CN113862383A - MNP (protein-binding protein) marker site of bacillus subtilis, primer composition and application of MNP marker site and primer composition - Google Patents
MNP (protein-binding protein) marker site of bacillus subtilis, primer composition and application of MNP marker site and primer composition Download PDFInfo
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Abstract
The invention discloses an MNP marker locus of bacillus subtilis, a primer composition, a kit and application thereof, wherein the MNP marker locus refers to a genome region which is screened on a bacillus subtilis genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker loci of MNP-1-MNP-12; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 24. The MNP marker site can specifically identify the bacillus subtilis and finely distinguish different subtypes; the primers are not interfered with each other, and by integrating multiple amplification and sequencing technologies, sequence analysis can be performed on all marked sites of multiple samples at one time, so that the method has the advantages of high throughput, multiple targets, high sensitivity and low-frequency variation detection, can be applied to identification and genetic variation detection of the bacillus subtilis of large-scale samples, and has important significance on scientific research and degradation monitoring of the bacillus subtilis.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to an MNP (protein-binding protein) marker locus of bacillus subtilis, a primer composition, a kit and application thereof.
Background
Bacillus subtilis (Bacillus subtilis) is a kind of Bacillus, gram-positive bacteria, widely distributed in soil and putrefactive organic substances, and is easy to reproduce in Sucus subtilis. Bacillus subtilis is a safe microbial strain with great development potential, and is widely applied to various fields of industry, agriculture, medicine, sanitation, food, animal husbandry, aquatic products and scientific research. However, in the practical application process, strains are often mutated, degenerated, have reduced fertility, have weakened or even lost resistance effect and the like, and particularly after the strains are subjected to multiple passages, the degeneration phenomenon is more serious, and the normal operation of production or scientific research is greatly influenced, so that the establishment of a rapid and accurate mutation detection method for the bacillus subtilis has important significance.
The classical bacillus subtilis 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, 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; 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 novel molecular markers with high polymorphism of the pathogenic microorganism bacillus subtilis and a detection technology thereof become technical problems to be solved urgently.
Disclosure of Invention
The invention aims to provide an MNP marker locus of bacillus subtilis, a primer composition, a kit and application thereof, which can be used for qualitative identification and mutation detection of bacillus subtilis 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 the first aspect of the invention, the MNP marker locus of the bacillus subtilis is provided, and the MNP marker locus is a genome region which is specific to a species screened on a bacillus subtilis genome and has a plurality of nucleotide polymorphisms in the species, and comprises marker loci of MNP-1-MNP-12 on an AL009126 genome.
In the above technical solution, the labeling sites of MNP-1 to MNP-12 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 AL009126 sequence.
In a second aspect of the invention, a multiplex PCR primer composition for detecting the MNP marker locus is provided, and the multiplex PCR primer composition comprises 12 pairs of primers, wherein the nucleotide sequences of the 12 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 24.
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 subtilis 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 MNP marker site of the bacillus subtilis or the multiple PCR primer composition or the detection kit is provided for the application in the detection of the bacillus subtilis.
In the fifth aspect of the invention, the MNP marker locus of the bacillus subtilis or the multiple PCR primer composition or the detection kit is provided for the application of detecting the genetic variation in the bacillus subtilis strain and among the strains.
In the sixth aspect of the invention, the application of the MNP marker site of the bacillus subtilis, the multiple PCR primer composition or the detection kit in the construction of a bacillus subtilis database is provided.
In the seventh aspect of the invention, the MNP marker site of the bacillus subtilis or the multiple PCR primer composition or the detection kit is provided for application in the fine typing detection of the bacillus subtilis.
In the bacillus subtilis detection, the detection of the genetic variation inside and among bacillus subtilis strains, the construction of a bacillus subtilis database and the fine typing detection application of the bacillus subtilis, 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 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 (3) comparing the sequencing result with the reference sequence of the bacillus subtilis 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 sequencing sequences of the bacillus subtilis and the number of detected MNP sites obtained from the total DNA and the blank control, so as 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 kit is used for identifying the bacillus subtilis, the bacillus subtilis DNA with known copy number or a mixture of the bacillus subtilis DNA and other DNA pathogens is used as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the bacillus subtilis are evaluated, and a quality control scheme and a judgment method for detecting the bacillus subtilis by the kit are established.
When used for the detection of the genetic variation of the bacillus subtilis, the detection of the genetic variation among strains and in the strains is included. The detection of genetic variation among strains comprises the steps of obtaining genotype data of each strain to be compared at 12 MNP sites by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared on the 12 MNP sites have difference or not through 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, 12 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. And if the to-be-detected strain has a minor genotype at least one MNP site, judging that the genetic variation exists in the to-be-detected strain.
When the method is used for constructing a DNA fingerprint database of the bacillus subtilis, the genotype data of the MNP locus of the bacillus subtilis identified from a sample is recorded into a database file to form the DNA fingerprint database of the bacillus subtilis; when different samples are identified each time, comparing with the DNA fingerprint database of the bacillus subtilis, identifying whether the bacillus subtilis in the samples has a main genotype difference (more than 50% of genotypes supported by sequencing fragments at one MNP site) with strains in the database at the MNP site, and collecting the bacillus subtilis with the main genotype difference at least 1 MNP site, namely a new variant type, into the DNA fingerprint database.
When the method is used for parting the bacillus subtilis, the bacillus subtilis in a sample to be detected is identified to obtain the genotype of each MNP locus. And comparing the sample with the DNA fingerprint database of the bacillus subtilis to identify whether the bacillus subtilis in the sample is the existing type or the new type, and recording the new type into the DNA fingerprint database. Therefore, the DNA fingerprint database can be continuously enriched by utilizing the primer combination.
The invention belongs to the initiative in the field of bacillus subtilis, and is not reported in related documents; MNP markers are mainly developed based on reference sequences, and are divided into MNP sites which are polymorphic in the bacillus subtilis and conserved in sequences at two sides of the MNP sites in other species on a large scale according to reported re-sequencing data of bacillus subtilis representative 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, 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 locus of bacillus subtilis, a primer composition, a kit and application thereof. The 12 MNP sites of the bacillus subtilis 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 high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on the bacillus subtilis and the requirement of accurately detecting the genetic variation among bacillus subtilis strains; the requirement for identifying the degradation of the bacillus subtilis population is met; the method meets the requirements of construction of shareable fingerprint data of the standard of the bacillus subtilis, and provides technical support for scientific research and degradation monitoring of the bacillus subtilis.
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 locus of Bacillus subtilis;
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 will be understood by those skilled in the art that the present embodiments and examples are illustrative of the present invention and are not to be construed as limiting the present 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 caused by multiple nucleotides over 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 locusnSpecies alleles, higher than SSR and SNP, are suitable for detection of microorganisms, a typical population of 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 the MNP label is fused with the ultra-multiplex PCR and the second-generation high-throughput sequencing technology, and has the following advantages: (1) the output is the base sequence, and a standardized database can be constructed for sharing without parallel experimentsSharing; (2) the efficiency is high, the sample DNA bar code is utilized, the limitation of the quantity of sequencing samples is broken through, and tens of thousands of MNP sites of hundreds of samples can be typed 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 a 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 foundation in plants.
Therefore, the invention develops the MNP marker locus of the bacillus subtilis, the MNP marker locus is a genome region which is screened on the bacillus subtilis genome, is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and the MNP marker locus comprises the marker loci of MNP-1-MNP-12 on the AL009126 genome.
Then, the invention develops a multiplex PCR primer composition for detecting the MNP marker locus of the bacillus subtilis, wherein the multiplex PCR primer composition comprises 12 pairs of primers, and the nucleotide sequences of the 12 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 24. 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 subtilis.
The kit provided by the invention can accurately and sensitively detect the 10 copies/reaction of the bacillus subtilis and detect the risk of false positive of the 1 copy/reaction of the bacillus subtilis.
In the reproducibility test of the invention, the logarithm of difference of the MNP labeling main gene type 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 present invention have high specificity in detecting a target microorganism in a complex template.
The marker and primer combinations developed by the present invention will be used to establish the national standards for pathogen detection (plan number 20201830-T-469), which will be released at the end of 2021.
The MNP marker site, primer composition, kit and application of the Bacillus subtilis of the present application will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1 screening of Bacillus subtilis MNP marker loci and design of multiplex PCR amplification primers
S1 screening of MNP marker sites of bacillus subtilis
Based on the complete or partial genome sequences of 381 representative strains belonging to different subspecies of the bacillus subtilis, 12 MNP marker loci are obtained by sequence comparison. 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 selected marker, the genomic sequence of at least 10 isolates is generally used as a reference. The 12 MNP marker sites screened are shown in table 1:
TABLE 1-starting position of the MNP marker site and detection primer on the reference sequence
The step S1 specifically includes:
selecting a genome sequence of one representative subtype of the bacillus subtilis 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 subtilis;
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 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.
S3 evaluation of detection efficiency of primer combination
Adding a bacillus subtilis counting standard with a known copy number into rice genome DNA to prepare a 1000-copy/reaction simulation template, detecting by the MNP marker detection method, constructing 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 out the primer compositions of 12 MNP sites in the table 1.
Example 2 detection of MNP sites and primers for Bacillus subtilis
1. Detection of MNP markers
Bacillus subtilis mock samples of 1 copy/reaction, 10 copies/reaction and 100 copies/reaction were prepared by adding to rice genomic DNA using Bacillus subtilis enumeration standards of known copy number. 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 flow of detection of MNP markers is shown in figure 3. According to the sequencing fragment number and the site number of MNP sites of the bacillus subtilis detected in a blank control and a bacillus subtilis nucleic acid standard substance in 12 repeated experiments, the repeatability, the accuracy and the sensitivity of the detection method are evaluated, and the threshold values of pollution of a quality control system and detection of target pathogens are set.
TABLE 2 detection sensitivity and stability analysis of MNP labeling method for Bacillus subtilis
2. Reproducibility and accuracy evaluation of detection of bacillus subtilis by MNP (MNP) marker detection kit
And evaluating the reproducibility and accuracy of the MNP marker detection method for detecting the bacillus subtilis based on whether the genotype of the co-detected site can be reproduced in the two repetitions. 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 evaluation of reproducibility and accuracy of the Bacillus subtilis MNP marker detection method
As can be seen from Table 3, the number of MNP sites differing in major genotypes was 0; the accuracy rate a is 1- (1-r)/2 is 0.5+0.5r, and r represents the reproducibility rate, i.e., the ratio of the number of sites where the major genotype is reproducible to the number of common sites, which is considered to be the principle of accuracy among 2 repeated experiments. In the reproducibility test of the invention, the logarithm of difference of the MNP labeling main gene type 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 of detection of bacillus subtilis by MNP (MNP) marker detection kit
The sequence aligned to Bacillus subtilis can be detected in 1 copy/reaction sample, and at least 1 MNP site is covered. While the Bacillus subtilis sequence was also detected in the partial blank control. 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 4 megabases. The measuring and calculating basis is that the number of MNP sites detected by each sample is 12, and the length of a sequencing fragment is 300 bases, so that when the data volume is more than 4 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 subtilis in the test sample and the noise index P of the bacillus subtilis in the blank control, wherein:
the blank control noise index P ═ Nc/Nc, where Nc and Nc represent the number of sequencing fragments and the total number of sequencing fragments of bacillus subtilis, respectively, 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 sequencing fragments of the bacillus subtilis and the total number of sequencing fragments in the test sample.
3) And calculating the detection rate of the MNP marker locus in the test sample, which is the ratio of the number of the detected locus to the number of the total design locus.
TABLE 4 Signal to noise ratio of Bacillus subtilis in samples to be tested
As shown in Table 4, the noise index of Bacillus subtilis in the blank was 0.04% on average, while the signal index in the 10-copy sample was 2.56% on average, and the signal-to-noise ratio of the 10-copy sample and the blank was 65.7 on average, and at least 7 MNP sites were stably detected, accounting for 58.3% of the total sites. Therefore, the kit provided by the invention can accurately and sensitively detect the 10 copies/reaction of the bacillus subtilis and detect the risk of false positive of the 1 copy/reaction of the bacillus subtilis. 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 30. And when the signal-to-noise ratio of the bacillus subtilis in the sample is more than 30 and the site detection rate is more than or equal to 30%, judging that the nucleic acid of the bacillus subtilis is detected in the sample.
4. Specificity evaluation of MNP (MNP) marker detection kit for detecting bacillus subtilis
Artificially mixing the DNAs of bacillus subtilis, bacillus anthracis, bacillus subtilis, acinetobacter, adenovirus, bau hollisi, 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 in equal molar quantity to prepare a mixed template, and detecting the bacillus subtilis 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 analyzed according to the quality control scheme and the judgment threshold, only the bacillus subtilis in the mixed template can be specifically detected in the 3 repeated experiments, which shows that the MNP marker and the kit can detect the high specificity of the target microorganism in the complex template.
Example 3 detection of genetic variation among Bacillus subtilis strains
6 sub-strains of one collected bacillus subtilis strain 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 MNP locus reaches 1103 times, and all 15 MNP markers can be detected from each strain (Table 5). The fingerprint spectra of 6 strains are compared pairwise, and the results are shown in table 5, wherein 1 part (S-2) of bacillus subtilis and 5 parts of bacillus subtilis detected together in the same batch have main genotype difference of partial sites (table 5), and variation exists among strains.
TABLE 5-6 detection assays for Bacillus subtilis
As can be seen from Table 5, the kit can be used for ensuring the genetic consistency of the same named Bacillus subtilis 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 the Bacillus subtilis. In clinical settings, diagnostic protocols can be considered as to whether differential sites affect drug resistance.
Example 4 detection of genetic variation in Bacillus subtilis
The detection of the genetic variation of the bacillus subtilis comprises the variation among strains and the variation inside the strains. Because the bacillus subtilis is parasitic in the host, the genetic variation of the bacillus subtilis among and inside the hosts is detected. The variation between hosts is detected by comparing the major genotypes, the obtained fingerprint spectra of the bacillus subtilis are compared pairwise, the 100% reproducibility and accuracy of the major genotypes are identified based on an MNP marking method, and the major genotype difference of one site of the two strains can be detected.
And variation within the B.subtilis host is difficult to detect. Bacillus subtilis, a population organism, has a mutation in a host or in a population, and shows an allelic form outside the major genotype of a locus when a population is subjected to molecular marker detection. When the variant individuals have not accumulated, they account for 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 labeling is significantly lower than that of SNP labeling, based on the probability of multiple errors occurring simultaneously being lower than the probability of one error occurring. 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 proportion of sequences sequenced in table 6. Inv function calculation under a 99.9999% probability guarantee, emax(n-1) and emax(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 emax(n-1) and emax(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 the typemax(n-1) and emax(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 critical value for determination of sub-allelic genotypes at partial sequencing depth
According to the above parameters, DNA of Bacillus subtilis in different variants was mixed 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 of which was tested for 3 replicates to obtain a total of 24 sequencing data. By accurately comparing the gene types of the MNP sites of the two variants of the bacillus subtilis, the heterozygous gene type sites can be detected in 24 artificial heterozygous samples, thereby demonstrating the applicability of the developed MNP marker detection method of the bacillus subtilis in detecting the genetic variation of the strains.
Example 5 construction of Bacillus subtilis DNA fingerprint database
Extracting DNA of all strains or samples for constructing a DNA fingerprint database of the bacillus subtilis 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 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 a main genotype of each site of each strain, forming an MNP fingerprint of each strain, and recording a database file to form a Bacillus subtilis 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 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 subtilis
The primer combination and the MNP marker locus detection method described in example 2 are utilized to obtain the MNP fingerprint of each strain. 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, defining the variants as new variants with main genotype difference at least one MNP site, and realizing the fine typing of the bacillus subtilis.
The results of the detection of 6 parts of bacillus subtilis are shown in table 5, and are consistent with the expectations, 1 part of the 6 parts of bacillus subtilis detected and 5 other parts of bacillus subtilis detected have differences in major genotypes at 2 MNP sites, and the results of the genotype analysis differentiate 6 strains into 2 types. Therefore, the resolution of the method for the bacillus subtilis reaches the level of a single base, and the bacillus subtilis in a sample can be finely typed.
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 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, if 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 also intended to encompass such modifications and variations.
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Claims (8)
1. The MNP marker locus of the bacillus subtilis is a genome region which is screened from a bacillus subtilis genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker loci of MNP-1-MNP-12 on an AL009126 genome.
2. A multiplex PCR primer composition for detecting the MNP marker locus of the bacillus subtilis as claimed in claim 1, wherein the multiplex PCR primer composition comprises 12 pairs of primers, and the nucleotide sequences of the 12 pairs of primers are shown as SEQ ID No. 1-SEQ ID No. 24.
3. A test kit for detecting the MNP marker site of the Bacillus subtilis of claim 1, wherein the kit comprises 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 bacillus subtilis according to claim 1 or the primer composition according to claim 2 or the detection kit according to any one of claims 3 to 4 for the detection of bacillus subtilis.
6. Use of the MNP marker site of bacillus subtilis according to claim 1 or the primer composition according to claim 2 or the detection kit according to any one of claims 3-4 for detecting genetic variations within and between strains of bacillus subtilis.
7. Use of the MNP marker site of bacillus subtilis according to claim 1 or the primer composition according to claim 2 or the detection kit according to any one of claims 3 to 4 for constructing a bacillus subtilis database.
8. Use of the MNP marker site of bacillus subtilis according to claim 1 or the primer composition according to claim 2 or the detection kit according to any one of claims 3 to 4 for the fine typing detection of bacillus subtilis.
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