CN113718057A

CN113718057A - MNP (MNP protein) marker site of EB (Epstein-Barr) virus, primer composition, kit and application

Info

Publication number: CN113718057A
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: 2021-11-30
Anticipated expiration: 2041-08-20
Also published as: CN113718057B

Abstract

The invention discloses an MNP marker locus of EB virus, a primer composition, a kit and application thereof, wherein the MNP marker locus refers to a genome region which is screened on an EB virus genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker loci of MNP-1-MNP-15; the primers are shown as SEQ ID NO. 1-SEQ ID NO. 30. The MNP marker locus can specifically identify EB virus and accurately detect variation; the primers are not interfered with each other, and by integrating multiple amplification and sequencing technologies, sequence analysis can be performed on all marked sites of multiple samples at one time, so that the kit has the detection advantages of high throughput, multiple targets, high sensitivity, high accuracy and monitoring variation, can be applied to 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 protein) marker site of EB (Epstein-Barr) virus, primer composition, kit and application

Technical Field

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

Background

Epstein-Barr virus (EB virus) is a DNA virus of the genus lymphotropic virus of the family Herpesviridae. The human is a host infected by the EB virus, is mainly transmitted through saliva, asymptomatic infection mostly occurs in children, more than 90% of children 3-5 years old are infected with the EB virus, and more than 90% of adults have virus antibodies. EB virus is the causative agent of infectious mononucleosis, which is an acute lymphoproliferative disease with good prognosis in normal patients and death in patients with immunodeficiency. In addition, EB virus is closely related to the occurrence of nasopharyngeal carcinoma and lymphoma of children, and is classified as one of human tumor viruses which are possible to cause cancers. The vaccine is the most effective method for preventing EB virus infection, but the genetic recombinant vaccine developed in China is under observation, and currently, antiviral drugs with definite curative effect on EB virus infection are not available. Therefore, the rapid and accurate detection of the EB virus has important significance for diagnosing the cause of disease in time, finding early treatment and reducing the disease deterioration.

In addition, EB virus is also a common model pathogenic microorganism for laboratory research. As a group organism, the individual in the group can be mutated in the interaction with a host and the environment. For laboratory studies, such inconspicuous variations can result in different laboratories or the same laboratory differing in the fact that identically named strains are different at different times, resulting in irreproducible and incomparable experimental results. Heterogeneity among human Hella cell laboratories has resulted in a large number of incomparable experimental results and wasted data. Therefore, the development of a rapid and accurate EB virus detection and analysis method capable of monitoring the variation has important significance for scientific research and application of the EB virus.

Classical EB virus detection methods, including isolation culture, PCR technology, whole and metagenome sequencing, have one or more limitations in terms of length of time, complexity of operation, detection throughput, accuracy and sensitivity of detection variation, cost, and the like. The targeted molecular marker detection technology combining the ultra-multiplex PCR amplification and the high-throughput sequencing can be used for enriching 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 whole genome and metagenome sequencing, and has the advantages of less 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 (single nucleotide polymorphism) markers and SSR (simple sequence repeat) markers. SSR markers are generally accepted as the most polymorphic markers, but are few in microorganisms; the SNP markers are large in number, densely distributed and are binary markers, and the polymorphism of a single SNP marker is insufficient to capture the potential allelic diversity in a microbial population. Therefore, the development of a novel molecular marker for EB virus and a detection technique thereof has become a technical problem to be solved urgently.

Disclosure of Invention

The invention aims to provide an MNP (MNP) marker locus of EB virus, a primer composition, a kit and application thereof, which can perform qualitative identification and variation 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 purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, MNP marker sites of EB virus are provided, the MNP marker sites are genome regions which are specific to species screened on the genome of EB virus and have a plurality of nucleotide polymorphisms in the species, and the MNP marker sites comprise marker sites of MNP-1-MNP-15 taking NC-007605.1 as a reference genome.

In the above technical solution, the labeling sites of MNP-1 to MNP-15 are specifically shown in table 1 of the specification, and the starting and ending positions of the MNP label marked in table 1 are determined based on the reference sequence of NC _007605.1 in table 1.

In a second aspect of the invention, a multiplex PCR primer composition for detecting the MNP marker locus is provided, and the multiplex PCR primer composition comprises 15 pairs of primers, wherein the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID No. 1-SEQ ID No. 30.

In the above technical scheme, the primer of each MNP marker site includes an upper primer and a lower primer, which are specifically shown in table 1 of the specification.

In a third aspect of the invention, a detection kit for detecting the MNP marker locus of the EB virus is provided, and the kit comprises the primer composition.

Further, the kit also comprises a multiplex PCR premix.

In the fourth aspect of the invention, the application of the MNP marker site of the EB virus, the multiple PCR primer composition or the detection kit in the identification of the EB virus is provided.

In the fifth aspect of the invention, the application of the MNP marker locus of the EB virus, the multiple PCR primer composition or the detection kit in the detection of genetic variation inside and among EB virus strains is provided

In the sixth aspect of the invention, the application of the MNP marker site of the EB virus, the multiple PCR primer composition or the detection kit in constructing an EB virus database is provided.

In the seventh aspect of the invention, the application of the MNP marker site of the EB virus, the multiple PCR primer composition or the detection kit in the EB virus typing detection is provided.

In the application, firstly, the total bacterial DNA of a sample to be detected is obtained; carrying out first round of multiplex PCR amplification on the total DNA and a blank control by using the kit disclosed by the invention, wherein the cycle number is not higher than 25; after purifying the amplification product, adding a sample label based on the second round of PCR amplification and a second-generation sequencing adaptor; purifying and quantifying the second round amplification product; when detecting a plurality of strains, performing high-throughput sequencing by equivalently mixing the amplification products of the second round; and comparing the sequencing result with the reference sequence of the EB virus to obtain the number and genotype data of the detection sequences of the total DNA. And performing data quality control and data analysis on the sequencing data of the total DNA according to the number of the EB virus sequencing sequences and the number of detected MNP sites obtained from the total DNA and the blank control, so as to obtain the number of the detected MNP sites, the number of the sequencing sequences covering each MNP site and the genotype data of the MNP sites.

When the kit is used for EB virus identification, an EB virus nucleic acid standard substance with known copy number or a mixture of the EB virus nucleic acid standard substance and other DNA pathogens is used 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 of the kit for detecting the EB virus are established.

When used for detecting the genetic variation of the EB virus, the genetic variation detection among strains and in the strains is included. The detection of genetic variation among strains comprises the steps of obtaining genotype data of strains to be compared at 15 MNP sites by using the kit and the method. And analyzing whether the major genotypes of the strains to be compared on the 15 MNP sites have difference or not through genotype comparison. If the strains to be compared have variation in the major genotype of at least one MNP site, the strains are judged to have genetic variation. As an alternative, 15 sites of the strains to be compared can be amplified respectively through single PCR, and then the amplification products are subjected to Sanger sequencing, and after the sequences are obtained, the genotypes of all MNP sites of the strains to be compared are compared. If there are MNP sites that are not identical in major genotypes, there is variation between the strains to be compared. When detecting the genetic variation in the strain, judging whether a secondary genotype other than the main genotype is detected at the MNP site of the strain to be detected or not through a statistical model. And if the to-be-detected strain has a minor genotype at least one MNP site, judging that the genetic variation exists in the to-be-detected strain.

When the method is used for constructing the EB virus DNA fingerprint database, the genotype data of the MNP sites of the EB virus identified from the sample is recorded into a database file to form the EB virus DNA fingerprint database; when different samples are identified each time, whether the EB virus in the samples has the difference of the major genotypes (more than 50% of genotypes supported by sequencing fragments at one MNP site) with the strains in the database at the MNP site is identified by comparing with the DNA fingerprint database of the EB virus, and the EB virus with the major genotype difference at least 1 MNP site is a new variation type and is included in the DNA fingerprint database.

When the method is used for EB virus typing detection, EB viruses in a sample to be detected are identified, and the genotype of each MNP locus is obtained. And comparing the EB virus with the DNA fingerprint database of the EB virus to identify 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 initiative in the EB virus field, and has no related literature report; MNP marks are mainly developed based on reference sequences, and MNP sites which are largely distinguished from other species, are polymorphic in EB virus species and have conserved sequences on two sides can be mined according to reported re-sequencing data of representing small species of EB viruses; MNP locus detection primers suitable for multiplex PCR amplification can be designed through conserved sequences on two sides of the MNP locus; and then according to the test result of the standard product, a primer combination and a detection kit with the maximum polymorphism, high specificity, the best compatibility and the set of MNP sites can be screened out.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the invention provides MNP (MNP) marker sites of EB (Epstein-Barr) virus, a primer composition, a kit and application thereof, wherein the 15 MNP sites of EB virus and the primer composition thereof can carry out multiple PCR (polymerase chain reaction) amplification, and are fused with a second-generation sequencing platform to carry out sequencing on an amplification product, so that the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on EB virus are met, and the requirements of standard and sharable fingerprint data construction of EB virus are met; accurately detecting the genetic variation among EB virus strains; the method identifies the needs of EB virus homozygosity and heterozygosity, and provides technical support for scientific research, scientific monitoring and prevention of EB virus.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

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

FIG. 2 is a flow chart of screening and primer design of the MNP marker sites of EB virus;

FIG. 3 is a flow chart of detection of MNP marker sites.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the embodiments of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that the present embodiments and examples are illustrative of the present invention and are not to be construed as limiting the present invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.

In order to solve the technical problems, the general idea of the embodiment of the application 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 over a region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) abundant alleles, 2 at a single MNP locusⁿSpecies alleles, higher than SSR and SNP, are suitable for identification of group organisms; (2) the species distinguishing capability is strong, species identification can be realized only by a small amount of MNP marks, and the detection error rate is reduced. Ultra-multiplex PCR-based bindingThe MNP labeling method for detecting the MNP label by the second-generation high-throughput sequencing technology has the following advantages: (1) the output is a base sequence, parallel experiments are not needed, and a standardized database can be constructed for sharing and sharing; (2) the efficiency is high, the sample DNA bar code is utilized, the limitation of the quantity of sequencing samples is broken through, and tens of thousands of MNP sites of hundreds of samples can be typed at one time; (3) the sensitivity is high, multiple targets are detected at one time by utilizing multiple PCR, and high false negative and low sensitivity caused by amplification failure of a single target are avoided; (4) high accuracy, using a second generation high throughput sequencer to sequence the amplification product hundreds of times.

In view of the advantages and the characteristics, the MNP marker and the detection technology MNP marking method thereof can realize the classification and the tracing of the multi-allelic genotypes of the population organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, detection of genetic variation and the like. At present, no report on MNP labeling exists in microorganisms, and corresponding technologies are lacked. The development, screening and application of the MNP marking method have better application foundation in plants.

Therefore, the invention develops the MNP marker sites of the EB virus, the MNP marker sites are genome regions which are screened on the genome of the EB virus, are distinguished from other species and have a plurality of nucleotide polymorphisms in the species, and the MNP marker sites comprise the marker sites of MNP-1-MNP-15 taking NC-007605.1 as a reference genome.

Then, the invention develops the multiplex PCR primer composition of the EB virus MNP marker locus, 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 conflict 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 MNP marker locus of the EB virus.

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

In the reproducibility test of the invention, the logarithm of difference of the MNP marking main gene type among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100 percent, and the accuracy a is 100 percent.

The MNP markers and the kits of the invention detect high specificity of target microorganisms in complex templates.

The marker and primer combinations developed by the present invention will be used to establish the national standards for pathogen detection (plan number 20201830-T-469), which will be released at the end of 2021.

The MNP marker site, primer composition, kit and application of EB virus of the present application will be described in detail with reference to examples, comparative examples and experimental data.

Example 1 screening of MNP marker sites of EB Virus and design of multiplex PCR amplification primers

S1 screening of MNP marker sites of EB virus

Based on the complete or partial genome sequences of 10864 EB virus isolates disclosed on the network, 15 specific MNP marker loci of EB virus are obtained by sequence alignment. For species without genomic data on the net, the genomic sequence information of the representative microspecies of the microbial species to be detected can also be obtained by high-throughput sequencing, wherein the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected marker, the genomic sequence of at least 10 isolates is generally used as a reference. The 15 MNP marker sites screened are shown in table 1:

TABLE 1-starting position of the MNP marker site and detection primer 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 performing sequence comparison on the genome sequence and the reference genome to obtain single nucleic acid polymorphic sites of each strain of the EB virus;

on the reference genome, performing window translation by taking 100-300 bp as a window and 1bp as a step length, and screening to obtain a plurality of candidate MNP site regions, wherein the candidate MNP site regions 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 two ends;

screening a region with the division DP of more than or equal to 0.2 in the candidate polynucleotide polymorphic site region as an MNP marker site; wherein, DP ═ d/t, t is the comparison logarithm of all the minor species in the region of the candidate polynucleotide polymorphic site when compared pairwise, d is the sample logarithm of at least two single nucleic acid polymorphisms that differ in the region of the candidate polynucleotide polymorphic site.

As an optional implementation mode, when the reference genome is screened by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, which is beneficial to comprehensive screening.

S2 design of multiplex PCR amplification primers

And designing the multiplex PCR amplification primers of the MNP sites through primer design software, wherein 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 normally amplified in one amplification reaction.

S3 evaluation of detection efficiency of primer combination

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 are constructed, primer combinations with even amplification and optimal compatibility are screened according to the detection condition of MNP sites in the 4 libraries, and finally primer compositions of 15 MNP sites disclosed in the invention table 1 are screened.

Example 2 identification of Epstein-Barr Virus by MNP sites and primers threshold setting and Performance evaluation

1. Detection of MNP markers

In this example, EB virus nucleic acid standards with known copy numbers were added to human genomic DNA to prepare 1 copy/reaction, 10 copy/reaction, and 100 copy/reaction EB virus mock samples. An equal volume of sterile water was also set as a blank. A total of 4 samples were obtained, each sample was constructed into 3 duplicate libraries each day, and the assay was continued for 4 days, i.e. 12 sets of sequencing data were obtained for each sample, as shown in table 2. According to the sequencing fragment number and the site number of the MNP site of the EB virus detected in the blank control and the nucleic acid standard substance of the EB virus in 12 repeated experiments, the repeatability, the accuracy and the sensitivity of the detection method are evaluated, and the threshold value for detecting the pollution of a quality control system and the target pathogen is established. The flow of detection of MNP markers is shown in figure 3.

TABLE 2 analysis of detection sensitivity and stability of MNP labeling method for EB Virus

2. Evaluation of reproducibility and accuracy of MNP (MNP) marker detection kit for detecting EB (Epstein-Barr) virus

And evaluating the reproducibility and accuracy of the MNP marker detection method for detecting the EB virus based on whether the genotype of the co-detected site can be reproduced in two times of repetition. Specifically, two-by-two comparisons were made for each of 12 sets of data for 100 copies of the sample, and the results are shown in table 3.

TABLE 3 evaluation of reproducibility and accuracy of MNP marker detection method for EB virus

As can be seen from Table 3, the number of MNP sites differing in major genotypes was 0; the accuracy rate a is 1- (1-r)/2 is 0.5+0.5r, and r represents the reproducibility rate, i.e., the ratio of the number of sites where the major genotype is reproducible to the number of common sites, which is considered to be the principle of accuracy among 2 repeated experiments. In the reproducibility test of the invention, the logarithm of difference of the MNP marking main gene type among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100 percent, and the accuracy a is 100 percent. Therefore, the kit can accurately detect EB virus with less than 10 copies/reaction.

3. Threshold judgment of EB virus detected by MNP (MNP) marker detection kit

Sequences aligned to epstein barr virus can be detected in 1 copy/reaction sample, covering at least 1 MNP site. The EB virus sequences were also detected in the partial blank control, as shown in Table 2. Due to the extreme sensitivity of MNP marker detection methods, contamination of the data during detection is likely to result in the generation of false positives. Therefore, the following quality control schemes are prepared in this example.

The quality control scheme is as follows:

1) the amount of sequencing data was greater than 4.5 megabases. The measuring and calculating basis is that the number of MNP sites detected by each sample is 15, and the length of a sequencing fragment is 300 bases, so that when the data volume is more than 4.5 million bases, the number of the sequencing fragments covering each site can be ensured to reach 1000 times by one-time experiment of most samples, and the accurate analysis of the base sequence of each MNP site is ensured.

2) And judging whether the pollution is acceptable according to the signal index S of the EB virus in the test sample and the noise index P of the EB virus in the blank control, wherein:

the blank control noise index P ═ Nc/Nc, where Nc and Nc represent the number of sequencing fragments of EB virus and the total number of sequencing fragments in the blank control, respectively.

3) The signal index S of the test sample is Nt/Nt, where Nt and Nt represent the number of sequencing fragments of EB virus and the total number of sequencing fragments in the test sample, respectively.

And calculating the detection rate of the MNP marker locus in the test sample, which is the ratio of the number of the detected locus to the number of the total design locus.

TABLE 4 SNR of EB Virus in samples to be tested

As shown in Table 4, the sequence of EB virus was not detected in the blank control, the noise index was 0, and the average value of the signal index in the sample of 1copy was 0.02%, but in order to ensure the accuracy of the detection result, when EB virus sequence was detected in the blank control, it was judged whether contamination was acceptable according to the signal-to-noise ratio of the measured sample and the blank control. The present invention provides that contamination in the detection system can be judged to be acceptable when the signal-to-noise ratio is greater than 10 times.

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

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

4. Specificity evaluation of MNP (MNP) label detection method for detecting EB (Epstein-Barr) virus

The EB virus and mycobacterium tuberculosis, acinetobacter strains, Bordetella pertussis, Bordetella Hotellae, 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 equimolar amount to prepare a mixed template, and the EB virus in the mixed template is detected by using the method provided by the invention with the internal standard DNA as a blank control. And 3 repeated experiments are carried out, and after analysis is carried out according to the quality control scheme and the judgment threshold, EB viruses in the mixed template can be specifically detected in the 3 repeated experiments, so that the MNP marker and the kit can detect the high specificity of the target microorganism in the complex template.

Example 3 detection of genetic variation between strains of EB Virus

6 copy strains of 1 collected EB strain are detected by using the kit and the MNP marker locus detection method, samples are named as S1-S6 in sequence, the average coverage factor of sequencing of each sample reaches 1310 times, and all 15 MNP markers can be detected on average in each strain (Table 5). The fingerprint spectra of 6 strains are compared pairwise, and the results are shown in table 5, wherein 1 EB virus and 5 EB viruses detected together in the same batch have main genotype difference of partial sites (table 5), and variation among strains exists.

TABLE 5-6 detection assay for EB Virus

The kit can be used for ensuring the genetic consistency of the same named EB virus strains in different laboratories by detecting the MNP marker and identifying the genetic variation among the strains, thereby ensuring the comparability of the research result, and having important significance for the scientific research of the EB virus. In clinical settings, diagnostic protocols can be considered as to whether differential sites affect drug resistance.

Example 4 detection of genetic variation within Epstein-Barr Virus strains

As a colony organism, the individual variation of a part of the interior of an EB virus colony causes the colony to be not homozygous any more, and a heterogeneous heterozygous colony is formed, thereby influencing the stability and consistency of the phenotype of the microorganism for testing in particular. Such variants exhibit an allelic profile outside the major genotype of the locus when tested for molecular markers in a population. When the variant individuals have not accumulated, they account for a very small fraction of the population and are characterized by a low frequency of alleles. Low frequency alleles tend to be confused with technical errors, making prior art techniques difficult to distinguish. The present invention detects highly polymorphic MNP markers. The technical error rate of MNP labeling is significantly lower than that of SNP labeling, based on the probability of multiple errors occurring simultaneously being lower than the probability of one error occurring.

The authenticity assessment of the sub-allelic genotypes of this example was performed as follows: allelic types with strand bias (ratio of the number of sequencing sequences overlaid on a DNA double strand) were first excluded according to the following rule: the strand preference is greater than 10-fold, or the difference from the strand preference of the dominant allele is greater than 5-fold.

Genotypes without strand preference were judged for authenticity based on the number and proportion of sequences sequenced in table 6. Inv function calculation under a 99.9999% probability guarantee, e_max(n-1) and e_max(n.gtoreq.2) 1.03% and 0.0994%, respectively, the number of sequenced sequences of the sub-allelic gene in each locus is a critical value, and only when the number of sequenced sequences of the sub-allelic gene exceeds the critical value, the true sub-allelic gene is determined. When multiple candidate sub-alleles are present, multiple corrections are made to the P-value for each candidate allele, FDR<0.5% of the candidate alleles were judged to be true sub-allelic genotypes.

TABLE 6 critical value for determination of sub-allelic genotypes at partial sequencing depth

Table 6 relates to the parameter e_max(n-1) and e_max(n.gtoreq.2) means that the highest proportion of the number of sequencing sequences carrying the wrong allele of n SNPs to the total number of sequencing sequences at that site. e.g. of the type_max(n-1) and e_max(n.gtoreq.2) 1.03% and 0.0994%, respectively, were obtained from the frequency of all the minor alleles detected at 930 homozygous MNP sites.

Nucleic acids of two strains having differences 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 according to the above parameters to prepare artificial heterozygous samples, and each sample was examined for 3 replicates to obtain a total of 24 sequencing data. By accurately comparing the gene types of the MNP sites of the two strains, the sites with heterozygous gene types are detected in 24 artificial heterozygous samples, thereby demonstrating the applicability of the developed MNP marker detection method of the EB virus in detecting the genetic variation in the strain population.

Example 5 construction of EB Virus DNA fingerprint database

Extracting DNA of all strains or samples for constructing an EB virus DNA fingerprint database by using a conventional CTAB method, a commercial kit and other methods, and detecting the quality of the DNA by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm to 230nm is more than 2.0, the ratio of the absorbance values of 260nm to 280nm is between 1.6 and 1.8, the main band of the DNA electrophoresis is obvious, and no obvious degradation or RNA residue exists, the genome DNA reaches the relevant quality requirement, and subsequent experiments can be carried out.

And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain a main genotype of each site of each strain, and forming the MNP fingerprint of each strain. The MNP fingerprint of each strain is compared with an MNP fingerprint database constructed on the basis of the existing genome data, and the MNP fingerprints of the strains with the main genotypes having differences are recorded into the constructed MNP fingerprint database. The constructed MNP fingerprint database is based on the gene sequences of detected strains, is compatible with all high-throughput sequencing data, and has the characteristics of complete co-construction and sharing and update at any time.

Example 6 application in Epstein-Barr Virus typing

The primer combination and MNP marker locus detection method described in the embodiment 2 are used for detecting the 6 EB virus strains, and an MNP fingerprint map of each strain is obtained. And the DNA fingerprint of each strain is pairwise compared with the constructed fingerprint database, the DNA fingerprint is the same as the constructed fingerprint database and is defined as an existing variant, and the DNA fingerprint of at least one MNP site has main genotype difference and is defined as a new variant, so that the EB virus is classified. The detection of 6 EB virus samples is shown in Table 5, and 1 EB virus and other 5 EB viruses detected in the 6 EB virus samples have differences of major genotypes at 2 MNP sites, and the major genotypes are possibly different variants. Therefore, the resolution of the method for EB virus reaches the level of single base, and the EB virus in a sample can be classified.

Finally, it should also be 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. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments 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 in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such 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 equivalents, the embodiments of the present invention are also intended to encompass such modifications and variations.

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<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cggcctaggt gtgccataag 20

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gttatcatgt gttgcttgag gagat 25

<210> 12

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caaggcttgg gtctccacg 19

<210> 13

<211> 23

<212> DNA

<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. An EB virus MNP marker locus is a genome region which is screened on an EB virus genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and the MNP marker locus comprises marker loci of MNP-1-MNP-15 taking NC-007605.1 as a reference genome.

2. The multiplex PCR primer composition for detecting the MNP marker sites of the EB virus according to claim 1, which is characterized by comprising 15 pairs of primers, wherein the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID No. 1-SEQ ID No. 30.

3. A test kit for detecting the MNP marker site of epstein-barr virus according to claim 1, comprising the primer composition 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 marker site of epstein-barr virus according to claim 1, or the primer composition according to claim 2, or the detection kit according to any of claims 3-4, for the identification of epstein-barr virus.

6. Use of the MNP marker site of epstein-barr virus of claim 1, the primer composition of claim 2, or the detection kit of any one of claims 3-4 for detecting genetic variations within and among epstein-barr virus strains.

7. Use of the MNP marker site of epstein-barr virus according to claim 1, or the primer composition according to claim 2, or the detection kit according to any one of claims 3-4, for constructing epstein-barr virus databases.

8. Use of the MNP marker site of epstein-barr virus according to claim 1, or the primer composition according to claim 2, or the detection kit according to any one of claims 3-4, in epstein-barr virus typing detection.