CN114790494B - MNP (varicella-zoster virus) marking site, primer composition, kit and application thereof - Google Patents
MNP (varicella-zoster virus) marking site, primer composition, kit and application thereof Download PDFInfo
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Abstract
The invention discloses an MNP (MNP) marking site, a primer composition, a kit and application thereof of varicella-zoster virus, wherein the MNP marking site refers to a genome region which is screened on varicella-zoster virus genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the interior of 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 varicella-zoster virus 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 detection advantages of high flux, multiple targets, high sensitivity, high precision and culture free are achieved, the method can be applied to the identification and genetic variation detection of varicella-zoster virus of large-scale samples, and the method has important significance on the scientific research and epidemic prevention monitoring of varicella-zoster virus.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marking site of varicella-zoster virus, a primer composition, a kit and application thereof.
Background
Varicella-zoster virus (varicella-zoster virus), human herpesvirus type 3 (HHV-3), is the causative agent of varicella or zoster. Varicella-zoster virus is quite common and enters the body by respiratory tract or contact infection, almost all people have been exposed to the virus before adulthood, and the virus has only one serotype, which can cause two different conditions, namely primary infection varicella (variella) and recurrent infection of herpes zoster (zoter). The varicella does not leave scars after disappearance, the illness is generally lighter, but the varicella is complicated with interstitial pneumonia and encephalitis after infection. When adults suffer from varicella, 20-30% of pneumonia is complicated, the illness is serious, and the death rate is high. The pregnant women also have severe varicella manifestations and may cause fetal malformation, abortion or stillbirth. For the prevention of this virus, healthy children aged 1-12 years are commonly vaccinated with attenuated vaccines. Detection of varicella-zoster virus includes electron microscopy of the herpesfluid, or cell culture to isolate the virus, or immunofluorescence assays to detect herpesvirus antigens in herpesvirus basal material smears and biopsy tissue sections, or PCR to amplify DNA of cerebrospinal fluid. Varicella-zoster virus is taken as a group organism, and individuals in the group can be mutated in interaction with a host and the environment, so that a 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, the development of a rapid, accurate and monitorable varicella-zoster virus detection and analysis method has important significance for clinical treatment, epidemic prevention detection and scientific research of varicella-zoster virus.
Existing methods for detecting varicella-zoster virus have one or more limitations in terms of duration, complexity of operation, throughput of detection, accuracy and sensitivity of detecting variations, 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 detectable variation. 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 for high polymorphism of varicella-zoster virus and detection technology thereof becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a MNP (MNP) marking site of varicella-zoster virus, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on varicella-zoster virus and have the effects of multiple targets, high flux, high sensitivity and fine 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 a MNP-marker locus of varicella-zoster virus, which is a genomic region screened on varicella-zoster virus genome which is distinguished from other species and has a plurality of nucleotide polymorphisms within the species, comprising marker loci of MNP-1 to MNP-15 on varicella-zoster virus reference sequence.
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 sequences corresponding to the same row of MNPs 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 specific primer sequences being shown in SEQ ID NO.1-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 varicella-zoster 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 said varicella-zoster virus MNP marker locus or said multiplex PCR primer composition or said detection kit for the identification of varicella-zoster virus of non diagnostic interest.
In a fifth aspect of the invention, there is provided the use of said varicella-zoster virus MNP labeling site or said multiplex PCR primer composition or said detection kit for detecting genetic variation within and between varicella-zoster virus strains.
In a sixth aspect of the present invention, there is provided the use of said varicella-zoster virus MNP marker locus or said multiplex PCR primer composition or said detection kit for constructing a varicella-zoster virus database.
In a seventh aspect of the present invention, there is provided the use of said varicella-zoster virus MNP marker locus or said multiplex PCR primer composition or said detection kit in varicella-zoster virus fine-division detection.
In the above-mentioned varicella-zoster virus identification, intra-strain and inter-strain genetic variation, database, and fine-segment detection applications, the specific operation steps are as follows: firstly, obtaining total bacterial DNA of a sample to be detected; 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; the sequencing results are aligned to the reference sequence of the varicella-zoster virus, and the number of detection sequences and genotype data of the total DNA are obtained. And performing data quality control and data analysis on the sequencing data of the total DNA according to the number of varicella-zoster virus sequencing sequences and the number of detected MNP sites obtained from 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 used for varicella-zoster virus identification, the quality control is carried out according to the number of the sequencing sequences of varicella-zoster virus detected in the sample to be detected and the blank control and the number of MNP sites detected, and then whether the sample to be detected contains nucleic acid of varicella-zoster virus or not is judged. The quality control scheme and the judging method are characterized in that DNA of varicella-zoster virus with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting varicella-zoster virus are evaluated, and the quality control scheme and the judging method when the kit detects varicella-zoster virus are formulated.
When used for varicella-zoster virus 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 used for constructing a varicella-zoster virus DNA fingerprint database, genotype data of the MNP locus of varicella-zoster virus identified from a sample is entered into a database file to form the varicella-zoster virus DNA fingerprint database; each time a different sample is identified, it is identified whether varicella-zoster virus in the sample differs from strains in the database in the main genotype (with more than 50% of the genotypes supported by the sequencing fragments at one MNP site) at the MNP site by comparison with the DNA fingerprint database of the varicella-zoster virus, and the varicella-zoster virus with the main genotype difference at least 1 MNP site is a new variant type and is recorded in the DNA fingerprint database.
When used for varicella-zoster virus typing, the varicella-zoster virus in a sample to be tested is identified, and the genotype of each MNP locus is obtained; collecting genome sequences of varicella-zoster viruses disclosed on the net and constructing a varicella-zoster virus DNA fingerprint database to form a varicella-zoster virus reference sequence library; comparing the genotype of varicella-zoster virus in the sample to be tested with a reference sequence library of varicella-zoster virus. And identifying whether varicella-zoster virus in the sample is of an existing type or a new mutation type according to the comparison result with the reference sequence library, and realizing the fine typing of the varicella-zoster virus.
The invention is initiated in the varicella-zoster virus field, and is not reported in related documents; MNP markers were developed based primarily on reference sequences, and large scale MNP sites that are differentiated from other species, polymorphic within varicella-zoster virus species, conserved flanking sequences could be mined from reported resequencing data for varicella-zoster virus representative races; 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 a MNP (varicella-zoster virus) marking site, a primer composition, a kit and application thereof. The provided 15 MNP loci of varicella-zoster virus and primer combination thereof can be subjected to multiplex PCR amplification, and a second generation sequencing platform is fused to sequence amplification products, so that the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on varicella-zoster virus are met, and the requirements of standard and sharable fingerprint data construction of varicella-zoster virus are met; the need to accurately detect genetic variation between varicella-zoster virus strains; the identification of the requirements of varicella-zoster virus homozygosity and heterozygosity provides technical support for the scientific research, scientific monitoring and prevention and control of varicella-zoster 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 showing screening of varicella zoster virus MNP labeling site and primer design;
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 which is suitable for detecting group organisms and is 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 Species alleles higher than SSR and SNP; (2) The species distinguishing capability is strong, the species identification can be realized by only a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting MNP labels based on the combination of super multiplex PCR and a second generation high throughput sequencing technology has the following advantages: (1) The output is a base sequence, a standardized database can be constructed for sharing without parallel experiments; (2) The method has high efficiency, breaks through the limitation of the number of sequencing samples by using the sample DNA bar code, and can type tens of thousands of MNP 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 MNP mark is reported in varicella-zoster virus, and corresponding technology is also lacked. Thus, the present invention developed MNP-marker loci of varicella-zoster virus, which are genomic regions screened on varicella-zoster virus genome that are differentiated from other species and have a plurality of nucleotide polymorphisms within the species, including marker loci of MNP-1 to MNP-15 of NC_001348.1 as reference genome.
Next, the present invention has developed a multiplex PCR primer composition for detecting the varicella-zoster virus MNP marker locus, 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. 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 detecting the varicella-zoster virus MNP labeling site.
The kit of the invention can accurately and sensitively detect varicella-zoster virus with the concentration as low as 10 copies/reaction.
The MNP markers and the kits of the invention have high specificity in detecting target microorganisms in complex templates.
The MNP marker loci, primer compositions, kits and uses thereof of one varicella-zoster virus of the present application will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1 screening of varicella zoster Virus MNP marker locus and design of multiplex PCR amplification primers
S1, screening of varicella-zoster virus MNP labeling site
Based on the complete or partial sequences of the genomes of the 3794 varicella-zoster virus different isolates disclosed on the net, 15 MNP marking sites 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 varicella-zoster 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 varicella-zoster 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. In this embodiment, the primers used to identify the MNP marker sites are shown in table 1.
S3, evaluating detection efficiency of primer combination
The detection method of the MNP marker is that all MNP loci are amplified at one time through multiplex PCR, amplification products are sequenced through second-generation high-throughput sequencing, sequencing data are analyzed, and compatibility of the primer combination is evaluated according to the detected loci.
The template of 1000 copies/reaction was prepared by adding varicella-zoster virus DNA with known copy number to human genome DNA, and the primer combination was used to screen the primer combination with optimal compatibility and uniformity of amplification according to detection of MNP sites in 4 libraries, and finally the primer combination of 15 MNP sites according to Table 1 of the present invention was selected.
Threshold settings and Performance evaluation for MNP site and primer identification of varicella-zoster Virus described in example 2
In this example, varicella-zoster virus nucleic acid standard with known copy number was added to human genomic DNA to prepare varicella-zoster virus mimetic 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. According to the number of sequencing fragments and the number of sites of varicella-zoster virus MNP sites detected in a blank control and varicella-zoster virus nucleic acid standard in 12 repeated experiments, preparing a threshold value for detecting the pollution of a quality control system and a target pathogen, evaluating the reproducibility, the accuracy and the sensitivity of a detection method, and preparing the threshold value for detecting the pollution of the quality control system and the target pathogen. The detection flow of MNP markers is shown in fig. 3.
1. Sensitivity and stability assessment of MNP-labeled detection kit for detecting varicella-zoster virus
As shown in Table 2, the kit can stably detect more than 9 MNP sites in a 10-copy/reaction sample, and can detect 2 MNP sites at most 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.
TABLE 2 detection sensitivity and stability analysis of MNP labeling method of varicella-zoster Virus
2. Reproducibility and accuracy assessment of MNP (MNP) marker detection kit for detecting varicella-zoster virus
Based on whether the genotype of the co-detection site is reproducible or not in the two replicates, the reproducibility and accuracy of detection of varicella-zoster virus by the MNP marker detection method are evaluated. Specifically, the paired comparison was performed on 12 sets of data for 100 copies of the sample, respectively, and the results are shown in table 3.
TABLE 3 reproducibility and accuracy assessment of detection method of varicella zoster virus MNP marker
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 project reproducibility test, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.
3. Threshold value judgment for detecting varicella-zoster virus by MNP (MNP) mark detection kit
As shown in Table 2, the sequences aligned to varicella-zoster virus could be detected in 1 copy/reaction sample, covering at least 1 MNP site. The varicella zoster virus sequence was also detected in a partial blank. Because of the extreme sensitivity of MNP marker detection methods, contamination of the data in the detection is prone to false positives. Therefore, the quality control scheme is formulated in this example, and is specifically 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 varicella-zoster virus in the test sample and the noise index P of varicella-zoster 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 number of sequenced fragments of varicella zoster virus, respectively, in the control.
The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments and the total number of sequenced fragments, respectively, of varicella-zoster virus in the test sample.
3) 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 varicella zoster Virus in samples to be tested
As a result, as shown in Table 4, the average value of the noise index of varicella-zoster virus in the control was 0.04%, the average value of the signal index in the 1-copy sample was 0.31%, and the average value of the signal to noise ratio of the 1-copy sample and the control was 8.1, 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 77.5, 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. Thus, the present standard specifies that the signal-to-noise ratio judgment threshold for varicella-zoster virus is 35, i.e., when the signal-to-noise ratio of varicella-zoster virus in the sample is greater than 35 and the site detection rate is 30% or more, it is judged that the nucleic acid of varicella-zoster virus is detected in the sample. Based on the above, the kit provided by the invention can accurately and sensitively detect varicella-zoster virus with the concentration as low as 10 copies/reaction.
4. Specific evaluation of detection of varicella-zoster virus by MNP marker detection method
The varicella-zoster virus, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao termates, chlamydia pneumoniae, mycoplasma pneumoniae, EB virus, haemophilus influenzae, cytomegalovirus, herpes simplex virus, human bocavirus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, streptococcus pneumoniae and streptococcus pyogenes are artificially mixed together according to the equimolar amount to prepare a mixed template, and a blank template is used as a control, so that varicella-zoster virus in the mixed template is detected by adopting the method provided by the invention, and 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 varicella-zoster virus 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 varicella zoster Virus strains
6 varicella-zoster virus strains provided by the Hubei province disease control prevention control center are detected by using the kit and the MNP marking site detection method, samples are sequentially named as S-1 to S-6, wherein S-2 to S-5 are offspring strains of the same strain in different periods. The average coverage of sequencing per sample was 2034 fold, and all 15 MNP markers could be detected per strain (table 5). The fingerprint patterns of 6 strains are subjected to pairwise comparison, and the results are shown in table 5, wherein the difference between S1 and other strains is at least the difference between the main genotypes of 3 MNP loci; s-2 and S3-S5 also present major genotype differences at 1 MNP site (Table 5), indicating that the same named strains have inter-strain variation.
TABLE 5 detection assay for 6 varicella zoster Virus
As can be seen from Table 5, the application of the kit of the invention in identifying genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of varicella-zoster virus strains named identically in different laboratories, thereby ensuring the comparability of research results, which has important significance for scientific research of varicella-zoster viruses. 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 varicella zoster Virus strains
As a group organism, the varicella-zoster virus group has mutation of partial 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, and the true hypogenotype is judged only when the number of sequences of the hypogenotype exceeds the critical value. When a plurality of candidate minor alleles exist, multiple correction is carried out on the P value of each candidate allele type, and FDR is carried out<0.5% of candidate alleles are judged to be true minor genotypes.
Parameter e related to Table 6 max (n=1) and e max (n.gtoreq.2) 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.
TABLE 6-threshold for determining the hypo-isogenotypes at partial sequencing depth
According to the above parameters, nucleic acids of two strains having a difference in genotype shown in Table 5 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 mycoplasma pneumoniae in detecting genetic variation inside a strain population is demonstrated.
EXAMPLE 5 construction of varicella zoster Virus DNA fingerprint database
All strains or DNA of samples used for constructing varicella-zoster virus DNA fingerprint database are extracted by using the conventional CTAB method, commercial kit and other methods, and the quality of the DNA is detected by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm and 230nm is more than 2.0, the ratio of the absorbance values of 260nm and 280nm is between 1.6 and 1.8, the DNA electrophoresis main band is obvious, no obvious degradation and RNA residues exist, the genome DNA reaches the relevant quality requirements, and the subsequent experiments can be carried out.
And (3) comparing the sequencing data of the 6 strains with the reference genotype in sequence, and obtaining the main genotype of each site of each strain to form the MNP fingerprint of each strain. And inputting the obtained MNP fingerprint of each strain into a database file to form a varicella-zoster virus DNA 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. And comparing the MNP fingerprint of the strain obtained by each detection with an MNP fingerprint database constructed based on the existing genome data, and inputting the MNP fingerprint of the strain with the main genotype difference into the constructed MNP fingerprint database to achieve real-time updating and co-construction sharing of the database.
Example 6 use in varicella zoster Virus Fine details
And detecting the 6 varicella-zoster virus strains by using the primer combination and MNP labeling site detection method, thereby obtaining MNP fingerprint of each strain. And comparing the DNA fingerprint of each strain with a constructed reference sequence library in pairs, wherein the DNA fingerprint is the same as the existing fingerprint database, has the main genotype difference at least one MNP locus, is a new variation type, and realizes the fine typing of varicella-zoster virus.
The detection results of 6 varicella-zoster viruses are shown in table 5, and after the 6 varicella-zoster viruses are compared in pairs, the 6 varicella-zoster viruses are divided into 3 types according to the difference of main genotypes, and the 3 types are different from strains in a reference sequence library and are new mutation types. Thus, the resolution of the varicella-zoster virus by the method reaches the level of single base, and the fine typing of varicella-zoster 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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Claims (7)
1. A multiplex PCR primer composition for detecting varicella-zoster virus MNP marking site, 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; the MNP marking site is a genome region which is screened on varicella-zoster virus genome and is distinguished from other species and has a plurality of nucleotide polymorphisms inside the species, and comprises MNP-1 to MNP-15 marking sites taking NC_001348.1 as a reference genome.
2. A detection kit for detecting varicella-zoster virus MNP marker locus, 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 the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 for the identification of varicella-zoster virus for non-diagnostic purposes.
5. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 for detecting varicella-zoster virus strain internal and inter-strain genetic variation for non-diagnostic purposes.
6. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 for constructing varicella-zoster virus database.
7. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 for the non-diagnostic purpose of finely divided detection of varicella-zoster virus.
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| CN105132584A (en) * | 2015-08-03 | 2015-12-09 | 中国人民解放军成都军区总医院 | Kit for genotyping VZV, production method of kit and application of kit |
| WO2020072409A1 (en) * | 2018-10-01 | 2020-04-09 | Gen-Probe Incorporated | Compositions and methods for amplifying or detecting varicella-zoster virus |
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| CN105132584A (en) * | 2015-08-03 | 2015-12-09 | 中国人民解放军成都军区总医院 | Kit for genotyping VZV, production method of kit and application of kit |
| WO2020072409A1 (en) * | 2018-10-01 | 2020-04-09 | Gen-Probe Incorporated | Compositions and methods for amplifying or detecting varicella-zoster virus |
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| 上海地区水痘带状疱疹病毒基因型研究;杨吉星;居丽雯;施强;李峰;吕锡宏;蒋露芳;;中国卫生检验杂志(第02期) * |
| 四川地区水痘-带状疱疹病毒临床分离株基因型分析研究;刘毓刚;杨川江;吴丽娟;曾平;吴艾霖;;中华医院感染学杂志(第20期) * |
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