced sequences in table 6. Table 6 lists e calculated based on binom. Inv function under the probability guarantee of α=99.9999% max (n=1) and e max (n.gtoreq.2) is 1.03% and 0.0994%, respectively, and the true hypogenotype is judged only when the number of sequences of the hypogenotype exceeds the critical value. When a plurality of candidate minor alleles exist, multiple correction is carried out on the P value of each candidate allele type, and FDR is carried out<0.5% of candidate alleles are judged to be true minor genotypes.
Parameter e related to Table 6 max (n=1) and e max (n.gtoreq.2) refers to the highest proportion of the total sequence of the locus of the sequence of the wrong allele carrying n SNPs. e, e max (n=1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, are obtained from the frequency of all minor genotypes detected at 930 homozygous MNP sites.
TABLE 6-threshold for determining the hypo-isogenotypes at partial sequencing depth
According to the above parameters, the DNAs of different variants of Bacillus anthracis were mixed in the following 8 ratios of 1/1000,3/1000,5/1000,7/1000,1/100,3/100,5/100,7/100 to prepare artificial heterozygous samples, each sample was tested 3 times for duplicate, and a total of 24 sequencing data were obtained. By accurately comparing the genotypes of MNP loci of two modified bacillus anthracis, heterozygous genotype loci can be detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marker detection method of bacillus anthracis in detecting genetic variation of strains is demonstrated.
Example 5 construction of Bacillus anthracis DNA fingerprint database
All strains or DNA of samples used for constructing the bacillus anthracis DNA fingerprint database are extracted by using the conventional CTAB method, commercial kit and other methods, and the quality of the DNA is detected by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm and 230nm is more than 2.0, the ratio of the absorbance values of 260nm and 280nm is between 1.6 and 1.8, the DNA electrophoresis main band is obvious, no obvious degradation and RNA residues exist, the genome DNA reaches the relevant quality requirements, and the subsequent experiments can be carried out.
And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain the main genotype of each site of each strain, forming MNP fingerprint of each strain, and recording a database file to form the bacillus anthracis DNA fingerprint database. 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 of the strain obtained by each detection is compared with the constructed MNP fingerprint database, and the MNP fingerprint database constructed by the MNP fingerprint of the strain with the main genotype difference is realized, so that the co-construction sharing and the random updating of the database are realized.
Example 6 use in Bacillus anthracis Fine subdivision
The MNP fingerprint of each strain was obtained using the primer combination and MNP marker locus detection method described in example 2. And comparing the DNA fingerprint of each strain with the constructed fingerprint database in pairs, defining the existing variant as the same as the existing fingerprint database, defining the new variant as the main genotype difference at least one MNP locus, and realizing the fine typing of bacillus anthracis.
The results of the detection of 6 parts of Bacillus anthracis are shown in Table 5, and are consistent with expectations, 1 part of the detected 6 parts of Bacillus anthracis has a difference from other 5 parts of the detected 6 parts of Bacillus anthracis in the main genotypes at 2 MNP sites, and the genotype analysis results distinguish 6 strains into 2 types. Therefore, the resolution of the method for bacillus anthracis reaches the level of single base, and the bacillus anthracis in the sample can be finely typed.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiment and all such alterations and modifications as fall within the scope of the embodiments of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, the embodiments of the present invention are intended to include such modifications and alterations insofar as they come within the scope of the embodiments of the invention as claimed and the equivalents thereof.
Sequence listing
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Claims (7)
1. A multiplex PCR primer composition for detecting bacillus anthracis, which is characterized by comprising 11 pairs of primers, wherein the nucleotide sequences of the 11 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 22.
2. A test kit for detecting bacillus anthracis, comprising the primer composition of claim 1.
3. The test kit of claim 2, wherein the kit further comprises a multiplex PCR premix.
4. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2-3 for qualitative detection of bacillus anthracis for non-diagnostic purposes and for the preparation of a bacillus anthracis detection product.
5. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for detecting genetic variation within and among bacillus anthracis strains.
6. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2-3 for constructing a bacillus anthracis database.
7. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 in the finely divided detection of bacillus anthracis.
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