CN113862384B - MNP (MNP) marking site of Francisella tularensis, primer composition, kit and application - Google Patents
MNP (MNP) marking site of Francisella tularensis, primer composition, kit and application Download PDFInfo
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Abstract
The invention discloses MNP (MNP) marking sites of Francisella tularensis, a primer composition, a kit and application thereof, wherein the MNP marking sites are genome regions which are screened on Francisella tularensis genome and are distinguished from other species and have a plurality of nucleotide polymorphisms in the species, and comprise marking sites of MNP-1-MNP-15; the primer is shown as SEQ ID NO.1-SEQ ID NO. 30. The MNP marker locus can specifically identify Francisella tularensis and finely distinguish different species; 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 detection advantages of high flux, multiple targets, high sensitivity, high precision and monitoring variation are achieved, the method can be applied to the identification and genetic variation detection of Francisco tularensis of large-scale samples, and the method has important significance on scientific research and epidemic prevention monitoring of Francisco tularensis.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to MNP (MNP) marking sites of Francisella tularensis, a primer composition, a kit and application thereof.
Background
Francisella tularensis (Francisella tularensis) is a gram-negative bacterium, a pathogenic bacterium of Tularemia, one of the most infectious pathogenic bacteria, and it has been found that more than one hundred animals can infect this bacterium. Francisella tularensis has special tolerance to low temperature, can survive for 9 months in water below 0 ℃ and 1-2 months in water at 20-25 ℃, does not change toxicity, is easy to spread through aerosol and has strong toxicity, and has been classified as a bioterrorism pathogen by the American CDC. In addition, for experimental studies of Francisella tularensis, variations in strains can result in strains that are not actually identical in different laboratories or in the same laboratory at different times and that are named identically, resulting in irreproducible and incomparable experimental results. Heterogeneity between human hela cell laboratories has resulted in a significant amount of incomparable experimental results and wasted data. Variation of strains can also affect clinical diagnostic protocols. Therefore, the development of the quick, accurate and variation-monitoring detection and analysis method for Francisella tularensis has important significance for scientific research and application of Francisella tularensis.
Classical detection methods of Francisella tularensis, including isolation and culture, PCR techniques, whole genome and metagenome sequencing, etc., have one or more limitations in terms of duration, operational complexity, 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 Francisella tularensis and a detection technology thereof becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide MNP (MNP) marking sites of Francisella tularensis, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on Francisella tularensis and have the effects of multiple targets, high flux, high sensitivity and fine typing.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a MNP marker locus of Francisella tularensis, the MNP marker locus being a genomic region screened on the Francisella tularensis genome that is distinct from other species and has a plurality of nucleotide polymorphisms within the species, including the marker loci of MNP-1 to MNP-15 on a Francisella tularensis reference sequence.
In the above technical scheme, the marking sites of MNP-1 to MNP-15 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 reference sequences corresponding to the same row of MNPs in the table 1.
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 15 pairs of primers, the specific primer sequences being shown in SEQ ID NO.1-SEQ ID NO. 30.
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, a detection kit for detecting the Francisella tularensis MNP marker locus is provided, 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 the MNP marker locus of franciscensis tularensis or the multiplex PCR primer composition or the detection kit in the identification of franciscensis tularensis.
In a fifth aspect of the invention, there is provided the use of the MNP marker locus of Francisella tularensis or the multiplex PCR primer composition or the detection kit for detecting genetic variations within and among Francisella tularensis strains.
In a sixth aspect of the invention, there is provided the use of the MNP marker locus of franciscensis tularensis or the multiplex PCR primer composition or the detection kit in the construction of a franciscensis database.
In a seventh aspect of the invention, there is provided the use of the MNP marker locus of francis tularensis or the multiplex PCR primer composition or the detection kit in the detection of francis tularensis fine segments.
In the application, the specific operation steps are as follows:
firstly, obtaining total bacterial DNA of a sample to be detected; 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 Francisella tularensis to obtain the number of the 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 Francisella tularensis and the number of the detected MNP sites obtained from the total DNA and the blank control, 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 kit is used for detecting Francisella tularensis, the sensitivity, accuracy and specificity of the kit for detecting Francisella tularensis are evaluated by taking a Francisella tularensis nucleic acid standard with known copy number or a mixture of the Francisella tularensis nucleic acid standard and other DNA pathogens as a detection sample, and a quality control scheme and a judging method when the kit detects Francisella tularensis are formulated.
When used in Francisella tularensis 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 15 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 15 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, 15 loci of the strain 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 strain 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 DNA fingerprint database of Francisella tularensis, genotype data of the MNP locus of Francisella tularensis identified from a sample is input into a database file to form the DNA fingerprint database of Francisella tularensis; and (3) when different samples are identified, comparing the samples with a DNA fingerprint database of Francisella tularensis, identifying whether Francisella tularensis in the samples and strains in the database have differences of main genotypes (the MNP sites have genotypes supported by more than 50% of sequencing fragments) at the MNP sites, wherein Francisella tularensis with the main genotypes at least 1 MNP site is a new mutation type, and recording the mutation type into the DNA fingerprint database.
When the method is used for the Francisella tularensis typing, francisella tularensis in a sample to be tested is identified, and the genotype of each MNP locus is obtained. And comparing the DNA fingerprint database of the Francisella tularensis with the DNA fingerprint database, identifying whether the Francisella tularensis in the sample is an existing type or a 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 is initiated in the field of Francisella tularensis, and is not reported in related documents; MNP markers are mainly developed based on reference sequences, and MNP sites which are large-scale and are distinguished from other species, polymorphic in the Francisella tularensis species and conserved in sequence at two sides can be mined according to reported resequencing data of the Francisella tularensis representative race; MNP site detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of the MNP site; and then a set of MNP locus with the largest polymorphism and high specificity and a primer combination with the best compatibility can be screened out according to the test result of the standard substance.
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 Francisella tularensis, a primer composition, a kit and application thereof. The provided 15 MNP loci of Francisella tularensis 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 mycobacterium combination are met, and the requirements of Francisella tularensis standard and shared fingerprint data construction are met; the need to accurately detect genetic variation between strains of Francisella tularensis; the requirements of homozygosity and heterozygosity of Francisella tularensis are identified, and technical support is provided for scientific research, scientific monitoring and control of Francisella tularensis.
Drawings
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 Francisella tularensis;
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:
the invention develops a novel molecular marker-MNP marker which is suitable for detecting group organisms and is specific to species. 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; (2) Species of speciesThe identification capability is strong, 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 labels based on the combination of super multiplex PCR and a second generation high throughput sequencing technology has the following advantages: (1) The output is a base sequence, 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. Thus, the present invention developed MNP marker loci for Francisella tularensis that are genomic regions screened on Francisella tularensis genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including marker loci for MNP-1-MNP-15 of the reference genome with AJ 749949.
Next, the present invention has developed a multiplex PCR primer composition for detecting MNP marker loci of Francisella tularensis, characterized in that the multiplex PCR primer composition comprises 15 pairs of primers, the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO.1-SEQ ID NO. 30. The primers do not collide with each other, and efficient amplification can be performed by multiplex PCR.
The multiplex PCR primer composition can be used as a detection kit for detecting the MNP marker locus of Francisella tularensis.
The kit disclosed by the invention can accurately and sensitively detect Francisella tularensis with the concentration as low as 10 copies/reaction.
The MNP markers and the kits of the invention detect high specificity of target microorganisms in complex templates.
The marker and primer combination developed in the present invention 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 sites, primer compositions, kits and applications of the MNP-labeling sites, primer compositions, kits of Francisella tularensis of the present application will be described in detail with reference to examples, comparative examples and experimental data.
Example 1 selection of MNP marker loci of Francisella tularensis and design of multiplex PCR amplification primers
S1, screening MNP (MNP) marker locus of Francisella tularensis
Based on complete or partial sequences of genome of 320 different isolates of Francisella tularensis disclosed on the network, 15 MNP marker loci 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 15 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 of a representative strain of Francisella tularensis as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphism sites of each strain of Francisella tularensis;
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.
In this embodiment, the primers used to identify the MNP marker sites are shown in table 1.
S3, evaluating detection efficiency of primer combination
The detection method of the MNP marker is that all MNP loci are amplified at one time through multiplex PCR, amplification products are sequenced through second-generation high-throughput sequencing, sequencing data are analyzed, and compatibility of the primer combination is evaluated according to the detected loci.
The primer combination is used for screening and amplifying uniformly and optimally compatible primer combinations according to detection conditions of MNP loci in 4 libraries, and finally the primer combinations of 15 MNP loci in table 1 are screened out.
Threshold settings and Performance assessment for MNP locus and primer identification of Francisella tularensis described in example 2
1. Detection of MNP markers
In this example, francisella tularensis nucleic acid standards of known copy numbers were added to human genomic DNA to prepare 1-, 10-, and 100-copy Francisella tularensis simulated samples. 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. And according to the number of sequencing fragments and the number of sites of MNP sites of Francisella tularensis detected in a blank control and a Francisella tularensis nucleic acid standard in 12 repeated experiments, evaluating the reproducibility, the accuracy and the sensitivity of a detection method, and formulating a quality control system pollution and a threshold value for detecting a target pathogen. The detection flow of MNP markers is shown in fig. 3.
TABLE 2 detection sensitivity and stability analysis of MNP labeling method of Francisella tularensis
2. Reproducibility and accuracy assessment of MNP (MNP) marker detection kit for detecting Francisella tularensis
Based on whether the genotype of the co-detected locus is reproducible in the two repetitions, the reproducibility and accuracy of detection of Francisella tularensis by the MNP marker detection method are evaluated. Specifically, the paired comparison was performed on 12 sets of data for 100 copies of the sample, respectively, and the results are shown in table 3.
TABLE 3 reproducibility and accuracy assessment of Francisella tularensis MNP marker detection method
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 project reproducibility test, 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%. Therefore, the kit of the invention can accurately detect Francisella tularensis as low as 10 copies/reaction.
3. Threshold value judgment for detecting Francisella tularensis by MNP (MNP) mark detection kit
As shown in Table 2, sequences aligned to Francisella tularensis can be detected in 1 copy/reaction samples, covering at least 1 MNP site. Sequences of Francisella tularensis were 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 4.5 megabases. The measurement and calculation basis is that the number of MNP loci detected by each sample is 15, and the length of one sequencing fragment is 300 bases, so that when the data size is more than 4.5 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 franciscensis in the test sample and the noise index P of franciscensis in the blank, wherein:
the blank noise index p=nc/Nc, where Nc and Nc represent the number of sequenced fragments and total number of sequenced fragments of francissampsonii, respectively, in the blank.
The signal index of the test sample s=nt/Nt, where Nt and Nt represent the number of sequenced fragments and the total number of sequenced fragments of francissampsonii, 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 Francisella tularensis in samples to be tested
As shown in Table 4, the average noise index of Francisella tularensis in the control was 0.04%, the average signal index in the 1-copy sample was 0.26%, and the average signal-to-noise ratio of the 1-copy sample and the control was 6.5, so that the present invention provides that when the signal-to-noise ratio was more than 10-fold, it was judged that the contamination in the detection system was acceptable.
The average signal to noise ratio of the 10 copies of the sample and the blank was 67.2, and at least 7 MNP sites were stably detected in the 10 copies/reaction 12 sets of data, accounting for 46.7% of the total sites. Therefore, under the condition of ensuring accuracy, the standard prescribes that the Francisella tularensis positive judgment standard is: when the signal-to-noise ratio of Francisella tularensis in the sample is more than 30 and the site detection rate is more than or equal to 30%, judging that the Francisella tularensis nucleic acid is detected in the sample.
Therefore, the kit provided by the invention can accurately and sensitively detect Francisella tularensis with the copy/reaction as low as 10.
4. MNP marker detection method for detecting specificity evaluation of Francisella tularensis
The DNA of Francisella tularensis, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao termates, 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 are artificially mixed together according to the equimolar amount to prepare a mixed template, and the blank template is used as a control to detect Francisella tularensis in the mixed template 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, francisella tularensis in the mixed template can be detected specifically 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 Francisella tularensis strains
The kit and MNP marker locus detection method are utilized to detect 6 sub-generation strains of one Francisella tularensis strain stored in the same laboratory, samples are sequentially named as S1-S6, the average coverage of sequencing of each sample is 1416 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, wherein 1 part (S-2) of the total genotype of 5 parts of Francisella tularensis detected together with the same batch are different from each other at partial loci (Table 5), and strain-to-strain variation exists.
TABLE 5 detection analysis of 6 Francisella tularensis
As can be seen from Table 5, the application of the kit in identifying genetic variation among strains by detecting MNP markers can be used for guaranteeing genetic consistency of Francisella tularensis strains named identically in different laboratories, so that comparability of research results is guaranteed, and the kit has important significance for scientific research of Francisella tularensis. In clinical terms, one can take into account the diagnostic regimen as to whether the site of the difference affects resistance.
Example 4 detection of genetic variation inside Francisella tularensis Strain
As a group organism, partial individuals in the Francisella tularensis group are mutated, so that the group is no longer homozygous to form a heterogeneous heterozygous group, and the stability and consistency of the phenotype of the microorganism for the test are influenced. Such variants, when detected by molecular marker detection on the population, appear as alleles outside the major genotype of the locus. 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 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, nucleic acids of two strains having a difference in genotype 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 the two strains, loci with heterozygous genotypes are detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marker detection method for mycoplasma pneumoniae in detecting genetic variation inside strain groups is demonstrated.
Example 5 construction of Francisella tularensis DNA fingerprint database
All strains or DNA of samples used for constructing DNA fingerprint databases of Francisella tularensis 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) comparing the sequencing data of the 6 strains with the reference genotype, and obtaining the main genotype of each site of each strain to form the MNP fingerprint of each strain. And recording the obtained MNP fingerprint of each strain into a database file to form a Francisella tularensis DNA fingerprint database.
The constructed MNP fingerprint database is based on the gene sequence of the detected strain, is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared and being updated at any time. And comparing the MNP fingerprint of the strain obtained by each detection with an MNP fingerprint database constructed based on the existing genome data, and inputting the MNP fingerprint of the strain with the main genotype difference into the constructed MNP fingerprint database to achieve real-time updating and co-construction sharing of the database.
Example 6 use in Francisella tularensis fine powder
And detecting the 6 parts of Francisella tularensis strains by using the primer combination and MNP marking site detection method, and obtaining MNP fingerprint of each strain. And comparing the DNA fingerprint of each strain with the constructed fingerprint database in pairs, defining the existing variants as the same as the existing fingerprint database, defining the variants as new variants with main genotype differences at least one MNP locus, and realizing the fine typing of Francisco tularensis. Detection of 6 samples of Francisella tularensis As shown in Table 5, 1 part of 6 samples of Francisella tularensis was detected with 5 other major genotypes at 2 MNP sites, possibly in different variants. Therefore, the resolution of the method on Francisella tularensis reaches the level of single base, and the fine typing of Francisella tularensis in the sample can be realized.
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.
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Claims (5)
1. The multiplex PCR primer composition for detecting the MNP marking site of Francisella tularensis is characterized in that the multiplex PCR primer composition is 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO.1-SEQ ID NO. 30.
2. A detection kit for detecting a MNP marker locus of francisco, 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 the non-diagnostic purpose identification of franciscensis terrestris.
5. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 for constructing a francisco database.
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