CN115029478B

CN115029478B - MNP (human cytomegalovirus) marker locus, primer composition, kit and application of MNP marker locus

Info

Publication number: CN115029478B
Application number: CN202111354822.6A
Authority: CN
Inventors: 周俊飞; 李论; 李甜甜; 肖华锋; 方治伟; 彭海; 高利芬; 陈利红
Original assignee: Jianghan University
Current assignee: Jianghan University
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2023-12-22
Anticipated expiration: 2041-11-16
Also published as: CN115029478A

Abstract

The invention discloses MNP (MNP) marking sites of human cytomegalovirus, a primer composition, a kit and application thereof, wherein the MNP marking sites refer to genome regions which are screened on human cytomegalovirus genome and are separated from other species and have a plurality of nucleotide polymorphisms in the species, and the MNP marking sites comprise MNP-1-MNP-15; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 30. The MNP marker locus can specifically identify human cytomegalovirus and monitor 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 throughput, multiple targets, high sensitivity and culture-free detection, can be applied to the identification and genetic variation detection of human cytomegalovirus of large-scale samples, and has important significance for scientific research and epidemic prevention monitoring of human cytomegalovirus.

Description

MNP (human cytomegalovirus) marker locus, primer composition, kit and application of MNP marker locus

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to MNP (MNP) marking sites of human cytomegalovirus, a primer composition, a kit and application thereof.

Background

Cytomegalovirus (Cytomegalovirus) is a herpesvirus DNA virus characterized by an infected cell enlargement and has large nuclear inclusion bodies. CMV is very widely infected in the population, and chinese adult infection rate reaches more than 95%. Can expel viruses from saliva, milk sweat, urine, semen and uterine secretions in a plurality of places for a long time or with intervals. CMV is discharged from the urinary tract and cervix of the parturient, and the infant may become infected through the birth canal during delivery. Maternal CMV infection can cause congenital infections by placenta invading the fetus, a minority of which result in premature birth, abortion, stillbirth or post-natal death. The infant may develop jaundice, hepatosplenomegaly, thrombocytopenic purpura and hemolytic anemia. Surviving children often leave permanent mental retardation, neuromuscular dyskinesia, deafness, chorioretinitis, etc. Therefore, cytomegalovirus screening during gestation has become one of the measures for prenatal and postnatal care. In addition, human cytomegalovirus is used as a group organism, and individuals in the group can be mutated in interaction with hosts and the environment, so that the detection or treatment method is disabled; for experimental studies, such undetectable variations can result in the same named strain being virtually different in different laboratories or different times in the same laboratory, resulting in irreproducible and incomparable experimental results. Therefore, developing a rapid, accurate and variation-monitoring human cytomegalovirus detection and analysis method has important significance for scientific research and application of human cytomegalovirus.

The existing screening technology is that through microscopic examination, separation culture, immunological detection or in-situ hybridization through a DNA probe, one or more molecular markers which limit the detection of the existing targeted detection technology mainly comprise SNP and SSR markers in terms of detection period, complexity, cross reaction, detection variation, accuracy, sensitivity and the like. 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, developing a novel molecular marker of human cytomegalovirus and a detection technology thereof becomes a technical problem to be solved.

Disclosure of Invention

The invention aims to provide a specific MNP (MNP) marker locus of human cytomegalovirus, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on the human cytomegalovirus 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 invention there is provided a human cytomegalovirus specific MNP marker locus which is species specific for a species selected on the human cytomegalovirus genome and has a plurality of nucleotide polymorphisms within the species, comprising the marker locus of MNP-1 to MNP-15 of the reference genome with NC_ 006273.2.

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_006273.2 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 invention, there is provided a detection kit for detecting the MNP marker locus of human cytomegalovirus, the kit comprising the primer composition.

Further, the kit further comprises a multiplex PCR premix.

In a fourth aspect of the invention, there is provided the use of the MNP marker locus of human cytomegalovirus or the multiplex PCR primer composition or the detection kit for qualitative detection of human cytomegalovirus for non-diagnostic purposes and for preparing a qualitative detection product of human cytomegalovirus.

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

In a sixth aspect of the invention, there is provided the use of the MNP marker locus of human cytomegalovirus or the multiplex PCR primer composition or the detection kit in the construction of human cytomegalovirus database.

In a seventh aspect of the invention, there is provided the use of the MNP marker locus of human cytomegalovirus or the multiplex PCR primer composition or the detection kit in human cytomegalovirus typing detection.

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

When the method is used for identifying the human cytomegalovirus, whether the sample to be detected contains nucleic acid of the human cytomegalovirus is judged after quality control according to the number of sequencing sequences of the human cytomegalovirus detected in the sample to be detected and the blank control and the number of MNP sites detected. The quality control scheme and the judging method are characterized in that DNA of the human cytomegalovirus with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the human cytomegalovirus are evaluated, and the quality control scheme and the judging method when the kit detects the human cytomegalovirus are formulated.

When used for human cytomegalovirus genetic variation detection, it includes 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 a human cytomegalovirus MNP fingerprint database, genotype data of MNP loci of human cytomegaloviruses identified from a sample are input into a database file to form the MNP fingerprint database of the human cytomegaloviruses; each time a different sample is identified, comparing with the MNP fingerprint database of the human cytomegalovirus, identifying whether the human cytomegalovirus in the sample has a difference of main genotype (with a genotype supported by more than 50% of sequencing fragments at one MNP site) with strains in the database at the MNP site, and recording the human cytomegalovirus with the difference of main genotype at least 1 MNP site as a new mutation type in the MNP fingerprint database.

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

The invention is initiated in the field of human cytomegalovirus, and is not reported in related documents; MNP markers are developed mainly based on reference sequences, and MNP sites which are large-scale and are distinguished from other species, polymorphic in the interior of the human cytomegalovirus species and conserved in sequence at two sides can be mined according to reported resequencing data of the human cytomegalovirus 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 compositions, kits and applications thereof of human cytomegalovirus, and the provided 15 MNP sites and primer compositions thereof of human cytomegalovirus 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 human cytomegalovirus are met, and the requirements of human cytomegalovirus standard and sharable fingerprint data construction are met; the need to accurately detect genetic variation between human cytomegalovirus strains; the requirement of homozygosity and heterozygosity of human cytomegalovirus is identified, and technical support is provided for scientific research, scientific monitoring and control of human cytomegalovirus.

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 the screening and primer design of MNP marker loci of human cytomegalovirus;

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 developed MNP marker loci for human cytomegalovirus that are genomic regions screened on the human cytomegalovirus genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including marker loci for MNP-1-MNP-15 of NC_006273.2 as a reference genome.

Next, the present invention developed a multiplex PCR primer composition for the MNP marker loci of human cytomegalovirus, comprising 15 pairs of primers, the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO.1 to 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 MNP (human cytomegalovirus) marker loci.

The kit provided by the invention can sensitively detect 10 copies/reaction of human cytomegalovirus.

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 the high specificity of human cytomegalovirus in complex templates.

The MNP marker loci, primer compositions, kits and uses thereof of one of the human cytomegaloviruses of the present application will be described in detail below in conjunction with examples, comparative examples and experimental data.

Example 1 screening of MNP marker loci of human cytomegalovirus and design of multiplex PCR amplification primers

S1, screening of MNP (human cytomegalovirus) marker loci

Based on complete or partial sequences of genomes of different isolates of 3794 human cytomegalovirus disclosed on the net, 15 MNP marker loci are obtained through sequence alignment. 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 genetically representative 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 human cytomegalovirus as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphic sites of each strain of the human cytomegalovirus;

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 copy number of the human cytomegalovirus DNA provided by the disease prevention control center of Hubei province is used to be added into human genome DNA to prepare a 1000 copy/reaction template, and the primer combination is used to detect 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 settings and Performance assessment of human cytomegalovirus

1. Detection of MNP markers

In this example, human cytomegalovirus nucleic acid standards of known copy number were added to human genomic DNA to prepare 1 copy/reaction, 10 copy/reaction and 100 copy/reaction human cytomegalovirus mimetic samples. An equal volume of sterile water was set at the same time as a blank. A total of 4 samples, each of which was constructed as 3 replicate libraries per day, were tested continuously for 4 days, i.e., 12 sets of sequencing data were obtained per sample, as shown in table 2. And evaluating the reproducibility, accuracy and sensitivity of the detection method according to the number of sequencing fragments and the number of sites of the MNP site of the human cytomegalovirus detected in the blank control and the human cytomegalovirus nucleic acid standard in 12 repeated experiments, 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 human cytomegalovirus

As shown in Table 2, the kit can stably detect more than 9 MNP sites in a 10-copy/reaction sample, and can detect at most 1 MNP site in a 0-copy/reaction sample, and the kit can clearly distinguish between a 10-copy/reaction sample and a 0-copy/reaction sample, and has technical stability and detection sensitivity as low as 10-copy/reaction.

2. Reproducibility and accuracy assessment of MNP (MNP) marker detection kit for detecting human cytomegalovirus

Based on whether the genotype of the co-detected site is reproducible in the two replicates, the reproducibility and accuracy of detection of human cytomegalovirus by the MNP marker detection method is evaluated. Specifically, the data of 12 sets of 100 copies/reaction sample were compared in pairs, and the results are shown in Table 3.

TABLE 3 reproducibility and accuracy assessment of human cytomegalovirus MNP marker detection method

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

3. Threshold value judgment for detecting human cytomegalovirus by MNP (MNP) mark detection kit

Sequences aligned to human cytomegalovirus can be detected in 1 copy/reaction samples, covering at least 1 MNP site. In the partial blank, the sequence of human cytomegalovirus 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 human cytomegalovirus in the test sample and the noise index P of human cytomegalovirus 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 human cytomegalovirus, respectively, in the control.

3) The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequencing fragments and total number of sequencing fragments, respectively, of human cytomegalovirus 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 human cytomegalovirus in samples to be tested

As a result, as shown in Table 4, the average value of the noise index of human cytomegalovirus in the control was 0.03%, the average value of the signal index in the 1-copy sample was 0.26%, and the average value of the signal to noise ratio of the 1-copy sample and the control was 7.9, and therefore, the present invention provides that when the signal to noise ratio is more than 10 times, it can be judged that the contamination in the detection system is acceptable. The average signal to noise ratio of the 10 copies of the sample and the blank was 76.5, and at least 9 MNP sites were stably detected in the 10 copies/reaction 12 sets of data, accounting for 60.0% of the total sites. Therefore, under the condition of ensuring the accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold of the human cytomegalovirus is 40, namely when the signal-to-noise ratio of the human cytomegalovirus in the sample is more than 38 and the site detection rate is more than or equal to 30 percent, the nucleic acid of the human cytomegalovirus is judged to be detected in the sample.

Therefore, the kit provided by the invention can sensitively detect human cytomegalovirus with the concentration as low as 10 copies/reaction.

4. MNP marker detection method for detecting specificity evaluation of human cytomegalovirus

Human cytomegalovirus, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao terbacteria, chlamydia pneumoniae, mycoplasma pneumoniae, haemophilus influenzae, varicella zoster virus, 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 same molar amount to prepare a mixed template, internal standard DNA is used as a blank control, and the human cytomegalovirus in the mixed template is detected by adopting the method provided by the invention, so that 3 repeated experiments are carried out. After sequence comparison and analysis according to the quality control scheme and the judgment threshold, 15 MNP sites of the human cytomegalovirus can be specifically detected in 3 repeated experiments, which shows that the MNP markers and the kit detect the high specificity of target microorganisms in complex templates.

Example 3 detection of genetic variation between human cytomegalovirus strains

6 copies of EB strains provided by the Hubei province disease control are detected by using the kit and the MNP marking site detection method, samples are sequentially named as S-1 to S-6, the average coverage multiple of sequencing of each sample is 1310 times, and all 15 MNP marks can 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 the major genotype differences (Table 5) of partial loci of 1 human cytomegalovirus and 5 human cytomegaloviruses detected together in the same batch indicate the presence of inter-strain variation.

TABLE 5-6 detection assay for human cytomegalovirus

The application of the kit for identifying the genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of the same named human cytomegalovirus strains in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of human cytomegaloviruses. In clinical terms, one can take into account the diagnostic regimen as to whether the site of the difference affects resistance.

Example 4 detection of genetic variation inside human cytomegalovirus strains

As a group organism, partial individuals in the human cytomegalovirus group are mutated, so that the group is no longer homozygous to form a heterogeneous heterozygous group, and the stability and consistency of the phenotype of the microorganism for test are influenced. Such variants, when detected by molecular marker detection on the population, appear as alleles outside the major genotype of the locus. When variant individuals have not accumulated, they occupy a very small proportion of the population and exhibit a low frequency of allelic forms. Low frequency alleles tend to mix with technical errors, making the prior art indistinguishable. The present invention detects MNP markers with high polymorphism. Based on the fact that the probability of occurrence of a plurality of errors is lower than that of one error, the technical error rate of MNP markers is significantly lower than that of SNP markers.

The authenticity assessment of the secondary isogenotypes of this example was performed as follows: the allelotype with strand preference (ratio of the number of sequencing sequences covered on the DNA duplex) is first excluded according to the following rule: the strand preference is greater than 10-fold, or the difference from the strand preference of the major allele is greater than 5-fold.

Genotypes without strand preference were judged for authenticity based on the number and proportion of sequenced sequences in table 6. Table 6 lists e calculated based on binom. Inv function under the probability guarantee of α=99.9999% _max (n=1) and e _max (n.gtoreq.2) is 1.03% and 0.0994%, respectively, and the true hypogenotype is judged only when the number of sequences of the hypogenotype exceeds the critical value. When a plurality of candidate minor alleles exist, multiple correction is carried out on the P value of each candidate allele type, and FDR is carried out<0.5% of candidate alleles are judged to be true minor genotypes.

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. By accurately comparing 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 human cytomegalovirus in detecting genetic variation inside a strain population is demonstrated.

EXAMPLE 5 construction of human cytomegalovirus MNP fingerprint database

The DNA of all strains or samples used for constructing a human cytomegalovirus MNP fingerprint database is extracted by using a conventional CTAB method, a commercial kit and the like, and the quality of the DNA is detected by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm and 230nm is more than 2.0, the ratio of the absorbance values of 260nm and 280nm is between 1.6 and 1.8, the DNA electrophoresis main band is obvious, no obvious degradation and RNA residues exist, the genome DNA reaches the relevant quality requirements, and the subsequent experiments can be carried out.

And (3) comparing the 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 use in human cytomegalovirus typing

The 6 human cytomegalovirus strains were detected by the primer combination and MNP marker locus detection method described in example 2, and MNP fingerprint of each strain was obtained. Comparing MNP fingerprint of each strain with a reference sequence library formed by the published human cytomegalovirus genome sequence and the constructed fingerprint database, wherein the MNP fingerprint is identical to the existing reference sequence, is a very similar strain of the existing strain, has a main genotype difference at least one MNP locus, and is defined as a new variant strain. Detection of 6 human cytomegalovirus samples as shown in table 5, 1 part of detected 6 human cytomegaloviruses were different from the other 5 major genotypes at 5 MNP sites, and were classified into 2 types. These 2 types of strains, in turn, differ from all strains in the reference sequence pool in major genotypes at more than one MNP marker locus, and are therefore judged as new variants. Therefore, the resolution of the method on the human cytomegalovirus reaches the level of single base, and the typing of the human cytomegalovirus 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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<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cttctgcttc gtcaccactt tc 22

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tggttcgttc taagatgagt atcgt 25

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gccaacgaga aaaccgaaaa gtaaa 25

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cgtgcgaccg agcttgtg 18

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

cttaggatcc acaaaaacaa caacc 25

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tagaccgtgt ctgtcttgag cat 23

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gaacaaggtt ctccatcagg atct 24

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

aatcgcacct gaaaagacac g 21

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

caagattttt ggagcgcaca tg 22

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gattatgccg agacaagatt gcatt 25

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

tggaatacag ttttaattgc acgta 25

<210> 17

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ggagcaatgc gacggagat 19

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

cggtgtacgt cgctctac 18

<210> 19

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

cgggataatt tcatctacct atacgga 27

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

tttatcctca tcgtcaacaa caact 25

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

gaagctgtgc aggtgctctt c 21

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

cccagaatac caccaagatc atctt 25

<210> 23

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

caggcgattg cagaaggtg 19

<210> 24

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

aaagtaggca ttcccactga ctt 23

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

cttgaaagga cgcgtggttt t 21

<210> 26

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

acaacccact cttgcagcg 19

<210> 27

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

ctgttgtaga cgtttaaccg cc 22

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

tccgatctgg agtgtgccaa 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

aagcggaaag acgtgtgtga 20

<210> 30

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

ttgcttttta tacacacatg gagac 25

Claims

1. A multiplex PCR primer composition for detecting MNP marker loci of human cytomegalovirus, which is characterized in that 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 MNP marker loci are genomic regions which are screened on the human cytomegalovirus genome and are distinguished from other species and have a plurality of nucleotide polymorphisms inside the species, and comprise the marker loci of MNP-1 to MNP-15 of a reference genome with NC_ 006273.2.

2. A detection kit for detecting a MNP marker locus of human cytomegalovirus, comprising the primer composition of claim 1.

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

4. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for qualitative detection of human cytomegalovirus for non-diagnostic purposes and for preparing a qualitative detection product of human cytomegalovirus.

5. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for the detection of genetic variation within and between human cytomegalovirus strains for non-diagnostic purposes.

6. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for the construction of a human cytomegalovirus database.

7. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for the non-diagnostic genotyping of human cytomegalovirus.