CN113862383B - MNP (MNP) marking site of bacillus subtilis, primer composition and application of MNP marking site - Google Patents

MNP (MNP) marking site of bacillus subtilis, primer composition and application of MNP marking site Download PDF

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CN113862383B
CN113862383B CN202110963590.8A CN202110963590A CN113862383B CN 113862383 B CN113862383 B CN 113862383B CN 202110963590 A CN202110963590 A CN 202110963590A CN 113862383 B CN113862383 B CN 113862383B
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CN113862383A (en
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彭海
李论
李甜甜
陈利红
方治伟
周俊飞
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Abstract

The application discloses MNP (MNP) marking sites of bacillus subtilis, a primer composition, a kit and application thereof, wherein the MNP marking sites refer to genome regions which are screened on bacillus subtilis genome and are separated from other species and have a plurality of nucleotide polymorphisms in the species, and the MNP marking sites comprise MNP-1-MNP-12; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 24. The MNP marker locus can specifically identify bacillus subtilis 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 marker loci of multiple samples at one time, the method has the advantages of high flux, multiple targets, high sensitivity and low-frequency variation detection, can be applied to the identification and genetic variation detection of bacillus subtilis of large-scale samples, and has important significance on scientific research and degradation monitoring of the bacillus subtilis.

Description

MNP (MNP) marking site of bacillus subtilis, primer composition and application of MNP marking site
Technical Field
The embodiment of the application relates to the technical field of biology, in particular to MNP (MNP) marking sites of bacillus subtilis, a primer composition, a kit and application thereof.
Background
Bacillus subtilis (Bacillus subtilis), a species of Bacillus, is widely distributed in soil and spoilage organisms and is readily propagated in the juice of the herb. Bacillus subtilis is a safe and extremely potential microbial strain 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, the strain is subjected to mutation, degradation, fertility decline, resistance effect weakening and even losing, and the like, and particularly after the strain is subjected to multiple passages, the degradation phenomenon is more serious, so that the normal operation of production or scientific research work is greatly influenced, and therefore, the establishment of a rapid and accurate bacillus subtilis mutation detection method has important significance.
Classical bacillus subtilis 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, development of a novel molecular marker of a pathogenic microorganism bacillus subtilis with high polymorphism and a detection technology thereof becomes a technical problem to be solved urgently.
Disclosure of Invention
The application aims to provide MNP (MNP) marking sites of bacillus subtilis, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on the bacillus subtilis and have the effects of multiple targets, high flux, high sensitivity and fine typing.
In order to achieve the above purpose, the application adopts the following technical scheme:
in a first aspect of the application there is provided a MNP marker locus of Bacillus subtilis, the MNP marker locus being a species-specific genomic region screened on the Bacillus subtilis genome and having a plurality of nucleotide polymorphisms within the species, comprising the marker locus of MNP-1 to MNP-12 on the AL009126 genome.
In the above technical scheme, the marking sites of MNP-1 to MNP-12 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 the AL009126 sequence.
In a second aspect of the present application, there is provided a multiplex PCR primer composition for detecting the MNP marker loci, the multiplex PCR primer composition comprising 12 pairs of primers, the nucleotide sequences of the 12 pairs of primers being shown as SEQ ID NO.1 to SEQ ID NO. 24.
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 application, there is provided a detection kit for detecting the MNP marker locus of bacillus subtilis, the kit comprising the primer composition.
Further, the kit further comprises a multiplex PCR premix.
In a fourth aspect of the application, there is provided the use of the MNP marker locus of bacillus subtilis or the multiplex PCR primer composition or the detection kit in the detection of bacillus subtilis.
In a fifth aspect of the application, there is provided the use of the MNP marker locus of bacillus subtilis or the multiplex PCR primer composition or the detection kit for detecting genetic variation within and among bacillus subtilis strains.
In a sixth aspect of the application, there is provided the use of said MNP marker locus of bacillus subtilis or said multiplex PCR primer composition or said detection kit in the construction of a bacillus subtilis database.
In a seventh aspect of the application, there is provided the use of the MNP marker locus of bacillus subtilis or the multiplex PCR primer composition or the detection kit in the detection of bacillus subtilis in a finely divided form.
In the detection and detection of bacillus subtilis, the genetic variation inside and among bacillus subtilis strains, and the construction of a bacillus subtilis database and the application of bacillus subtilis fine-segment detection, the total DNA of bacteria of a sample to be detected is firstly 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; purifying the amplified product, and then adding a sample tag and a second generation sequencing joint based on the second-round 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 subtilis to obtain the number of detection sequences and genotype data of the total DNA. And according to the number of the bacillus subtilis sequencing sequences and the number of the detected MNP sites obtained in the total DNA and the blank control, carrying out data quality control and data analysis on the sequencing data of the total DNA to obtain the number of the detected MNP sites, the number of the sequencing sequences covering each MNP site and the MNP site genotype data.
When the kit is used for identifying the bacillus subtilis, the sensitivity, accuracy and specificity of the kit for detecting the bacillus subtilis are evaluated by taking the bacillus subtilis DNA with known copy number or a mixture of the bacillus subtilis DNA and other DNA pathogens as a detection sample, and a quality control scheme and a judging method for the kit for detecting the bacillus subtilis are formulated.
When used in bacillus subtilis genetic variation detection, it includes inter-and intra-strain genetic variation detection. The detection of genetic variation among strains comprises the steps of obtaining genotype data of 12 MNP sites 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 12 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, the 12 loci of the strains to be compared may be amplified by single PCR, and the amplified products may be subjected to Sanger sequencing to obtain sequences, and the genotypes of each MNP locus of the strains to be compared may be 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 subtilis DNA fingerprint database, genotype data of the MNP locus of bacillus subtilis identified from a sample is input into a database file to form the bacillus subtilis DNA fingerprint database; and (3) when different samples are identified, comparing the samples with a DNA fingerprint database of the bacillus subtilis, identifying whether the bacillus subtilis in the samples has a difference of a main genotype (a genotype supported by more than 50% of sequencing fragments at one MNP site) with strains in the database, wherein the bacillus subtilis with the main genotype difference at least 1 MNP site is a new mutation type, and recording the new mutation type in the DNA fingerprint database.
When the method is used for bacillus subtilis typing, bacillus subtilis in a sample to be tested is identified, and the genotype of each MNP site is obtained. And identifying whether the bacillus subtilis in the sample is an existing type or a new type by comparing the bacillus subtilis with the DNA fingerprint database of the bacillus subtilis, wherein the new type is recorded in the DNA fingerprint database. Therefore, the DNA fingerprint database can be continuously enriched by utilizing the primer combination.
The application is initiated in the field of bacillus subtilis, and is not reported in related documents; MNP markers are developed mainly based on reference sequences, and are characterized in that MNP sites which are polymorphic in the interior of bacillus subtilis species and conserved in sequence at two sides are distinguished from other species on a large scale according to reported resequencing sequences of bacillus subtilis representative species; MNP site detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of the MNP site; 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 application at least have the following technical effects or advantages:
the application provides MNP (MNP) marking sites of bacillus subtilis, a primer composition, a kit and application thereof. The provided 12 MNP loci of the bacillus subtilis 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 the bacillus subtilis are met, and the requirements of accurate detection of genetic variation among bacillus subtilis strains are met; the requirement of identifying the degradation of the bacillus subtilis population is met; meets the requirements of the standard and sharable fingerprint data construction of the bacillus subtilis, and provides technical support for the scientific research and degradation monitoring of the bacillus subtilis.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, 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 application, 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 subtilis;
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 application 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 application.
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 application 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 application 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, and a standardized database can be constructed for sharing without parallel experiments; (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 application developed MNP marker loci for Bacillus subtilis that are genomic regions screened on the Bacillus subtilis genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including marker loci for MNP-1-MNP-12 on the AL009126 genome.
Next, the present application developed a multiplex PCR primer composition for detecting the MNP marker loci of Bacillus subtilis, comprising 12 pairs of primers, the nucleotide sequences of the 12 pairs of primers are shown as SEQ ID NO.1 to SEQ ID NO. 24. 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 MNP labeling sites of bacillus subtilis.
The kit provided by the application can accurately and sensitively detect 10 copies/reaction of bacillus subtilis, and detect 1 copy/reaction of bacillus subtilis with false positive risk.
In the reproducibility test of the application, 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 application have high specificity in detecting target microorganisms in complex templates.
The marker and primer combination developed in the present application will be used to formulate the national standard for pathogen detection (program number 20201830-T-469) which will be released at the end of 2021.
The MNP-labeling site, primer composition, kit and use thereof of Bacillus subtilis 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 subtilis and design of multiplex PCR amplification primers
S1, screening MNP (MNP) marker locus of bacillus subtilis
Based on the complete or partial sequences of 381 genome belonging to different subspecies of bacillus subtilis, 12 MNP marking sites are obtained through sequence comparison. 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, genomic sequences of at least 10 isolates are generally used as reference. The 12 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 the bacillus subtilis as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphic sites of each strain of the bacillus subtilis;
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 subtilis counting standard with known copy number into rice genome DNA to prepare a 1000-copy/reaction simulated template, detecting by using the MNP mark detection method, constructing 4 repeated sequencing libraries, screening a primer combination with uniform amplification and optimal compatibility according to detection conditions of MNP sites in the 4 libraries, and finally screening the primer combination of 12 MNP sites in the table 1.
Example 2 detection of Bacillus subtilis by MNP site and primer
1. Detection of MNP markers
Bacillus subtilis simulants of 1 copy/reaction, 10 copy/reaction and 100 copy/reaction were prepared using a known copy number of a Bacillus subtilis count standard, added to rice 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 (3) evaluating the reproducibility, accuracy and sensitivity of the detection method according to the number of sequenced fragments and the number of sites of the MNP site of the bacillus subtilis detected in the blank control and the bacillus subtilis nucleic acid standard substance in 12 repeated experiments, and formulating a threshold value for pollution of a quality control system and detection of a target pathogen.
TABLE 2 detection sensitivity and stability analysis of MNP labeling method of Bacillus subtilis
2. Reproducibility and accuracy assessment of detection of bacillus subtilis by MNP (MNP) marker detection kit
Based on whether the genotype of the co-detected site is reproducible in the two replicates, the reproducibility and accuracy of detection of bacillus subtilis by the MNP marker detection method are evaluated. 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 the MNP marker detection method of Bacillus subtilis
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 application, 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 judgment for detecting bacillus subtilis by MNP (MNP) mark detection kit
The sequence aligned to Bacillus subtilis can be detected in 1 copy/reaction sample, covering at least 1 MNP site. The sequence of Bacillus subtilis was also detected in part of the 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 4 megabases. The measurement and calculation basis is that the number of MNP loci detected by each sample is 12, and the length of one sequencing fragment is 300 bases, so that when the data size is more than 4 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 the contamination is acceptable based on the signal index S of bacillus subtilis in the test sample and the noise index P of bacillus subtilis 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 subtilis, respectively, in the control.
The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments and the total number of sequenced fragments of bacillus subtilis, 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.
TABLE 4 SNR of Bacillus subtilis in samples to be tested
As shown in Table 4, the average noise index of Bacillus subtilis in the control was 0.04%, the average signal index in the 10 copies was 2.56%, and the average signal-to-noise ratio of the 10 copies and the control was 65.7, so that at least 7 MNP sites were stably detected, accounting for 58.3% of the total sites. Therefore, the kit provided by the application can accurately and sensitively detect 10 copies/reaction of bacillus subtilis, and the detection of 1 copy/reaction of bacillus subtilis has the risk of false positive. The application provides that when the signal to noise ratio is greater than 30, it can be determined that the contamination in the detection system is acceptable. 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. Specific evaluation for detecting bacillus subtilis by MNP (MNP) mark detection kit
Artificially mixing bacillus subtilis with equal molar amounts of bacillus anthracis, bacillus subtilis, acinetobacter, adenovirus, huo Shibao termyces, bordetella pertussis, chlamydia pneumoniae, mycoplasma pneumoniae, epstein barr 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, preparing a mixed template, and detecting the bacillus subtilis in the mixed template by adopting the method provided by the application by taking sterile water as blank control. After 3 repeated experiments are carried out and analysis is carried out according to the quality control scheme and the judgment threshold, only bacillus subtilis in the mixed template can be specifically detected in the 3 repeated experiments, which shows that MNP marks and the kit detect high specificity of target microorganisms in complex templates.
Example 3 detection of genetic variation between Bacillus subtilis strains
The kit and MNP marker locus detection method are used for detecting 6 sub-generation strains of one bacillus subtilis strain, samples are sequentially named as S1-S6, the average coverage of sequencing of each MNP locus is 1103 times, and all 15 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 subtilis detected in the same batch all had a major genotype difference at part of the sites (Table 5), and there was a variation between strains.
TABLE 5-6 detection assay for Bacillus subtilis
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 subtilis strains in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for the scientific research of bacillus subtilis. 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 subtilis
Genetic variation detection of bacillus subtilis includes inter-and intra-strain variation. Since bacillus subtilis is parasitic in the host, i.e., genetic variation of bacillus subtilis is detected between and within the host. The variation among hosts is detected by comparing the main genotypes, the obtained fingerprints of the bacillus subtilis are compared pairwise, 100% reproducibility and accuracy of the main genotypes are identified based on an MNP labeling method, and the main genotype difference of two strains with one site can be detected.
And it is the variation inside the bacillus subtilis host that is difficult to detect. As a group organism, bacillus subtilis 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 application 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 application 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 number of sequences sequenced in each locus is determined to be true only when the number of sequences in the hypo-genotype exceeds the thresholdAllelic type. 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 subtilis 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 repetition, and 24 sequencing data were obtained in total. Through the accurate comparison with the genotypes of MNP loci of two modified bacillus subtilis, heterozygous genotype loci can be detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marker detection method of bacillus subtilis in detecting genetic variation of strains is demonstrated.
EXAMPLE 5 construction of Bacillus subtilis DNA fingerprint database
All strains or DNA of samples used for constructing a bacillus subtilis DNA fingerprint database are extracted by using a conventional CTAB method, a 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 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 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 subtilis 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 subtilis.
The results of the detection of 6 parts of Bacillus subtilis are shown in Table 5, and are consistent with expectations, 1 part of the detected 6 parts of Bacillus subtilis is different from the other 5 parts of the detected 6 parts of Bacillus subtilis 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 subtilis reaches the level of single base, and the method can realize the fine typing of bacillus subtilis in a sample.
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 application 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 application.
It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments of the present application without departing from the spirit or scope of the embodiments of the application. Thus, the embodiments of the present application are intended to include such modifications and alterations insofar as they come within the scope of the embodiments of the application as claimed and the equivalents thereof.
Sequence listing
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agattacata atacaaatcg ctcgga 26
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gttcatgaat tttcccgtat gctcc 25
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ctcggatcag ttttcaacca ttcat 25
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Claims (7)

1. A multiplex PCR primer composition for detecting a MNP marker locus of bacillus subtilis, wherein the MNP marker locus is a genomic region screened on the bacillus subtilis genome that is distinguished from other species and has a plurality of nucleotide polymorphisms inside the species, and comprises marker loci of MNP-1 to MNP-12 on an AL009126 genome, and 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 to SEQ ID No. 24.
2. A detection kit for detecting the MNP marker locus of bacillus subtilis according to claim 1, characterized in that the kit comprises the primer composition according to 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 to 3 for the detection of bacillus subtilis for non-diagnostic purposes.
5. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 for detecting genetic variation within and among bacillus subtilis strains for non-diagnostic purposes.
6. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 for constructing a bacillus subtilis database.
7. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for the finely divided detection of bacillus subtilis for non-diagnostic purposes.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN107937502A (en) * 2017-12-07 2018-04-20 江汉大学 A kind of method for screening the high polymorphic molecular marker site of microorganism

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Publication number Priority date Publication date Assignee Title
CN107937502A (en) * 2017-12-07 2018-04-20 江汉大学 A kind of method for screening the high polymorphic molecular marker site of microorganism

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多核苷酸多态性分析在异基因造血干细胞移植后嵌合状态检测中的应用;吕晓东 等;中华血液学杂志;第40卷(第8期);662-666 *
靶向测序基因型检测（GBTS）技术及其应用;徐云碧 等;中国农业科学;第53卷(第15期);2983-3004 *

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