CN114277165B - MNP (MNP) marking combination of plague bacillus, primer pair combination, kit and application of MNP marking combination - Google Patents

Abstract

The invention belongs to the technical field of molecular biology, and discloses MNP (MNP) marking combination, primer pair combination, kit and application thereof of plague bacillus, wherein MNP marking loci refer to genome regions which are screened on staphylococcus aureus genome and are separated from other species and have a plurality of nucleotide polymorphisms in the species, and the MNP marking loci comprise MNP-1-MNP-15; the primer pair combination comprises 15 pairs of primers, and the specific nucleotide sequence is shown as SEQ ID NO.1-SEQ ID NO. 30. The MNP marker combination can specifically identify plague bacillus and finely distinguish different species; the primers are not interfered with each other, and the sequence analysis can be carried out on all the mark combinations of multiple samples at one time by integrating multiple amplification and sequencing technologies, so that the method has the detection advantages of high flux, multiple targets, high sensitivity, high precision and culture avoidance, can be applied to the identification and genetic variation detection of the pestis of a large scale of samples, and has important significance on scientific research and epidemic prevention monitoring of the pestis.

Description

MNP (MNP) marking combination of plague bacillus, primer pair combination, kit and application of MNP marking combination

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to MNP (MNP) marking combination, primer pair combination, kit and application thereof of plague bacillus.

Background

Plague bacillus (Yersinia pestis) belongs to the genus Yersinia, which is a pathogenic bacterium causing pestis of virulent infectious disease, and is also one of the lethal bacterial warfare agents. Plague is a type A infectious disease regulated by the infectious disease control method in China, has strong infectivity and mortality, and people are infected by contacting infected animals, eating polluted food or being bitten by rat fleas animals and the like.

In addition, when the plague bacillus is used as a colony organism, individuals in the plague bacillus colony often have variation in interaction with hosts and environment, so that the detection method and the prevention method are ineffective; for laboratory studies of plague bacillus, this variation also results in the same named strain being virtually different in different laboratories or different times in the same laboratory, resulting in irreproducible and incomparable experimental results. Therefore, the development of a rapid, accurate and mutation-monitoring plague bacillus detection and analysis method has important significance for scientific research and epidemic prevention of plague bacillus.

Classical methods for pestis detection, 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 molecular marker 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 with high polymorphism and a detection technology thereof is a technical problem to be solved urgently.

The invention develops a novel molecular marker-MNP marker 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 allele type is rich, and single MNP mark has 2 ⁿ The species allelic type is higher than SSR and SNP; (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 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 labels 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. Therefore, there is a need to develop MNP markers and detection primers for pathogenic microorganisms plague bacillus. 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.

Disclosure of Invention

The invention aims to provide MNP (MNP) marking combination, primer pair combination, kit and application thereof of pestis, which can identify and variant detection of pestis and has the effects of multiple targets, high flux, high sensitivity and fine typing.

In a first aspect of the invention, there is provided a combination of MNP markers of plague bacillus, the MNP markers being genomic regions screened on the plague bacillus genome that are distinct from other species and have a plurality of nucleotide polymorphisms within the species, the combination of MNP markers comprising 15 markers of MNP-1 to MNP-15 on a reference sequence of plague bacillus AL 590842. The specific nucleotide sequences of MNP-1-MNP-15 markers in the technical scheme are shown as SEQ ID NO.1-SEQ ID NO. 15. Description table 1 further details that the start and end positions of the MNP markers noted in table 1 are determined based on the reference sequence AL590842 in table 1.

In a second aspect of the invention, there is provided a multiplex PCR primer pair combination for detecting said MNP marker combination, said multiplex PCR primer pair combination comprising 15 pairs of primers, the specific primer sequences being shown in SEQ ID NO.16-SEQ ID NO. 45. In the above technical solution, each MNP-labeled primer includes an upper primer and a lower primer, and is specifically shown in table 1 of the specification.

In a third aspect of the invention, there is provided a detection kit for detecting said combination of MNP markers of plague bacteria, said kit comprising said combination of primer pairs.

Further, the kit further comprises a multiplex PCR premix.

And the MNP mark combination of the plague bacillus or the primer pair combination or the application of the detection kit in qualitative detection of the plague bacillus of non-eruption purpose, and the application in preparation of a plague bacillus qualitative detection product.

In a fourth aspect of the invention, the MNP marker combination of the plague bacillus or the multiplex PCR primer pair combination or the application of the detection kit in the identification of the plague bacillus, the construction of a DNA fingerprint database and the detection of genetic variation are provided.

In the above application, firstly, the total DNA of the bacteria of the sample to be tested is obtained; performing a first round of multiplex PCR amplification on the total DNA and the blank control by using the kit, wherein the number of cycles is not higher than 25; 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 plague bacillus 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 the plague bacillus and the number of the detected MNP markers obtained in the total DNA and the blank control, and obtaining the number of the detected MNP markers, the number of the sequencing sequences covering each MNP marker and the MNP marker genotype data.

When the method is used for the identification of the plague bacillus, whether the sample to be detected contains the nucleic acid of the plague bacillus or not is judged after quality control according to the number of the sequencing sequences of the plague bacillus and the number of the MNP loci detected in the sample to be detected and the blank control. The quality control scheme and the judging method are characterized in that DNA of plague bacillus with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting plague bacillus are evaluated, and the quality control scheme and the judging method when the kit detects plague bacillus are formulated.

When used in the detection of genetic variation of pestis, genetic variation detection between strains and within strains is included. The detection of genetic variation among strains comprises the steps of obtaining genotype data of 15 MNP markers of each strain to be compared by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared are different from each other on the 15 MNP labels through genotype comparison. If the strain to be compared has a variation in the main genotype of at least one MNP marker, it is determined that there is a genetic variation in both. Alternatively, the 15 markers of the strain to be compared may be amplified by single PCR, respectively, and then Sanger sequencing may be performed on the amplified products to obtain sequences, and then the genotypes of each MNP marker of the strain to be compared may be aligned. If MNP markers of non-identical major genotypes are present, variations are present between the strains to be compared. When detecting genetic variation inside the strain, determining whether the MNP marker of the strain to be detected detects a secondary genotype other than the primary genotype through a statistical model. If the strain to be tested has the subgenotype in at least one MNP mark, judging that the strain to be tested has genetic variation.

When the method is used for constructing a DNA fingerprint database of the plague bacillus, genotype data of the MNP mark of the plague bacillus identified from a sample is recorded into a database file to form the DNA fingerprint database of the plague bacillus; and (3) when different samples are identified, comparing the samples with a DNA fingerprint database of the plague bacillus, identifying whether the plague bacillus in the samples has a difference of a main genotype (the MNP mark has a genotype supported by more than 50% of sequencing fragments) with strains in the database, wherein the plague bacillus with the main genotype difference in at least 1 MNP mark is a new mutation type, and recording the new mutation type in the DNA fingerprint database.

When the method is used for typing the plague bacillus, the plague bacillus in a sample to be tested is identified, and the genotype of each MNP locus is obtained; collecting genome sequences of plague bacillus disclosed on the net and constructing a plague bacillus DNA fingerprint database to form a plague bacillus reference sequence library; comparing the genotype of the plague bacillus in the sample to be tested with a reference sequence library of the plague bacillus, and screening strains which are genetically identical or closest to each other to obtain the typing of the plague bacillus in the sample to be tested. And identifying whether the plague bacillus in the sample is an existing type or a new variant according to the comparison result with the reference sequence library, and realizing the fine typing of the plague bacillus.

Compared with the prior art, the invention has the following advantages:

the invention provides MNP (MNP) marking combination of plague bacillus, primer pair combination, kit and application thereof. The provided 15 MNP markers of the plague bacillus 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 on the plague bacillus are met, and the requirements of standard and sharable fingerprint data construction of the plague bacillus are met; the need to accurately detect genetic variation between strains of plague bacillus; the need for homozygosity and heterozygosity of plague bacillus was identified.

The invention belongs to the first creation in the field of plague bacillus, and is not reported in related documents; MNP markers are mainly developed based on reference sequences, and MNP markers which are distinguished from other species on a large scale, polymorphic in the species of the plague bacillus and conserved in sequences at two sides can be mined according to reported resequencing data of the plague bacillus representing the race; MNP marker detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of MNP markers; and then a set of MNP (MNP) marks with maximum polymorphism and high specificity, a primer combination with the best compatibility and a detection method can be screened out according to the test result of the standard substance, and the method is used for detecting pestis, constructing DNA fingerprint, detecting genetic variation in strains and among strains and other related applications, and providing technical support for scientific research, scientific monitoring and prevention and control of the pestis.

Drawings

FIG. 1 is a schematic diagram of MNP marker polymorphism;

FIG. 2 is a flow chart of screening and primer design for MNP markers of P.pestis;

FIG. 3 is a flow chart of detection of MNP markers;

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

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 this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

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.

Example 1 screening of MNP marker combinations of Phytophthora and design of multiplex PCR amplification primers

S1, screening of MNP marker combinations of plague bacillus

Based on complete or partial sequences of genomes of 419 different isolates of plague bacillus disclosed on the net, 15 MNP markers 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 markers screened are shown in table 1:

MNP markers and detection primers starting positions on the reference sequence as described in Table 1

The step S1 specifically includes:

selecting a genome sequence of a representative strain of the plague bacillus as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleotide polymorphism markers of each strain of the plague bacillus;

on the reference genome, carrying out window translation by taking 100-300bp as a window and taking 1bp as a step length, and screening to obtain a plurality of candidate MNP (MNP) marker areas, wherein the candidate MNP marker areas contain more than or equal to 2 single nucleotide variation markers, and the single nucleotide polymorphism markers do not exist on sequences of 30bp at both ends;

screening a region with the discrimination DP of more than or equal to 0.2 from the candidate polynucleotide polymorphism marking regions as MNP marks; wherein dp=d/t, t is the log of comparisons when all the minor species in the candidate polynucleotide polymorphism marker region are compared pairwise, and d is the log of samples of at least two single nucleotide polymorphism differences in the candidate polynucleotide polymorphism marker region.

As an optional implementation mode, when screening is performed on the reference genome by taking 100-300bp 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 MNP marked multiplex PCR amplification primers 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 detection method of the MNP markers comprises the steps of amplifying all MNP markers at one time through multiplex PCR, sequencing amplified products through second-generation high-throughput sequencing, analyzing sequencing data, and evaluating the compatibility of the primer combination according to the detected markers.

The method comprises the steps of adding pestilence bacillus DNA with known copy number into human genome DNA to prepare a 1000-copy/reaction template, detecting by the MNP mark detection method, constructing 4 repeated sequencing libraries, screening the designed primer combination according to detection results, and finally screening to obtain the primer pair combination of 15 MNP marks which can be efficiently detected in the 4 libraries and 15 MNP marks with the best compatibility, wherein the primer pair combination is shown in the table 1.

Threshold setting and Performance evaluation for MNP marker and primer identification of Phytophthora plague 1 described in example 2, sensitivity and stability evaluation for detection of Phytophthora plague by MNP marker detection kit

In this example, a known copy number of a nucleotide standard of plague bacillus was added to human genomic DNA to prepare 1 copy/reaction, 10 copy/reaction, and 100 copy/reaction plague bacillus 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 (3) according to the number of sequencing fragments and the number of markers of the MNP markers of the plague bacillus detected in the blank control and the plague bacillus nucleotide standard substance in 12 repeated experiments, preparing a threshold value for pollution of a quality control system and detection of a target pathogen, and evaluating the reproducibility, the accuracy and the sensitivity of the detection method.

The detection flow of MNP markers is shown in fig. 3.

TABLE 2 detection sensitivity and stability analysis of MNP labeling method of pestis

As shown in Table 2, the kit can stably detect more than 8 MNP sites in 10 copies/reaction samples, and at most 1 MNP site in a few samples of 0 copies/reaction, can clearly distinguish between 10 copies/reaction and 0 copies/reaction samples, and has technical stability and detection sensitivity as low as 10 copies/reaction.

2. Reproducibility and accuracy assessment of MNP (MNP) marker detection kit for detecting plague bacillus

Based on whether the genotype of the marker is reproducible or not in the two repetitions, the reproducibility and accuracy of detection of plague bacillus by the MNP marker detection method are evaluated. Specifically, the data of 12 groups of 100 copies of the sample were compared in pairs, and the result is shown in Table 3, wherein the number of MNP markers with differences in the main genotypes is 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, r represents the reproducibility, i.e. the ratio of the number of reproducible markers of the main genotype to the number of common markers. 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%. Based on this, the kit can accurately and sensitively detect pestis with as low as 10 copies/reaction.

TABLE 3 reproducibility and accuracy assessment of MNP marker detection method of plague bacillus

3. Threshold judgment for detection of plague bacillus by MNP (MNP) mark detection kit

As shown in Table 2, the sequences aligned to pestis were detected in 1 copy/reaction samples, covering at least 1 MNP tag. The sequence of pestis was also detected in a partial blank. Because of the extreme sensitivity of MNP marker detection methods, contamination of the data in the detection is prone to false positives. The following quality control scheme is formulated in this example.

The quality control scheme is 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 labels 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 label reaches 1000 times by one experiment, and the accurate analysis of the base sequence of each MNP label is ensured.

2) Determining whether the contamination is acceptable based on the signal index S of plague bacillus in the test sample and the noise index P of plague bacillus 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 plague bacillus, 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 plague bacillus, respectively, in the test sample.

3) The detection rate of MNP markers in the test sample is calculated and refers to the ratio of the number of detected markers to the total number of designed markers.

TABLE 4 SNR of pestis in samples to be tested

As shown in Table 4, the average noise figure of pestis in the blank is 0.04%, the average signal figure in the 1 copy sample is 0.29%, and the average signal to noise ratio of the 1 copy sample and the blank is 6.9, so the present invention provides that when the signal to noise ratio is greater than 10 times, it can be judged that the contamination in the detection system is acceptable.

As shown in Table 4, the average signal-to-noise ratio of the 10 copies of the sample and the blank was 65.3, and at least 8 MNP markers were stably detected in the 10 copies/reaction 12 sets of data, accounting for 53.3% of the total markers. Therefore, under the condition of ensuring accuracy, the standard prescribes that the plague bacillus positive judgment standard is: when the signal to noise ratio of the plague bacillus in the sample is more than 30 and the mark detection rate is more than or equal to 30%, judging that the nucleotide of the plague bacillus is detected in the sample. Therefore, the kit provided by the invention can sensitively detect pestis with the concentration as low as 10 copies/reaction.

4. Specific evaluation of MNP marker detection method for detecting plague bacillus

The method comprises the steps of artificially mixing DNA of plague bacillus, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao termitis bacteria, 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 according to the equimolar amount, preparing a mixed template, detecting plague bacillus in the mixed template by adopting the method provided by the invention by adopting internal standard DNA as a blank control, and carrying out 3 repeated experiments. Results the sequencing sequences obtained in 3 replicates were aligned only to 14 MNP sites in plague. After analysis according to the quality control scheme and the judgment threshold, the nucleic acid of the plague bacillus is detected specifically in 3 repeated experiments, which shows that the MNP mark and the kit detect the high specificity of the plague bacillus in complex templates.

Example 3 detection of genetic variation between strains of Phytophthora

The kit and MNP mark detection method are utilized to detect 6 sub-generation strains of the same pestilence bacterial strain provided by the Hubei province disease control and prevention control center in different periods, samples are sequentially named as S1-S6, the average coverage multiple of sequencing of each sample is 1300 times, and all 15 MNP marks can be detected by each bacterial strain (table 5). The fingerprints of 6 strains are compared pairwise, the results are shown in table 5, the fingerprints of S2 and S4 are consistent, and the fingerprints of the other 4 parts are consistent, which indicates that the strains named in the same way have inter-strain variation due to genetic variation or genetic drift in the long-term propagation and preservation processes.

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 pestis strains in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of the pestis.

Table 3 6 detection and analysis of pestis

Example 4 detection of genetic variation inside a pestilence Strain

As a group organism, partial individuals in the plague bacillus group mutate, 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 markers on a population, appear as alleles outside the main genotype of the marker. 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) means that the number of sequences of the wrong allele carrying n SNPs is the highest proportion of the total number of sequences of the marker. 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 markers.

TABLE 6 critical values for determining the hypo-isogenotypes at partial sequencing depth

According to the above parameters, nucleotides 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 MNP marked genotypes of the two strains, the mark with the heterozygous genotype is detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marked detection method for the plague bacillus in detecting the genetic variation inside the strain group is demonstrated.

EXAMPLE 5 construction of DNA fingerprint database of pestis

All strains or DNA of samples used for constructing a DNA fingerprint database of the plague bacillus 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 marker 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 an MNP fingerprint database of the plague bacillus.

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. Comparing MNP fingerprint of each strain obtained by each detection with a constructed MNP fingerprint database, and inputting MNP fingerprint of strains with main genotypes different into the constructed MNP fingerprint database to achieve real-time updating and co-construction sharing of the database.

Example 6 use in the fine subdivision of pestis

And detecting the 6 strains of the plague bacillus by using the primer combination and the MNP mark detection method, so as to obtain MNP fingerprint of each strain. And (3) comparing the DNA fingerprints of each strain in pairs, then comparing the DNA fingerprints with the disclosed reference sequence of the plague bacillus and a constructed fingerprint database, and screening to obtain the strain with the closest fingerprint. As the existing fingerprint database, the MNP marker has main genotype difference in at least one MNP marker, and is a new variant, thus realizing the fine typing of the plague bacillus. Detection of 6 samples of pestis As shown in Table 5, 6 detected pestis were classified into 2 types, which are close to strain A112 and NCTC5923, respectively. Therefore, the resolution of the method for the plague bacillus reaches the level of single base, and the precise typing of the plague bacillus 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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<211> 148

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gctgagatcg gcaatctgta tgcgagaaat cgggccttgc tgatcaatta gccggtaaac 60

cgcggctcca ttgagttgtt ttaccagatc cacgttacca atttgtgctt gtccgccggt 120

gctcatcaat aaagttactc gcttatga 148

<210> 10

<211> 149

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ccgccgcctg actaacattg gaacgttgcg cgcctatctg gtggcttatc tgcgggcaca 60

tctggggatc catcagtcga tgacattaat ggtgcgccaa cttgccccaa cacccgaggg 120

cttgccgcta gaagtttatg cttttacca 149

<210> 11

<211> 146

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gcggtgaaac aattagccat ctgtctggcc agtgctagcg atatcagctt gcgggctaaa 60

aaatcggaaa aagccgcaca acagttgttc ggggcattac tcgcgaaagc attgcaattc 120

cctgatcaaa accatgttta tctggt 146

<210> 12

<211> 150

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

caccggttta tagcagcttg caatccctct caacgcagga gactggcttg tttatggcat 60

tgtctatctt cgccggcctt gtcgcccagt ttccactcag ttggttatca gaccgctata 120

accggacaca attgatccga attaacgctc 150

<210> 13

<211> 147

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

acagggtatg tgtcaggcag cgatttccgc cctgacagcc ttctttaccc tgacaacggg 60

ttgcatcaac ggattcgtca acggcagtga taaccgcgcc aaccgcgatt gctgatgcat 120

ctttgcccag cagataacca ccgcctg 147

<210> 14

<211> 150

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gatacagtac cgttcagttg gagaagggtt tccaactctt gttcaagatc tgccagacga 60

ggttccaggc aggcaataat tcgctcacca gcttctgtgg gagcgacact tcgcgttgtt 120

cgggtcagaa ggcgtaggtt taatctttct 150

<210> 15

<211> 150

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ttaaaatagc cccagtgtta ttgccattgc agctaggtag cgagtaaatc ctgaacagtg 60

aaaaccgcga ataacgcaat aacatcaagg atgatggaga tggcagttaa gcatctacat 120

aaacactttt gatatgagca tcgaatgagt 150

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

attacgtccc aataccggat ctatc 25

<210> 17

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gtaggttttt acgcaaccac agtg 24

<210> 18

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

gacgtgcggg ttcacattg 19

<210> 19

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

ccaaattagc cagtgccggt aa 22

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

cttccgcgtc aagctgtatg 20

<210> 21

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

agaaaacgat ccactcactg acg 23

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

gtacggttta ctcacataaa cgtca 25

<210> 23

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

tttcaccgcc cactcactct a 21

<210> 24

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

gctgagatcg gcaatctgta tg 22

<210> 25

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

ccgccgcctg actaacatt 19

<210> 26

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

gcggtgaaac aattagccat ctg 23

<210> 27

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

caccggttta tagcagcttg c 21

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

acagggtatg tgtcaggcag 20

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

gatacagtac cgttcagttg gagaa 25

<210> 30

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

ttaaaatagc cccagtgtta ttgcc 25

<210> 31

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

agcccgttgt acgatggtg 19

<210> 32

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

ccagtggctt cctgatttct g 21

<210> 33

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

cactacgact catgacaccg tg 22

<210> 34

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

ctatcggtgc agtgcaaaca tc 22

<210> 35

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

aatcaaaact ccgcgagtcc a 21

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

ccaagcgtgg caatattcat g 21

<210> 37

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

agaatggaat gtattgtgtc gataat 26

<210> 38

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

agcaaagaac atccttatta gcaaaga 27

<210> 39

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

tcataagcga gtaactttat tgatgag 27

<210> 40

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

tggtaaaagc ataaacttct agcgg 25

<210> 41

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

accagataaa catggttttg atcagg 26

<210> 42

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

gagcgttaat tcggatcaat tgt 23

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

caggcggtgg ttatctgctg 20

<210> 44

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 44

agaaagatta aacctacgcc ttctg 25

<210> 45

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 45

actcattcga tgctcatatc aaaagt 26

Claims (9)

1. The MNP labeling combination of the plague bacillus is characterized by comprising 15 labels, and the specific nucleotide sequence is shown as SEQ ID NO.1-SEQ ID NO. 15.

2. A multiplex PCR primer pair combination for detecting the plague MNP marker combination according to claim 1, wherein the multiplex PCR primer pair combination comprises 15 pairs of primers having the nucleotide sequence shown in SEQ ID No.16-SEQ ID No. 45.

3. A detection kit for detecting the MNP marker combination according to claim 1, wherein said kit comprises the primer pair combination according to claim 2.

4. The test kit of claim 3, wherein the kit further comprises a multiplex PCR premix.

5. Use of the MNP-labelled combination of plague bacillus according to claim 1 or the primer pair combination of claim 2 or the detection kit of any one of claims 3-4 for the qualitative detection of plague bacillus of non-diagnostic purpose.

6. Use of the MNP marker combination of plague bacillus according to claim 1 or the primer pair combination of claim 2 or the detection kit of any one of claims 3-4 for the preparation of a qualitative detection product of plague bacillus.

7. Use of the MNP marker combination of plague bacillus according to claim 1 or the primer pair combination of claim 2 or the detection kit of any one of claims 3-4 for the detection of genetic variations inside and between plague bacillus strains for non-diagnostic purposes.

8. Use of the MNP marker combination of plague bacillus according to claim 1 or the primer pair combination of claim 2 or the detection kit of any one of claims 3-4 for constructing a plague bacillus database.

9. Use of the MNP marker combination of plague bacillus according to claim 1 or the primer pair combination of claim 2 or the detection kit of any one of claims 3-4 for the non-diagnostic purpose of the fine-grained detection of plague bacillus.

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