WO2023077489A1

WO2023077489A1 - Mnp marker combination of yersinia pestis, primer pair combination, kit, and application thereof

Info

Publication number: WO2023077489A1
Application number: PCT/CN2021/129167
Authority: WO
Inventors: 万人静; 高利芬; 方治伟; 陈利红; 肖华峰; 李甜甜; 李论; 周俊飞; 彭海
Original assignee: 江汉大学
Priority date: 2021-11-06
Filing date: 2021-11-06
Publication date: 2023-05-11

Abstract

Disclosed in the present invention are an MNP marker combination of Yersinia pestis, a primer pair combination, a kit, and an application thereof. The MNP marker site refers to a genome region which is screened on a genome of Yersinia pestis, is distinguished from other species, and has a plurality of nucleotide polymorphism inside the species. A combination of marker sites MNP-1 to MNP-15 is comprised. The specific nucleotide sequences of the 15 marker sites are as shown in SEQ ID NO. 1 to SEQ ID NO. 15. The primer pair combination comprises 15 pairs of primers. The specific nucleotide sequences of the 15 pairs of primers are as shown as SEQ ID NO. 16 to SEQ ID NO. 45. The MNP marker combination can be used for identification and typing of Yersinia pestis, construction of a DNA fingerprint database, genetic variation detection, etc.

Description

A kind of MNP marker combination of Yersinia pestis, primer pair combination, kit and application thereof

technical field

The embodiment of the present invention relates to the field of biotechnology, in particular to a MNP marker combination of Yersinia pestis, a primer pair combination, a kit and applications thereof.

Background technique

Yersinia pestis (Yersinia pestis) belongs to the genus Yersinia, it is the pathogenic bacteria that causes the severe infectious disease plague, and it is also one of the deadly bacterial warfare agents. Plague is a Class A infectious disease stipulated in my country's Law on the Prevention and Control of Infectious Diseases. It is highly contagious and fatal. People are infected through contact with infected animals, eating contaminated food, or being bitten by rats and fleas.

In addition, Yersinia pestis is a group organism, and in the interaction with the host and the environment, individuals in the Yersinia pestis group often mutate, resulting in the failure of detection methods and prevention methods; for laboratory research on Yersinia pestis, this Variation can also cause strains with the same name in different laboratories or in different periods of the same laboratory to be actually different, resulting in non-reproducible and non-comparable experimental results. Therefore, the development of a rapid, accurate, and variable-monitoring detection and analysis method for Yersinia pestis is of great significance for the scientific research and epidemic prevention of Yersinia pestis.

Classical detection methods of Yersinia pestis, including isolation and culture, PCR technology, whole genome and metagenomic sequencing, etc., have one or more limitations in terms of time length, operational complexity, detection throughput, accuracy and sensitivity of detection of mutations, and cost. The targeted molecular marker detection technology that combines ultra-multiplex PCR amplification and high-throughput sequencing can enrich target microorganisms in samples with low microbial content, avoiding a large amount of data waste and background caused by whole genome and metagenomic sequencing Noise, which has the advantages of less sample requirement, accurate diagnosis results, saving data volume, and detecting low-frequency variation.

The molecular markers detected by the existing targeted molecular marker detection technology mainly include SNP and SSR markers. SSR markers are recognized as the most polymorphic markers, but their number is small in microorganisms; SNP markers are huge in number, densely distributed, and are dimorphic markers, and the polymorphism of a single SNP marker is not enough to capture potential alleles in microbial populations genetic diversity. Therefore, the development of new molecular markers with high polymorphism and their detection technology has become an urgent technical problem to be solved.

The present invention develops a species-specific novel molecular marker-MNP marker. MNP markers refer to polymorphic markers caused by multiple nucleotides in an upper region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) rich allelic types, with 2 ⁿ allele types on a single MNP marker, higher than SSR and SNP markers; (2) strong species discrimination ability , only a small amount of MNP markers are needed to achieve species identification, reducing the detection error rate. The MNP marker method based on ultra-multiplex PCR combined with next-generation high-throughput sequencing technology to detect MNP markers has the following advantages: (1) The output is base sequence, without parallel experiments, and a standardized database can be constructed for sharing; (2) High Efficiency, using sample DNA barcodes, breaking through the limitation of the number of sequencing samples, and can type tens of thousands of MNP markers in hundreds of samples at one time; (3) High sensitivity, using multiplex PCR to detect multiple targets at a time, avoiding single Target amplification failures lead to high false negatives and low sensitivity; (4) high accuracy, using a second-generation high-throughput sequencer to sequence the amplified product hundreds of times.

In view of the above advantages and characteristics, MNP marker and its detection technology MNP marker method can realize the classification and traceability of multi-allelic genotypes in populations, and has application potential in the identification of pathogenic microorganisms, fingerprint database construction, and genetic variation detection. At present, in microorganisms, there is no report on MNP labeling, and there is a lack of corresponding technology. Therefore, there is an urgent need to develop MNP markers and detection primers for the pathogenic microorganism Yersinia pestis. The combination of markers and primers developed in the present invention will be used to formulate national standards for pathogen detection (plan number 20201830-T-469), which will be released by the end of 2021.

Contents of the invention

The purpose of the present invention is to provide a MNP marker combination, primer pair combination, kit and application of Yersinia pestis, which can identify and detect mutations of Yersinia pestis, and have the effects of multi-target, high throughput, high sensitivity and fine typing .

In the first aspect of the present invention, a combination of MNP markers of Yersinia pestis is provided, the MNP markers refer to the MNP markers that are screened on the Yersinia pestis genome to distinguish them from other species and have multiple nucleotide polymorphisms within the species In the genome region, the MNP marker combination includes 15 markers from MNP-1 to MNP-15 on the reference sequence of Yersinia pestis AL590842.

The specific nucleotide sequence of the MNP-1～MNP-15 marker in the above technical scheme is shown in SEQ ID NO.1-SEQ ID NO.15, wherein ID NO.16-SEQ ID NO.30 is the upper primer, ID NO.31-SEQ ID NO.45 is the lower primer. Table 1 of the description further specifies that the start and end positions of the MNP marker marked in Table 1 are determined based on the reference sequence AL590842 in Table 1.

In a second aspect of the present invention, there is provided a combination of multiple PCR primer pairs for detecting the MNP marker combination, the multiple PCR primer pair combination includes 15 pairs of primers, and the specific primer sequences are as SEQ ID NO.1- Shown in SEQ ID NO.30.

In the above technical scheme, each MNP-labeled primer includes an upper primer and a lower primer, as shown in Table 1 of the specification.

In the third aspect of the present invention, a detection kit for detecting the Y. pestis MNP marker combination is provided, the kit includes the primer pair combination.

Further, the kit also includes a multiplex PCR master mix.

And the application of the MNP marker combination of Yersinia pestis or the primer pair combination or detection kit in the qualitative detection of Yersinia pestis of non-diagnosed species, and the application in the preparation of qualitative detection products of Yersinia pestis.

In the fourth aspect of the present invention, there is provided the MNP marker combination of Y. pestis or the combination of multiple PCR primer pairs or the detection kit in the identification of Y. pestis, the construction of DNA fingerprint database, and the detection of genetic variation. in the application.

In the above-mentioned application, at first be to obtain the bacterial total DNA of the sample to be tested; Utilize the kit of the present invention to carry out the first round of multiplex PCR amplification to the total DNA and the blank control, and the number of cycles is not higher than 25; After the amplified product is purified, add sample tags and next-generation sequencing adapters based on the second round of PCR amplification; quantify the second-round amplified product after purification; detect multiple strains by combining the second-round amplified product, etc. High-throughput sequencing was carried out after the amounts were mixed; the sequencing results were compared to the reference sequence of Yersinia pestis, and the number of detected sequences and genotype data in the total DNA were obtained. According to the number of sequenced sequences of Yersinia pestis and the number of detected MNP markers obtained in the total DNA and the blank control, data quality control and data analysis were performed on the sequencing data of the total DNA to obtain the number of detected MNP markers, coverage The sequence number of each MNP marker and the genotype data of the MNP marker.

When used for the identification of Yersinia pestis, according to the number of sequencing sequences of Yersinia pestis detected in the sample to be tested and the blank control and the number of detected MNP sites, after quality control, it is determined whether the nucleic acid of Yersinia pestis is contained in the sample to be tested . Wherein, the quality control scheme and determination method use the DNA of Yersinia pestis with a known copy number as the detection sample, evaluate the sensitivity, accuracy and specificity of the kit to detect Yersinia pestis, and formulate the kit to detect plague The quality control scheme and determination method for bacillus.

When used for the detection of genetic variation of Yersinia pestis, it includes the detection of genetic variation between strains and within strains. The detection of genetic variation between strains includes using the above kit and method to obtain the genotype data of each of the 15 MNP markers of the strains to be compared. Through genotype comparison, analyze whether there are differences in the main genotypes of the strains to be compared on the 15 MNP markers. If there is a variation in at least one main genotype of the MNP marker in the strain to be compared, it is determined that there is a genetic variation between the two. As an alternative, it is also possible to amplify the 15 markers of the strain to be compared by single-plex PCR, and then perform Sanger sequencing on the amplified products. After obtaining the sequence, compare the genotype of each MNP marker of the strain to be compared. right. If there are MNP markers with inconsistent main genotypes, there is variation among the strains to be compared. When detecting the genetic variation within the strain, it is judged by statistical model whether the MNP markers in the strain to be tested detect sub-genotypes other than the main genotype. If the strain to be tested has a subgenotype at least one MNP marker, it is determined that there is genetic variation within the strain to be tested.

When used to construct the Yersinia pestis DNA fingerprint database, the genotype data of the MNP marker of the Yersinia pestis identified from the sample is entered into the database file to form the DNA fingerprint database of the Yersinia pestis; when different samples are identified each time, by Compared with the DNA fingerprint database of Yersinia pestis, identify whether the Yersinia pestis in the sample and the strains in the database have a main genotype (a genotype supported by more than 50% sequencing fragments in an MNP marker) at the MNP marker Differences, Yersinia pestis with major genotype differences in at least one MNP marker is a new variant type, which is included in the DNA fingerprint database.

When used for Yersinia pestis typing, it is to identify the Yersinia pestis in the sample to be tested, and obtain the genotype of each MNP site; collect the genome sequence of Yersinia pestis published on the Internet and the constructed DNA fingerprint database of Yersinia pestis Constitute the reference sequence library of Yersinia pestis; compare the genotype of Yersinia pestis in the sample to be tested with the reference sequence library of Yersinia pestis, screen the genetically consistent or closest strains, and obtain the type of Yersinia pestis in the sample to be tested . According to the comparison result with the reference sequence library, it is identified whether the Yersinia pestis in the sample is an existing type or a new variant, and the fine typing of the Yersinia pestis is realized.

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

The invention provides a MNP marker combination of Yersinia pestis, a primer pair combination, a kit and applications thereof. The provided 15 MNP markers of Yersinia pestis and their primer combinations can be used for multiplex PCR amplification, and the next-generation sequencing platform can be used to sequence the amplified products, which meets the needs of high-throughput, high-efficiency, high-accuracy and The demand for high-sensitivity detection meets the requirements for the construction of standard and shareable fingerprint data for Y. pestis; the need for accurate detection of genetic variation among Y. pestis strains; the need for identifying homozygous and heterozygous Y. pestis.

The present invention is the first in the field of Yersinia pestis, and there are no related literature reports; MNP markers are mainly developed based on reference sequences, and according to the resequencing data of the reported Yersinia pestis representative races, large-scale identifications of Yersinia pestis species, which are different from other species, can be mined MNP markers with polymorphic and conserved sequences on both sides of the species; MNP marker detection primers suitable for multiplex PCR amplification can be designed through the conserved sequences on both sides of the MNP markers; and a set of multiple A set of MNP markers with the largest morphism and high specificity, the most compatible primer combination and detection method, and used in the detection of Yersinia pestis, the construction of DNA fingerprints, the detection of genetic variation within and between strains and other related applications, Provide technical support for scientific research, scientific monitoring and control of Yersinia pestis.

Description of drawings

Figure 1 is a schematic diagram of MNP marker polymorphism;

Fig. 2 is the screening and primer design flowchart of Yersinia pestis MNP marker;

Fig. 3 is the detection flowchart of MNP mark;

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

It should be noted that, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field of the present invention. Terms used in the description of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the examples of the present invention can be purchased from the market or prepared by existing methods.

Example 1 Screening of Yersinia pestis MNP marker combinations and design of multiplex PCR amplification primers

S1, Screening of Yersinia pestis MNP marker combinations

Based on the complete or partial genome sequences of 419 different isolates of Y. pestis published online, 15 MNP markers were obtained through sequence alignment. For species that do not have genomic data on the Internet, the genome sequence information of representative races of the microbial species to be detected can also be obtained through high-throughput sequencing, where high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure the polymorphism of the selected markers, the genome sequences of at least 10 isolates are generally used as references.

The 15 MNP markers screened are shown in Table 1:

The starting positions of the MNP markers and detection primers on the reference sequence in Table 1

The step S1 specifically includes:

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

On the reference genome, use 100-300bp as the window, and perform window translation with 1bp as the step size, and screen to obtain multiple candidate MNP marker regions, wherein the candidate MNP marker regions contain ≥ 2 of the single nucleotides Variation markers, and the single nucleotide polymorphism markers do not exist on the 30bp sequences at both ends;

In the candidate polynucleotide polymorphic marker region, the region with a discrimination degree DP≥0.2 is selected as the MNP marker; wherein, DP=d/t, and t is all the small polynucleotide polymorphic marker regions in the candidate polynucleotide d is the logarithm of comparisons of at least two single nucleotide polymorphisms in the region marked by the candidate polynucleotide polymorphism.

As an optional implementation, other step lengths can also be used when screening on the reference genome with a window of 100-300 bp. In this embodiment, the step size is 1 bp, which is conducive to comprehensive screening.

S2, the design of multiple PCR amplification primers

The multiple PCR amplification primers labeled with MNP are designed by primer design software. The primer design follows that the primers do not interfere with each other. All primers can be combined into a primer pool for multiple PCR amplification, that is, all designed primers can be used in one amplification reaction. normal expansion.

S3. Evaluation of detection efficiency of primer combinations

The method for detecting the MNP markers is to amplify all the MNP markers at one time through multiplex PCR, sequence the amplified products through second-generation high-throughput sequencing, analyze the sequencing data, and evaluate the primers according to the detected markers. Combination compatibility.

Use Yersinia pestis DNA with a known copy number, add it to human genomic DNA, prepare a template of 1000 copies/reaction, detect it by the MNP marker detection method, construct 4 repeated sequencing libraries, and perform the sequencing according to the detection results. The designed primer combinations were screened, and finally the combination of 15 MNP markers provided by the present invention that could be detected efficiently in the 4 libraries and the 15 MNP markers with the best compatibility were finally screened, specifically as shown in Table 1 Show.

Example 2 Threshold setting and performance evaluation of MNP markers and primers identifying Yersinia pestis

1. Evaluation of the sensitivity and stability of the MNP marker detection kit for the detection of Yersinia pestis

In this example, the Y. pestis nucleotide standard with known copy number was added to human genomic DNA to prepare mock samples of Y. pestis with 1 copy/reaction, 10 copies/reaction and 100 copies/reaction. At the same time, an equal volume of sterile water was set as a blank control. A total of 4 samples were constructed, and 3 replicate libraries were constructed per day for each sample, and were tested continuously for 4 days, that is, 12 sets of sequencing data were obtained for each sample, as shown in Table 2. According to the number of sequenced fragments and markers of Y. pestis MNP markers detected in the blank control and Y. pestis nucleotide standards in 12 repeated experiments, the thresholds for contamination of the quality control system and detection of target pathogens were established, and the detection was evaluated. Method reproducibility, accuracy, sensitivity.

The detection process of MNP markers is shown in Figure 3.

Table 2 Detection sensitivity and stability analysis of Yersinia pestis MNP labeling method

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

2. Evaluation of the reproducibility and accuracy of the MNP marker detection kit for the detection of Yersinia pestis

Based on whether the genotypes of the co-detected markers were reproducible in the two replicates, the reproducibility and accuracy of the MNP marker detection method for the detection of Y. pestis were evaluated. Specifically, 12 sets of data of 100 copies of samples were compared in pairs. The results are shown in Table 3. The number of MNP markers with differences in main genotypes is 0; It is the principle of accuracy, the accuracy rate a=1-(1-r)/2=0.5+0.5r, r represents the recurrence rate, that is, the ratio of the number of reproducible markers of the main genotype to the number of common markers. In the reproducibility test of this project, the logarithm of the difference of the main genotype of the MNP marker between different libraries and different database 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 Yersinia pestis as low as 10 copies/reaction.

Table 3 Evaluation of the reproducibility and accuracy of the detection methods of Yersinia pestis MNP markers

3. Threshold determination for the detection of Yersinia pestis by the MNP marker detection kit

As shown in Table 2, the sequence aligned to Y. pestis can be detected in 1 copy/reaction sample, covering at least 1 MNP marker. In some blank controls, the sequence of Yersinia pestis was also detected. Due to the extreme sensitivity of the MNP marker detection method, data contamination during the detection process can easily lead to false positives. Therefore, the following quality control plan was formulated in this example.

The quality control plan is as follows:

1) The amount of sequencing data is greater than 4.5 million bases. The calculation is based on the fact that the number of MNP markers detected in each sample is 15, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 4.5 million bases, most samples can be guaranteed to cover each marker in one experiment. The number of sequencing fragments reaches 1000 times, ensuring accurate analysis of the base sequence of each MNP marker.

2) Determine whether the pollution is acceptable according to the signal index S of Yersinia pestis in the test sample and the noise index P of Yersinia pestis in the blank control, wherein:

Blank control noise index P=nc/Nc, wherein nc and Nc respectively represent the number of sequenced fragments and the total number of sequenced fragments of Yersinia pestis in the blank control.

The signal index of the test sample S=nt/Nt, wherein nt and Nt respectively represent the number of sequenced fragments and the total number of sequenced fragments of Yersinia pestis in the test sample.

3) Calculate the detection rate of MNP markers in the test sample, which refers to the ratio of the number of detected markers to the total number of designed markers.

Table 4 The signal-to-noise ratio of Yersinia pestis in the samples to be tested

As shown in Table 4, the mean value of the noise index of Yersinia pestis in the blank control is 0.04%, while the mean value of the signal index in the sample of 1 copy is 0.29%, the signal-to-noise ratio of the sample of 1 copy and the blank control The average value of is 6.9, therefore, the present invention stipulates 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 value of the signal-to-noise ratio of 10 copies of the sample and the blank control is 65.3, and in the 12 sets of data of 10 copies/reaction, at least 8 MNP markers can be stably detected, accounting for the total markers 53.3%. Therefore, in the case of ensuring the accuracy, this standard stipulates that the positive judgment standard of Yersinia pestis is: when the signal-to-noise ratio of Yersinia pestis in the sample is greater than 30, and the marker detection rate is greater than or equal to 30%, it is determined that plague has been detected in the sample Bacillus nucleotides. It can be seen that the kit provided by the present invention can sensitively detect Yersinia pestis as low as 10 copies/reaction.

4. Evaluation of the specificity of the MNP marker detection method for the detection of Yersinia pestis

Artificial Yersinia pestis and Mycobacterium tuberculosis, Acinetobacter strains, Bordetella pertussis, Bordetella hallii, Chlamydia pneumoniae, Mycoplasma pneumoniae, Epstein-Barr virus, Haemophilus influenzae, varicella-zoster virus, cytomegalovirus Viruses, Herpes Simplex Virus, Human Boca Virus, Klebsiella pneumoniae, Legionella, Moraxella catarrhalis, Pseudomonas aeruginosa, Rickettsia, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pneumoniae The DNA of Streptococcus pyogenes was mixed together in equimolar amounts to prepare a mixed template, and the internal standard DNA was used as a blank control, and the method provided by the present invention was used to detect Yersinia pestis in the mixed template, and three repeated experiments were carried out. Results The sequencing sequences obtained in the three replicates could only be aligned to 14 MNP sites of Y. pestis. After analysis according to the described quality control scheme and judgment threshold, the nucleic acid of Yersinia pestis was specifically detected in three repeated experiments, indicating that the MNP marker and the kit have high specificity in detecting Yersinia pestis in complex templates sex.

Example 3, Detection of Genetic Variation Between Yersinia pestis strains

Using the kit and the MNP marker detection method to detect 6 progeny strains of the same Yersinia pestis strain in different periods provided by the Hubei Provincial Center for Disease Control and Prevention, the samples were named S1-S6 in sequence, and the sequencing average of each sample was The coverage factor was up to 1300 times, and all 15 MNP markers could be detected for each strain (Table 5). Compare the fingerprints of the 6 strains in pairs, and the results are shown in Table 5. The fingerprints of S2 and S4 are consistent, and the fingerprints of the other 4 strains are consistent, indicating that during the long-term reproduction and preservation process, due to genetic variation Or genetic drift, where there is variation between strains of the same name.

The application of the kit to identify genetic variation between strains by detecting MNP markers can be used to ensure the genetic consistency of the same named Yersinia pestis strains in different laboratories, thereby ensuring the comparability of research results, which is of great significance to the scientific research of Yersinia pestis Significance.

Table 3 Detection and analysis of 6 Yersinia pestis

Example 4, Detection of Genetic Variations in Yersinia pestis Strains

As a group organism, some individuals within the Yersinia pestis group mutate, making the group no longer homozygous and forming a heterogeneous heterozygous group, which affects the stability and consistency of the microbial phenotype, especially in the test. Such variants appear as alleles outside of the marker's major genotype when the population is tested for molecular markers. When the variant individual has not yet accumulated, it only accounts for a very small part of the population, showing a low frequency allele type. Low-frequency allele types are often mixed with technical errors, making them difficult to distinguish with existing techniques. The present invention detects highly polymorphic MNP markers. The technical error rate of MNP markers is significantly lower than that of SNP markers, based on the fact that multiple errors are less likely to occur simultaneously than one error.

The authenticity evaluation of the secondary allelic type in this embodiment is carried out as follows: first, the allelic type with strand preference (the ratio of the number of sequencing sequences covered on the DNA double strand) is excluded according to the following rules: the strand preference is greater than 10 times, or more than 5 times different from the strand preference of the main allele type.

The authenticity of genotypes without strand preference was determined based on the number and proportion of sequenced sequences in Table 6. Table 6 lists the calculations based on the BINOM.INV function under the probability guarantee of α=99.9999%, when e _max (n=1) and e _max (n≥2) are 1.03% and 0.0994%, respectively, in each marker The critical value of the sequence number of the allele type, only when the sequence number of the secondary allele type exceeds the critical value, it is determined as the real secondary allele type. When there are multiple candidate minor alleles, the P value of each candidate allele type is multiple-corrected, and the candidate alleles with FDR<0.5% are judged to be true minor allele types.

The parameters e _max (n=1) and e _max (n≥2) involved in Table 6 refer to the highest ratio of the number of sequencing sequences carrying the wrong alleles of n SNPs to the total number of sequencing sequences of the marker. e _max (n=1) and e _max (n≥2) of 1.03% and 0.0994%, respectively, were obtained from the frequencies of all minor alleles detected at 930 homozygous MNP markers.

Table 6 The critical value for determining the suballelic genotype under partial sequencing depth

According to the above parameters, the nucleotides of the two strains with different genotypes are divided into the following 8 ratios: 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100, 7/100 was mixed to prepare artificial heterozygous samples, each sample was detected 3 times, and a total of 24 sequencing data were obtained. Through accurate comparison with the genotypes of the MNP markers of the two strains, markers of heterozygous genotypes were detected in 24 artificial heterozygous samples, indicating that the developed MNP marker detection method of Yersinia pestis is in Suitability for detecting genetic variation within strain populations.

Example 5 Construction of Yersinia pestis DNA fingerprint database

The DNA of all strains or samples used to construct the Y. pestis DNA fingerprint database was extracted by conventional CTAB method and commercial kits, and the quality of the DNA was detected by agarose gel and ultraviolet spectrophotometer. If the ratio of the absorbance value of the extracted DNA at 260nm to 230nm is greater than 2.0, the ratio of the absorbance value at 260nm to 280nm is between 1.6 and 1.8, the main band of DNA electrophoresis is obvious, and there is no obvious degradation and RNA residue, it means that the genomic DNA has reached Relevant quality requirements, follow-up experiments can be carried out.

After comparing the sequence data of the above six strains with the reference genotype, the main genotype of each marker of each strain was obtained to form the MNP fingerprint of each strain. The obtained MNP fingerprints of each bacterial strain were entered into the database file to form the MNP fingerprint database of Yersinia pestis.

The constructed MNP fingerprint database is based on the gene sequence of the detected strains, so it is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed, shared, and updateable at any time. Compare the MNP fingerprints of each strain obtained by each test with the established MNP fingerprint database, and enter the MNP fingerprints of strains with differences in main genotypes into the constructed MNP fingerprint database to achieve real-time update of the database and co-construction and sharing.

Embodiment 6, application in fine typing of Yersinia pestis

The above six Yersinia pestis strains were detected by using the primer combination and the MNP marker detection method, and the MNP fingerprints of each strain were obtained. The DNA fingerprints of each strain were compared in pairs, and then compared with the published reference sequence of Yersinia pestis and the constructed fingerprint database, and the strains with the closest fingerprints were screened. The ones that are the same as the existing fingerprint database are existing variants, and those that have a main genotype difference in at least one MNP marker are new variants to achieve fine typing of Yersinia pestis. Detection of 6 Yersinia pestis samples As shown in Table 5, the 6 Yersinia pestis detected were divided into 2 types, which were close to A112 and NCTC5923 strains respectively. Therefore, the resolution of the method for Yersinia pestis reaches the level of single base, and can realize the fine typing of Yersinia pestis in the sample.

Finally, it should also be noted that the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, but also Other elements not expressly listed, or inherent to the process, method, article, or apparatus are also included.

Having described preferred embodiments of embodiments of the present invention, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, the appended claims are intended to be construed to cover the preferred embodiment and all changes and modifications which fall within the scope of the embodiments of the present invention.

Apparently, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. In this way, if the modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalent technologies, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

A MNP marker combination of Yersinia pestis, characterized in that the MNP marker combination includes 15 markers, and the specific nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.30.
A multiplex PCR primer pair combination for detecting the Yersinia pestis MNP marker combination described in claim 1, characterized in that said multiplex PCR primer pair combination includes 15 pairs of primers, and the specific primer nucleotide sequence is as SEQ ID NO. Shown in 1-SEQ ID NO.30.
A detection kit for detecting the MNP marker combination of Yersinia pestis according to claim 1, characterized in that the kit includes the primer pair combination according to claim 2.
The detection kit according to claim 3, characterized in that the kit also includes a multiplex PCR master mix.
The application of the MNP marker combination of Y. pestis described in claim 1 or the primer pair combination described in claim 2 or the detection kit described in any one of claims 3-4 in the qualitative detection of Y. pestis for non-diagnostic purposes.
The application of the MNP marker combination of Y. pestis described in claim 1 or the primer pair combination described in claim 2 or the detection kit described in any one of claims 3-4 in the preparation of qualitative detection products for Y. pestis.
The MNP marker combination of Y. pestis described in claim 1 or the primer pair combination described in claim 2 or the detection kit described in any one of claims 3-4 in the detection of genetic variation within and between strains of Y. pestis application.
The application of the MNP marker combination of Y. pestis described in claim 1 or the primer pair combination described in claim 2 or the detection kit described in any one of claims 3-4 in constructing a Y. pestis database.
The application of the MNP marker combination of Y. pestis described in claim 1 or the primer pair combination described in claim 2 or the detection kit described in any one of claims 3-4 in fine typing detection of Y. pestis.