CN113718057B

CN113718057B - MNP (MNP) marking site of EB (Epstein-Barr) virus, primer composition, kit and application

Info

Publication number: CN113718057B
Application number: CN202110962190.5A
Authority: CN
Inventors: 李甜甜; 彭海; 周俊飞; 高利芬; 李论; 方治伟
Original assignee: Jianghan University
Current assignee: Jianghan University
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2024-03-22
Anticipated expiration: 2041-08-20
Also published as: CN113718057A

Abstract

The invention discloses an MNP marking site, a primer composition, a kit and application thereof of EB virus, wherein the MNP marking site refers to a genome region which is screened on EB virus genome and is separated from other species and has a plurality of nucleotide polymorphisms in the species, and comprises 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 the EB virus and accurately detect variation; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that the sequence analysis can be performed on all the marker loci of multiple samples at one time, the method has the advantages of high flux, multiple targets, high sensitivity, high precision and detection variation, can be applied to the identification and genetic variation detection of EB viruses of large-scale samples, and has important significance on scientific research and epidemic prevention monitoring of the EB viruses.

Description

MNP (MNP) marking site of EB (Epstein-Barr) virus, primer composition, kit and application

Technical Field

The embodiment of the invention relates to the field of biotechnology, in particular to an MNP (MNP) marking site of an EB (Epstein-Barr) virus, a primer composition, a kit and application thereof.

Background

EB virus (Epstein-Barr virus) is a DNA virus of the genus lymphotropic virus of the family Herpesviridae. The human is a host infected by EB virus, and mainly transmitted by saliva, asymptomatic infection occurs in infants, more than 90% of children 3-5 years old have infected EB virus, and more than 90% of adults have virus antibodies. Epstein barr virus is the causative agent of infectious mononucleosis, an acute lymphoproliferative disease, with a good prognosis for normal people and death in immunodeficiency patients. In addition, EB virus is closely related to the occurrence of nasopharyngeal carcinoma and childhood lymphoma, and is one of human tumor viruses that may be carcinogenic. The vaccine is the most effective method for preventing EB virus infection, but in the observation of gene recombination vaccine developed in China, antiviral drugs with definite curative effect on EB virus infection are not available at present. Therefore, the rapid and accurate detection of the EB virus has important significance for diagnosing the etiology in time, realizing early detection and early treatment and reducing the disease deterioration.

In addition, EB virus is also a common model pathogen for laboratory research. As a group organism, individuals in the group can be mutated in interaction with hosts and environments. For laboratory studies, such undetectable variations can result in the same named strain being virtually different from laboratory to laboratory or from different times in the same laboratory, 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. Therefore, the development of a rapid, accurate and mutation-monitoring EB virus detection and analysis method has important significance for scientific research and application of EB viruses.

Classical EB virus detection methods, including isolation and culture, PCR techniques, whole genome and metagenome sequencing, etc., have one or more limitations in terms of duration, complexity of operation, detection throughput, accuracy and sensitivity of detection variation, cost, etc. The targeted molecular marker detection technology integrating the ultra-multiplex PCR amplification and the high-throughput sequencing can enrich target microorganisms in a sample with low microorganism content in a targeted manner, avoids a large amount of data waste and background noise caused by sequencing of a whole genome and a metagenome, and has the advantages of small sample requirement, accurate diagnosis result, data quantity saving and low-frequency variation detection.

The molecular markers detected by the existing targeted detection technology mainly comprise SNP and SSR markers. SSR markers are the most well-accepted markers for polymorphism, but are small in number in microorganisms; the number of SNP markers is huge, the distribution is dense, and the polymorphism of single SNP marker is insufficient to capture the potential allelic diversity in microorganism population. Therefore, development of a novel molecular marker of EB virus and a detection technology thereof is a technical problem to be solved.

Disclosure of Invention

The invention aims to provide an MNP (MNP) marking site of an EB (Epstein-Barr) virus, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on the EB virus and have the effects of high flux, high accuracy, high specificity, high sensitivity and accurate typing.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided an MNP marker locus of EB virus, which is a genomic region specific to a species selected on the genome of EB virus and having a plurality of nucleotide polymorphisms within the species, comprising marker loci of MNP-1 to MNP-15 of NC_007605.1 as a reference genome.

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 sequence of NC_007605.1 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 nucleotide sequences of the 15 pairs of primers being shown as SEQ ID NO.1 to 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 present invention, there is provided a detection kit for detecting the epstein barr virus MNP marker locus, the kit comprising the primer composition.

Further, the kit further comprises a multiplex PCR premix.

In a fourth aspect of the present invention, there is provided the use of the MNP marker locus of epstein barr virus or the multiplex PCR primer composition or the detection kit in the identification of epstein barr virus.

In a fifth aspect of the present invention, there is provided the use of the MNP marker locus of EB virus or the multiplex PCR primer composition or the detection kit for detecting genetic variation within and between EB virus strains

In a sixth aspect of the present invention, there is provided an application of the MNP marker locus of epstein barr virus or the multiplex PCR primer composition or the detection kit in constructing epstein barr virus database.

In a seventh aspect of the present invention, there is provided an application of the MNP marker locus of epstein barr virus or the multiplex PCR primer composition or the detection kit in the genotyping detection of epstein barr virus.

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 EB virus to obtain the number of the detection sequences and genotype data of the total DNA. And according to the number of EB virus sequencing sequences and the number of detected MNP sites obtained in the total DNA and the blank control, carrying out data quality control and data analysis on the sequencing data of the total DNA to obtain the number of detected MNP sites, the number of sequencing sequences covering each MNP site and the MNP site genotype data.

When the kit is used for EB virus identification, the EB virus nucleic acid standard with known copy number or the mixture of the EB virus nucleic acid standard and other DNA pathogens is taken as a detection sample, the sensitivity, the accuracy and the specificity of the kit for detecting the EB virus are evaluated, and a quality control scheme and a judgment standard for the kit for detecting the EB virus are formulated.

When used for EB virus genetic variation detection, the method comprises inter-strain and intra-strain genetic variation detection. The detection of genetic variation among strains comprises the steps of obtaining genotype data of each strain to be compared at 15 MNP sites by using the kit and the method. By genotype comparison, whether the main genotypes of the strains to be compared are different at the 15 MNP sites is analyzed. If the strains to be compared have variation in the main genotype of at least one MNP site, then the two are judged to have genetic variation. Alternatively, 15 loci of strains to be compared can be amplified by single PCR, and then Sanger sequencing is performed on the amplified products to obtain sequences, and the genotypes of each MNP locus of the strains to be compared are aligned. If there are MNP sites of inconsistent major genotypes, there is variation 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 an EB virus DNA fingerprint database, genotype data of the MNP locus of the EB virus identified from a sample is recorded into a database file to form the DNA fingerprint database of the EB virus; and when different samples are identified each time, comparing the samples with a DNA fingerprint database of the EB virus, identifying whether the EB virus in the samples has a difference of a main genotype (the genotype supported by more than 50% of sequencing fragments at one MNP site) with strains in the database, and recording the EB virus with the main genotype difference at least 1 MNP site as a new mutation type into the DNA fingerprint database.

When the method is used for EB virus typing detection, the EB virus in a sample to be detected is identified, and the genotype of each MNP locus is obtained. And comparing the EB virus with a DNA fingerprint database of the EB virus, identifying whether the EB virus 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 belongs to the first creation in the field of EB virus, 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 EB virus species and conserved in sequences at two sides can be mined according to reported resequencing data of the EB virus representative species; MNP site detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of the MNP site; and then 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 marking sites, primer combination, kit and application thereof of EB virus, and the provided 15 MNP sites of EB virus and primer combination thereof can carry out multiplex PCR amplification, and a second generation sequencing platform is fused to carry out sequencing of amplification products, so that the requirements of high throughput, high efficiency, high accuracy and high sensitivity detection of EB virus are met, and the requirements of building sharable fingerprint data of EB virus standard are met; the need to accurately detect genetic variation between strains of EB virus; the requirements of the homozygous and heterozygous EB virus are identified, and technical support is provided for scientific research, scientific monitoring and prevention and control of the EB virus.

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 EB virus MNP marker locus;

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 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 The seed alleles, which are higher than SSR and SNP, are suitable for the identification of the population organisms; (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 sites of hundreds of samples at one time; (3) High sensitivity, multiple targets are detected at one time by using multiple PCR, and high false negative and low sensitivity caused by single target amplification failure are avoided; (4) High accuracy, and sequencing the amplified product hundreds of times by using a second-generation high-throughput sequencer.

In view of the advantages and the characteristics, the MNP marking and the detection technology thereof can realize classification and tracing of the multi-allele types of the group organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, genetic variation detection and the like. At present, no report about MNP labeling exists in microorganisms, and corresponding technology is lacking. The development, screening and application of MNP labeling method has better application foundation in plants.

Thus, the present invention has developed MNP marker loci of EB virus, which are genomic regions screened on EB virus genome that are discriminated from other species and have multiple nucleotide polymorphisms within the species, including marker loci of MNP-1 to MNP-15 of NC_007605.1 as reference genome.

Next, the invention develops a multiplex PCR primer composition of the EB virus MNP marking site, wherein the multiplex PCR primer composition comprises 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. 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 the EB virus MNP marking site.

The kit provided by the invention can sensitively detect the EB virus of 1 copy/reaction.

In the reproducibility test of the invention, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.

The MNP markers and the kits of the 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 marker locus, primer composition, kit and use thereof of the epstein barr virus of the present application will be described in detail below with reference to examples, comparative examples and experimental data.

Example 1 screening of MNP marker loci of Epstein Barr Virus and design of multiplex PCR amplification primers

S1, screening of MNP (Epstein Barr) virus marker loci

Based on the complete or partial sequences of genomes of different isolates of 10864 EB viruses disclosed on the net, 15 EB virus specific MNP marking sites are obtained through sequence comparison. For species on which no genomic data is present on the net, genomic sequence information representing a minispecies of the microorganism species to be detected may also be obtained by high throughput sequencing, which may be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected markers, genomic sequences of at least 10 isolates are generally used as reference. The 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 the EB virus as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphism sites of each strain of the EB virus;

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

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

As an optional implementation mode, when screening is performed on the reference genome by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, so that the comprehensive screening is facilitated.

S2, design of multiplex PCR amplification primer

The multiplex PCR amplification primers of the MNP locus are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.

S3, evaluating detection efficiency of primer combination

The EB virus DNA with known copy number is added into human genome DNA to prepare 1000 copies/reaction template, and the primer combination is used for detection by the MNP mark detection method. 4 repeated sequencing libraries were constructed, and the primer combinations of the 15 MNP sites described in Table 1 of the present invention were finally selected by screening for primer combinations of uniform amplification and optimal compatibility according to the detection conditions of MNP sites in the 4 libraries.

Example 2 MNP site and primer identification threshold setting and Performance evaluation of Epstein Barr Virus

1. Detection of MNP markers

In this example, EBV nucleic acid standards with known copy numbers were added to human genomic DNA to prepare EBV-mimic samples of 1 copy/reaction, 10 copy/reaction and 100 copy/reaction. 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 the MNP sites of the EB virus detected in the blank control and the EB virus nucleic acid standard in 12 repeated experiments, evaluating the repeatability, the accuracy and the sensitivity of the 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 EB Virus

2. Reproducibility and accuracy assessment of detection of EB virus by MNP (MNP) marker detection kit

Based on whether the genotype of the co-detected site is reproducible or not in the two repetitions, the reproducibility and accuracy of detection of EB virus by the MNP marker detection method are evaluated. Specifically, the data of 12 sets of 100 copies of the sample were compared in pairs, and the results are shown in Table 3.

TABLE 3 reproducibility and accuracy assessment of the method for detecting MNP markers of EB Virus

As can be seen from Table 3, the number of MNP sites having a difference in the main genotypes was 0; according to the principle that the reproducible genotypes are considered to be accurate between 2 repeated experiments, the accuracy a=1- (1-r)/2=0.5+0.5r, and r represents the reproducibility, namely the ratio of the reproducible site number of the main genotype to the common site number. In the reproducibility test of the invention, 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 can accurately detect EB virus of less than 10 copies/reaction.

3. Threshold value judgment for detecting EB virus by MNP (mammalian binding protein) mark detection kit

Sequences aligned to EB virus can be detected in 1 copy/reaction sample, covering at least 1 MNP site. In the case of the partial blank, the sequence of EB virus was also detected as shown in Table 2. 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 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 the epstein barr virus in the test sample and the noise index P of the epstein barr virus 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 epstein barr virus, respectively, in the control.

3) The signal index of the test sample s=nt/Nt, where Nt and Nt represent the number of sequencing fragments and the total number of sequencing fragments of epstein barr virus, respectively, in the test sample.

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 EB Virus in samples to be tested

As a result, as shown in Table 4, no EBV sequence was detected in the blank, the noise index was 0, and the signal index average value in the 1-copy sample was 0.02%, but in order to ensure the accuracy of the detection result, when the EBV sequence was detected in the blank, it was judged whether or not the contamination was acceptable based on the signal-to-noise ratios of the measured sample and the space-ratio control. The invention provides that when the signal to noise ratio is greater than 10 times, it can be determined that the contamination in the detection system is acceptable.

At least 3 MNP sites were detected in the 1-copy/reaction 12-set data, accounting for 20% of the total sites; all 15 MNP sites were stably detected in the 10 copies/reaction 12 sets of data. Therefore, under the condition of ensuring the accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold of the EB virus is 10, namely when the signal-to-noise ratio of the EB virus in the sample is more than 10 and the site detection rate is more than or equal to 20 percent, the detection of the nucleic acid of the EB virus in the sample is judged.

Therefore, the kit provided by the invention can sensitively detect the EB virus with the concentration as low as 1 copy/reaction.

4. Specific evaluation for detecting EB virus by MNP (MNP) mark detection method

The EB virus, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao termyces, chlamydia pneumoniae, mycoplasma pneumoniae, 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 internal standard DNA is used as a blank control, so that the EB virus in the mixed template is detected 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, only EB virus in the mixed template can be specifically detected in the 3 repeated experiments, which shows that MNP marks and the kit detect high specificity of target microorganisms in complex templates.

Example 3 detection of genetic variation between Epstein-Barr Virus strains

6 copies of the collected 1 EB strain were tested using the kit and MNP marker locus detection method, the samples were designated S1-S6 in sequence, the average coverage of sequencing of each sample was 1310-fold, and all 15 MNP markers could be detected on average for each strain (Table 5). The results of pairwise comparison of the fingerprints of 6 strains are shown in Table 5, and 1 part of EB virus detected together with the same batch has a major genotype difference (Table 5) of partial sites, and there is an inter-strain variation.

TABLE 5 detection and analysis of 6 EB viruses

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 strains of the EB virus named in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of the EB virus. 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 in Epstein-Barr Virus strain

As a group organism, the EB virus group is mutated in part of individuals inside, 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 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, the critical value of the number of hypo-isotyping sequences in each site is onlyA true suballele is determined when the number of sequenced sequences of the suballele exceeds a threshold. 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.

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

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.

According to the above parameters, nucleic acids of two strains having different genotypes were mixed in the following 8 ratios of 1/1000,3/1000,5/1000,7/1000,1/100,3/100,5/100,7/100, and artificial heterozygous samples were prepared, 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 the EB virus in detecting genetic variation inside a strain population is demonstrated.

EXAMPLE 5 construction of EB Virus DNA fingerprint database

Extracting DNA of all strains or samples for constructing EB virus DNA fingerprint database by using conventional CTAB method, commercial kit and other methods, and detecting the quality of the DNA by using agarose gel and 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 sequence of the sequencing data of the 6 strains to obtain the main genotype of each site of each strain, thereby forming the MNP fingerprint of each strain. The MNP fingerprint of each strain is compared with an MNP fingerprint database constructed based on the existing genome data, and the MNP fingerprint of the strain with the main genotype difference is input into the constructed MNP 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 capable of being updated at any time.

Example 6 application in EB Virus typing

The 6 EB virus strains are detected by using the primer combination and MNP labeling site detection method described in the example 2, and MNP fingerprint of each strain is obtained. 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 site, and realizing the typing of the EB virus. Detection of 6 EB virus samples As shown in Table 5, 1 part of EB virus was detected and 5 parts of EB virus were detected with a difference in the major genotypes at the 2 MNP sites, possibly in different variants. Therefore, the resolution of the method for the EB virus reaches the level of single base, and the typing of the EB virus 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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<213> Artificial sequence (Artificial Sequence)

<400> 13

aagtagtgcc tgtctgcaaa atg 23

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ccccacgctt caagggttt 19

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ggcagtggag gatcttcgtg 20

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

aaaaacatca tctggaagtg tttca 25

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gaaaatgtac ccatcccggc c 21

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

accgtcagct gtccctctag 20

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

cgcgtcgctt cctaggga 18

<210> 20

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

catgaatgtg ctccaggaga aga 23

<210> 21

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

acactaatct atacattttc tcagcact 28

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

ccggcctaaa actcacgacc 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

ccggatggcc tatgggttac 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

gcggaaaagg tcacgtgaca 20

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

ctcgtttgcg ctcgtgactt t 21

<210> 26

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

cttcgtagac tcgagaactt gctag 25

<210> 27

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

gccaccagat ctcgcttaga ac 22

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

gaggcagaaa gatatatggt ggacg 25

<210> 29

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

gtcacagacc agcccctc 18

<210> 30

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

aaaacagacc gcacaaccaa aaa 23

Claims

1. A multiplex PCR primer composition for detecting MNP marker loci of epstein barr virus, characterized in that said MNP marker loci are genomic regions screened on the genome of epstein barr virus that are distinct from other species and have multiple nucleotide polymorphisms within the species, comprising marker loci of MNP-1 to MNP-15 of nc_007605.1 as reference genome; the multiplex PCR primer composition comprises 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are shown in SEQ ID NO. 1-SEQ ID NO. 30.

2. A detection kit for detecting the MNP marker locus of epstein barr virus according to claim 1, characterized in that said kit comprises the primer composition according to claim 1.

3. The test kit of claim 2, wherein the kit further comprises a multiplex PCR premix.

4. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 for detecting genetic variation within and among strains of epstein barr virus.

5. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 in constructing an epstein barr virus database.

6. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 in the detection of epstein barr virus typing.