CN114836573B - MNP (MNP) marking site of measles virus, primer composition, kit and application of MNP marking site - Google Patents
MNP (MNP) marking site of measles virus, primer composition, kit and application of MNP marking site Download PDFInfo
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Abstract
The invention discloses a MNP (MNP) marking site of measles virus, a primer composition, a kit and application thereof, wherein the MNP marking site refers to a genome region which is screened on measles virus genome and is separated from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marking sites of MNP-1-MNP-5; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 10. The MNP marker locus can specifically identify measles virus and finely distinguish different subtypes; the primer is not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that the sequence analysis can be carried out on all the marker loci of multiple samples at one time, the method has the advantages of high flux, multiple targets, high sensitivity and culture free, can be applied to the identification and genetic variation detection of measles viruses of large-scale samples, and has important significance on scientific research and monitoring of measles viruses.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to a MNP (MNP) marking site of measles virus, a primer composition, a kit and application thereof.
Background
Measles virus belongs to the genus measles virus of the Paramyxoviridae and is an RNA virus. The only natural storage host is humans, mainly transmitted by the respiratory tract and direct contact with the patient's nasopharyngeal secretions. Measles is a common acute infectious disease in children caused by measles virus and is highly contagious and characterized by pimples, fever and respiratory symptoms. Measles is a legal infectious disease of class B in China, and the incidence and death rate of measles are obviously reduced since planned immunization is implemented. However, due to the increased population flow, partial measles vaccine of children is leaked or the immunity fails, and the like, measles occur in small-scale epidemic, and the harm to human health is still very serious. Because measles is very similar to the related symptoms of diseases such as acute rash, drug eruption, rubella and the like of infants, the identification difficulty is increased. Therefore, the rapid and accurate measles virus detection has important significance for timely diagnosis of etiology, early detection and treatment, reduction of disease deterioration and pathogen transmission control. In addition, measles virus is used as a colony organism, and individuals in the colony 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, the development of a rapid, accurate and mutation-monitoring measles virus detection and analysis method has important significance for the clinical treatment, epidemic prevention detection and scientific research of measles virus.
Classical measles virus detection methods, including isolation culture, PCR techniques, whole genome and metagenome sequencing, etc., suffer from one or more limitations in terms of duration, complexity of operation, throughput of detection, accuracy and sensitivity of detection of 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 low-frequency variation detection.
The molecular markers detected by the existing targeted detection technology mainly comprise SNP and SSR markers. SSR markers are the most well-accepted markers for polymorphism, but are small in number in microorganisms; the number of SNP markers is huge, the distribution is dense, and the polymorphism of single SNP marker is insufficient to capture the potential allelic diversity in microorganism population.
Therefore, development of a novel molecular marker with high polymorphism of pathogenic microorganism measles 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 measles virus, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on measles virus and have the effects of multiple targets, high flux, high sensitivity and fine typing.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention there is provided a MNP marker locus of measles virus, the MNP marker locus being a genomic region specific to the species selected on the measles virus genome and having a plurality of nucleotide polymorphisms within the species, comprising the marker locus of MNP-1 to MNP-5 on the AF266288.2 genome.
In the above technical scheme, the marking sites of MNP-1 to MNP-5 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 AF266288.2 sequence.
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 5 pairs of primers, the nucleotide sequences of the 5 pairs of primers being shown in SEQ ID NO.1 to SEQ ID NO. 10.
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 measles 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 invention, there is provided the use of said MNP marker locus of measles virus or of said multiplex PCR primer composition or of said detection kit for the qualitative detection of measles virus for non-diagnostic purposes.
In a fifth aspect of the invention, there is provided the use of said MNP marker locus of measles virus or of said multiplex PCR primer composition or of said detection kit for the detection of genetic variations both inside and between measles virus strains.
In a sixth aspect of the invention there is provided the use of said MNP marker locus of measles virus or said multiplex PCR primer composition or said detection kit for the construction of measles virus databases.
In a seventh aspect of the invention, there is provided the use of said MNP-tagged site of measles virus or of said multiplex PCR primer composition or of said detection kit for the detection of measles virus in a finely divided form.
In the above application, the method includes: firstly, obtaining virus total RNA of a sample to be tested; reverse transcription is carried out on the total RNA by using a reverse transcription kit to synthesize cDNA; carrying out a first round of multiplex PCR amplification on the cDNA and the blank control by using the kit, wherein the cycle number 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 compared with the reference sequence of measles virus to obtain the number of detection sequences and genotype data of the cDNA. And according to the number of measles virus sequencing sequences obtained from the cDNA and the blank control and the number of detected MNP sites, performing data quality control and data analysis on the sequencing data of the cDNA to obtain the number of detected MNP sites, the number of sequencing sequences covering each MNP site and the genotype data of the MNP sites.
When used for measles virus identification, the quality control is performed according to the number of measles virus sequencing sequences detected in the sample to be detected and the blank control and the number of MNP sites detected, and then whether measles virus nucleic acid is contained in the sample to be detected is judged. The quality control scheme and the judging method are characterized in that RNA of measles virus with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the measles virus detection by the kit are evaluated, and the quality control scheme and the judging method when the measles virus is detected by the kit are formulated. When used for measles 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 5 MNP sites by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared are different at the 5 MNP sites through genotype comparison. 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, 5 sites of the strains to be compared may be amplified by single PCR, and then Sanger sequencing is performed on the amplified products to obtain sequences, and then the genotypes of each MNP site 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 measles virus MNP fingerprint database, the genotype data of the MNP locus of measles virus identified from a sample is recorded into a database file to form the measles virus MNP fingerprint database; each time a different sample is identified, by comparing with the MNP fingerprint database of measles virus, whether measles virus in the sample has a difference of main genotype (with genotype supported by more than 50% sequencing fragment at one MNP site) with strains in the database at the MNP site is identified, and measles virus with the difference of main genotype at least 1 MNP site is a new mutation type and is recorded in the MNP fingerprint database.
When used for measles virus typing, the measles virus in the sample to be tested is identified, and the genotype of each MNP locus is obtained; collecting the genome sequence of measles virus disclosed on the internet and constructing a measles virus reference sequence library by the constructed measles virus MNP fingerprint database; and comparing the genotype of measles virus in the sample to be tested with a reference sequence library of measles virus. And identifying whether measles viruses in the sample are existing strain types or new variant strain types according to the comparison result with the reference sequence library, and realizing the fine typing of the measles viruses.
The invention is initiated in the measles virus field, and is not reported in related documents; MNP markers were developed based primarily on reference sequences, and large-scale discrimination of the reported resequencing sequences of measles virus representative minispecies from MNP sites of other species, polymorphic within measles virus species, flanking sequence conservation; MNP site detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of the MNP site; and then according to the test result of the standard substance, a set of primer combination with the largest polymorphism, high specificity MNP locus and the best compatibility and a detection kit are screened.
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 measles virus MNP (MNP) marking site, a primer composition, a kit and application thereof. The provided 5 MNP sites of measles virus and the primer combination thereof can be used for multiplex PCR amplification, and the amplification products are sequenced by fusing a second generation sequencing platform, so that the detection requirements of high throughput, high efficiency, high accuracy, high sensitivity and culture-free measles virus are met, and the requirements of accurately detecting genetic variation among measles virus strains and inside of a strain group are met; meets the requirements of measles virus standard and sharable fingerprint data construction, and provides technical support for scientific research and monitoring of measles virus.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of screening and primer design of measles virus MNP marker locus;
FIG. 3 is a flow chart of detection of MNP marker loci.
Detailed Description
The advantages and various effects of the embodiments of the present invention will be more clearly apparent from the following detailed description and examples. Those skilled in the art will appreciate that these specific implementations and examples are provided to illustrate, but not limit, examples of the present invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or may be prepared by existing methods.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
MNP markers suitable for detection of the population organisms are screened as detection targets. 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, are suitable for detection of microorganisms, a typical population organism; (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 labeling fuses the ultra-multiplex PCR and the second-generation high-throughput sequencing technology, and has the following advantages: (1) The output is a base sequence, and 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 measles virus that are genomic regions screened on measles virus genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including the marker loci for MNP-1-MNP-5 on the AF266288.2 genome.
Next, the present invention developed a multiplex PCR primer composition for detecting the MNP marker loci of measles virus, comprising 5 pairs of primers, the nucleotide sequences of the 5 pairs of primers are shown in SEQ ID NO.1 to SEQ ID NO. 10. The primers do not collide with each other, and efficient amplification can be performed through multiplex PCR;
the multiplex PCR primer composition can be used for a detection kit for detecting measles virus MNP labeling sites.
The kit provided by the invention can sensitively detect measles virus with 10 copies/reaction.
In the reproducibility test of the invention, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.
The MNP markers and the kits of the invention have high specificity in detecting target microorganisms in complex templates.
The MNP marker locus, primer composition, kit and use thereof of one measles virus of the present application will be described in detail with reference to examples, comparative examples and experimental data.
Example 1 screening of measles virus MNP marker loci and design of multiplex PCR amplification primers
S1, screening of measles virus MNP (MNP) marker loci
Based on complete or partial sequences of genomes of 3794 measles virus different isolates disclosed on the net, 5 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 5 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 representing a subtype of the measles 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 measles virus;
on the reference genome, carrying out window translation by taking 100-300 bp as a window and taking 1bp as a step length, and screening to obtain a plurality of candidate MNP site areas, wherein the candidate MNP site areas contain more than or equal to 2 single nucleotide variation sites, and the single nucleotide polymorphism sites do not exist on sequences of 30bp at both ends;
screening a region with the discrimination DP more than or equal to 0.2 from the candidate polynucleotide polymorphism site region as an MNP marking site; wherein dp=d/t, t is the log of comparisons when all the minor species are compared pairwise in the candidate polynucleotide polymorphic site region, and d is the log of samples of differences in at least two single nucleic acid polymorphisms in the candidate polynucleotide polymorphic site region.
As an optional implementation mode, when screening is performed on the reference genome by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, so that the comprehensive screening is facilitated.
S2, design of multiplex PCR amplification primer
The multiplex PCR amplification primers of the MNP locus are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.
S3, evaluating detection efficiency of primer combination
The measles virus RNA with known copy number provided by the disease prevention control center of Hubei province is used, is reversely transcribed into cDNA by a commercial reverse transcription kit, is added into human genome DNA to prepare a 1000-copy/reaction simulation template, is detected by the MNP mark detection method, 4 repeated sequencing libraries are constructed, and primer combinations with uniform amplification and optimal compatibility are screened according to the detection condition of MNP sites in the 4 libraries, and finally the primer combinations of 5 MNP sites are screened.
Example 2 detection of measles Virus by MNP site and primer
The measles virus RNA with known copy number provided by the Hubei province disease control and prevention control center is used, is reversely transcribed into cDNA by a commercial reverse transcription kit and then added into human genome DNA to prepare measles virus simulation 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. The detection flow of MNP markers is shown in fig. 3. And according to the number of sequencing fragments and the number of sites of measles virus MNP sites detected in the blank control and measles virus nucleic acid standard in 12 repeated experiments, evaluating the reproducibility, accuracy and sensitivity of the detection method, and formulating a quality control system pollution and a threshold value for detecting a target pathogen.
The detection flow of MNP markers is shown in fig. 3.
1. Sensitivity and stability evaluation of MNP (MNaphthyl) marked detection kit for detecting measles virus
As shown in Table 2, the kit can stably detect 1-3 MNP sites in a 1-copy/reaction sample, 6 MNP sites in a 10-copy/reaction sample, and 2 MNP sites at most in a 0-copy/reaction sample, and 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 measles virus
2. Reproducibility and accuracy assessment of detection of measles virus by MNP (MNaphthyl) marker detection kit
Based on whether the genotype of the co-detected site is reproducible or not in the two replicates, the reproducibility and accuracy of detection of measles virus by the MNP marker detection method is evaluated. Specifically, the data of 12 sets of 100 copies/reaction samples were compared in pairs, and the results are shown in Table 3.
TABLE 3 reproducibility and accuracy assessment of measles virus 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%.
3. Threshold value judgment for detection of measles virus by MNP (MNP) marker detection kit
Sequences aligned to measles virus can be detected in 1 copy/reaction samples, covering at least 2 MNP sites. The measles virus sequence was also detected in the 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 1.5 megabases. The measurement and calculation basis is that the number of MNP loci detected by each sample is 5, and the length of one sequencing fragment is 300 bases, so that when the data size is more than 1.5 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 measles virus in the test sample and the noise index P of measles virus in the blank, wherein:
the noise figure p=nc/Nc for the blank, where Nc and Nc represent the number of sequenced fragments and total sequenced fragments of measles virus, respectively, in the blank.
The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments of measles virus and the total number of sequenced fragments, respectively, 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. The results are shown in Table 4;
TABLE 4 SNR of measles Virus in test samples
As can be seen from Table 4, the mean value of the noise index of measles virus in the blank is 0.04%, the mean value of the signal index in the 1-copy sample is 0.30%, and the mean value of the signal to noise ratio of the 1-copy sample and the blank is 7.5, so that 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 70.0, and at least 6 MNP sites were stably detected in the 10 copies/reaction 12 sets of data, accounting for 40.0% of the total sites. Therefore, under the condition of ensuring the accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold value of measles virus is 35, namely when the signal-to-noise ratio of measles virus in a sample is more than 35 and the site detection rate is more than or equal to 30 percent, the measles virus nucleic acid is judged to be detected in the sample.
Therefore, the kit provided by the invention can sensitively detect measles virus of 10 copies/reaction.
4. Specific evaluation of MNP (MNaphthyl) marker detection kit for measles virus detection
The measles virus, human rhinovirus, coronavirus, influenza virus, mycobacterium tuberculosis, staphylococcus aureus, acinetobacter strain, human parainfluenza virus, huo Shibao termates, chlamydia pneumoniae, mycoplasma pneumoniae, haemophilus influenzae, epstein barr virus, varicella zoster virus, cytomegalovirus, herpes simplex virus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and streptococcus pyogenes are artificially mixed together to prepare a mixed template, and the measles virus in the mixed template is detected by adopting the method provided by the invention by using a blank template as a control, so that 3 repeated experiments are carried out. And 5 MNP sites of measles virus can be specifically detected in 3 repeated experiments, and after analysis is carried out according to the quality control scheme and the judgment threshold, the measles virus is judged to be detected in 3 mixed templates, which shows that MNP labels and the kit detect the high specificity of target microorganisms in complex templates.
Example 3 detection of genetic variation between measles Virus strains
The 6 measles virus strains collected are detected by using the kit and the MNP labeling site detection method, the samples are sequentially named as S1-S6, the average coverage of the sequencing of each MNP site is 4220 times, and all 5 MNP labels can be detected by each strain (Table 5). The fingerprints of 6 strains were aligned pairwise and the results are shown in Table 5, with 1 (S-2) and 5 measles viruses detected together in the same batch differing in major genotypes at all sites (Table 5), possibly other species with mislabeling.
Tables 5-6 parts measles virus assay
As can be seen from Table 5, 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 measles virus strains named identically in different laboratories, so that the comparability of research results is ensured, and the kit has great significance for scientific research of measles 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 of measles Virus
Genetic variation detection of measles virus, including inter-strain and intra-strain variation. Since measles virus is parasitic in the host, i.e. the genetic variation of measles virus is detected between and within the host. The variation among hosts is detected by comparing the main genotypes, the obtained fingerprint patterns of measles viruses are compared pairwise, 100% reproducibility and accuracy of the main genotypes are identified based on MNP labeling, and the main genotype difference of two strains with one site can be detected.
And it is the variation inside the measles virus host that is difficult to detect. As a group organism, measles virus varies in a host or in a group, and when the group is detected by molecular marker, it appears as an allele outside the main genotype of the site. 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 invention distinguishes true minor genotypes from error genotypes caused by technical errors through a statistical model. Specifically:
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, the RNA of measles virus of different variants was mixed according to the following 8 ratios 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 in duplicate, and a total of 24 sequencing data were obtained. By accurately comparing the genotypes of MNP loci of two modified measles viruses, heterozygous genotype loci can be detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marker detection method for measles viruses in detecting genetic variation of strains is demonstrated.
Example 5 construction of measles virus MNP fingerprint database
Extracting RNA of all strains or samples for constructing measles virus MNP fingerprint database by using conventional CTAB method, commercial kit and the like, and detecting the quality of the RNA by using agarose gel electrophoresis and an ultraviolet spectrophotometer.
And (3) comparing the sequence of the sequencing data of the 6 strains to obtain the main genotype of each site of each strain, forming MNP fingerprint of each strain, and recording a database file to form a measles virus MNP fingerprint database. The constructed MNP fingerprint database is based on the gene sequence of the detected strain and is therefore compatible with all high throughput sequencing data. The MNP fingerprint of the strain obtained by each detection is compared with the constructed MNP fingerprint database, and the MNP fingerprint database constructed by the MNP fingerprint of the strain with the main genotype difference is realized, so that the co-construction sharing and the random updating of the database are realized.
Example 6 use in measles Virus fine segment
Detecting measles virus in the sample to be detected by using the kit, and obtaining MNP fingerprint of the measles virus in each sample; constructing a reference sequence library consisting of the genomic sequence of the measles virus and the MNP fingerprint database of the measles virus; comparing MNP fingerprint of measles virus of each sample with a constructed reference sequence library, identifying the measles virus as a very similar strain with the same reference sequence, identifying the measles virus as a new variant strain with main genotype difference at more than one MNP locus, and realizing the fine typing of measles virus.
Detection of 6 measles virus samples as shown in Table 5, 1 measles virus and 5 other measles viruses were detected with different major genotypes at the 5 MNP sites, and the strains in the reference sequence library were also different, possibly as new variants or as false marks during the experiment or other strain contamination events. Therefore, the resolution of the measles virus by the method reaches the level of single base, and the fine typing of measles virus in a 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 measles virus, which is characterized by comprising 5 pairs of primers, wherein the nucleotide sequences of the 5 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 10.
2. A test kit for the detection of measles virus, characterized in that it comprises the primer composition according to claim 1.
3. The test kit of claim 2, wherein the kit further comprises a multiplex PCR premix.
4. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for the qualitative detection of measles virus for non-diagnostic purposes.
5. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for detecting genetic variations within and between measles virus strains.
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 measles virus database.
7. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 in the detection of measles virus in a finely divided form.
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