CN114836573A - MNP (protein-binding protein) marker locus of measles virus, primer composition, kit and application of MNP marker locus - Google Patents
MNP (protein-binding protein) marker locus of measles virus, primer composition, kit and application of MNP marker locus Download PDFInfo
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Abstract
The invention discloses an MNP marker locus of measles virus, a primer composition, a kit and application thereof, wherein the MNP marker locus refers to a genome region which is screened from a measles virus genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker loci of MNP-1-MNP-5; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 10. The MNP marker locus can specifically identify the measles virus and finely distinguish different subtypes; the primers are not interfered with each other, and by integrating multiple amplification and sequencing technologies, all marked sites of multiple samples can be subjected to sequence analysis at one time, so that the method has the advantages of high throughput, multiple targets, high sensitivity and no culture, can be applied to identification and genetic variation detection of measles viruses of large-scale samples, and has important significance for scientific research and monitoring of the measles viruses.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marker locus of measles virus, a primer composition, a kit and application thereof.
Background
Measles virus (measles virus) belongs to the genus measles virus of the family paramyxoviridae and is an RNA virus. The only natural storage host is human, primarily by airborne transmission via the respiratory tract and direct contact with the nasopharyngeal secretions of patients. Measles is a common acute infectious disease of children caused by measles virus, and is highly contagious and characterized by pimples, fever and respiratory symptoms. Measles is a legal infectious disease class B in China, and the incidence and fatality rate of measles are remarkably reduced since the planned immunization is implemented. However, due to the increase of population mobility, the small-scale epidemic of measles sometimes happens due to the reasons of the missing of measles vaccine or the failure of immunity of part of children and the like, and the damage to the health of human is still very serious. Because measles is very similar to the symptoms related to diseases such as acute eruption, drug eruption, rubella and the like of infants, the identification difficulty is increased. Therefore, the rapid and accurate measles virus detection is of great significance for diagnosing the cause of the disease in time, finding early treatment, reducing the disease deterioration and controlling the transmission of the pathogen. In addition, measles virus, as a population organism, can mutate in the interaction with the host and the environment, and cause the failure of detection or treatment methods; for experimental studies, such inconspicuous variations can result in different laboratories or the same laboratory differing in the fact that identically named strains are different at different times, resulting in non-reproducible and incomparable experimental results. Therefore, the development of a rapid and accurate measles virus detection and analysis method capable of monitoring the mutation has important significance for clinical treatment, epidemic prevention detection and scientific research of measles virus.
Classical measles virus detection methods, including isolation and culture, PCR techniques, whole and metagenomic sequencing, have one or more limitations in terms of duration, complexity of operation, detection throughput, accuracy and sensitivity of detection variation, cost, and the like. The targeted molecular marker detection technology combining the ultra-multiplex PCR amplification and the high-throughput sequencing can be used for enriching target microorganisms in a sample with low microorganism content in a targeted manner, avoids a large amount of data waste and background noise caused by whole genome and metagenome sequencing, and has the advantages of less sample requirement, accurate diagnosis result, data quantity saving and low-frequency variation detection.
The molecular markers detected by the existing targeted detection technology mainly comprise SNP (single nucleotide polymorphism) markers and SSR (simple sequence repeat) markers. SSR markers are generally accepted as the most polymorphic markers, but are few in microorganisms; the SNP markers are large in number, densely distributed and are binary markers, and the polymorphism of a single SNP marker is insufficient to capture the potential allelic diversity in a microbial population.
Therefore, development of a novel molecular marker with high polymorphism of pathogenic microorganism measles virus and a detection technique thereof have become technical problems to be solved urgently.
Disclosure of Invention
The invention aims to provide an MNP (MNP) marker locus of measles virus, a primer composition, a kit and application thereof, which can perform 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 purpose, the invention adopts the following technical scheme:
in a first aspect of the invention there is provided a MNP marker site for measles virus which is a region of the genome specific to the species screened for in the genome of measles virus and having a plurality of nucleotide polymorphisms within that species, including marker sites for MNP-1 to MNP-5 on the AF266288.2 genome.
In the above technical solution, the labeling sites of MNP-1 to MNP-5 are specifically shown in table 1 of the specification, and the starting and ending positions of the MNP label marked in table 1 are determined based on the AF266288.2 sequence.
In a second aspect of the invention, a multiplex PCR primer composition for detecting the MNP marker site is provided, the multiplex PCR primer composition comprises 5 pairs of primers, and the nucleotide sequences of the 5 pairs of primers are shown as SEQ ID No.1 to SEQ ID No. 10.
In the above technical scheme, the primer of each MNP marker site includes an upper primer and a lower primer, which are specifically shown in table 1 of the specification.
In a third aspect of the invention, there is provided a test kit for detecting the MNP marker site of measles virus, which kit comprises the primer composition.
Further, the kit also comprises a multiplex PCR premix.
In a fourth aspect of the invention, there is provided the use of said MNP marker site of measles virus or said multiplex PCR primer composition or said detection kit for the qualitative detection of measles virus for non-diagnostic purposes.
In the fifth aspect of the invention, the application of the MNP marker locus of the measles virus or the multiple PCR primer composition or the detection kit in detecting the genetic variation inside and among measles virus strains is provided.
In the sixth aspect of the present invention, there is provided the use of the MNP marker site of measles virus or the multiplex PCR primer composition or the detection kit for constructing a measles virus database.
In the seventh aspect of the present invention, there is provided the use of the MNP marker site of measles virus or the multiplex PCR primer composition or the detection kit for the detection of measles virus subtyping.
The application comprises the following steps: firstly, acquiring the virus total RNA of a sample to be detected; reverse transcription is carried out on the total RNA by utilizing a reverse transcription kit to synthesize cDNA; performing a first round of multiplex PCR amplification on the cDNA and a blank control by using the kit disclosed by the invention, wherein the cycle number is not higher than 25; after purifying the amplification product, adding a sample label based on the second round of PCR amplification and a second-generation sequencing adaptor; purifying and quantifying the second round amplification product; when detecting a plurality of strains, performing high-throughput sequencing by equivalently mixing the amplification products of the second round; the sequencing result is aligned to the reference sequence of the measles virus, and the number and genotype data of the detected sequences in the cDNA are obtained. And performing data quality control and data analysis on the sequencing data of the cDNA according to the number of the obtained measles virus sequencing sequences of the cDNA and the blank control and the number of detected MNP loci to obtain the number of the detected MNP loci, the number of the sequencing sequences covering each MNP locus and the MNP locus genotype data.
When the method is used for measles virus identification, the number of sequencing sequences of measles virus detected in a sample to be detected and a blank control and the number of MNP sites detected are subjected to quality control, and then whether the nucleic acid of the measles virus exists in the sample to be detected or not is judged. The quality control scheme and the determination method are characterized in that measles virus RNA with known copy number is used as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting measles virus are evaluated, and the quality control scheme and the determination method for detecting measles virus by the kit are established. When used for measles virus genetic variation detection, the detection of genetic variation between strains and within strains is included. The detection of genetic variation among strains comprises the steps of obtaining genotype data of strains to be compared at 5 MNP sites by using the kit and the method. And analyzing whether the major genotypes of the strains to be compared on the 5 MNP sites have difference or not through genotype comparison. If the strains to be compared have variation in the major genotype of at least one MNP site, the strains are judged to have genetic variation. As an alternative, 5 sites of the strains to be compared can be amplified respectively through single PCR, and then the amplification products are subjected to Sanger sequencing, and after the sequences are obtained, the genotypes of all MNP sites of the strains to be compared are compared. If there are MNP sites that are not identical in major genotypes, there is variation between the strains to be compared. When detecting the genetic variation in the strain, judging whether a secondary genotype other than the main genotype is detected at the MNP site of the strain to be detected or not through a statistical model. And if the to-be-detected strain has a minor genotype at least one MNP site, judging that the genetic variation exists in the to-be-detected strain.
When used for constructing a measles virus MNP fingerprint database, typing the genotype data of the MNP sites of measles virus identified from a sample into a database file to form the measles virus MNP fingerprint database; when different samples are identified, the measles virus in the samples is compared with the MNP fingerprint database of the measles virus to identify whether the measles virus in the samples has a main genotype difference (more than 50 percent of genotypes supported by a sequencing fragment at one MNP locus) with the strains in the database at the MNP locus, and the measles virus with the main genotype difference at least 1 MNP locus is a new variant type and is included in the MNP fingerprint database.
When the method is used for measles virus typing, the measles virus in a sample to be tested is identified to obtain the genotype of each MNP locus; collecting genome sequences of measles viruses disclosed on the Internet and a constructed measles virus MNP fingerprint database to construct a measles virus reference sequence database; and (3) comparing the measles virus genotype in the sample to be tested with the measles virus reference sequence library. And identifying whether the measles virus in the sample is an existing strain type or a new variant strain type according to the comparison result of the measles virus and the reference sequence library, thereby realizing the fine classification of the measles virus.
The invention belongs to the field of measles virus, and is not reported in related documents; MNP markers are mainly developed based on reference sequences, and are differentiated from MNP sites of other species, which are polymorphic in the measles virus species and conserved in sequences at two sides of the measles virus species on a large scale according to reported re-sequencing data of small measles virus representative species; MNP locus detection primers suitable for multiplex PCR amplification can be designed through conserved sequences on two sides of the MNP locus; and then according to the test result of the standard product, a set of MNP sites with the largest polymorphism and high specificity, a primer combination with the best compatibility and a detection kit are screened.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the invention provides a MNP marker locus of measles virus, a primer composition, a kit and application thereof. The provided 5 MNP sites of the measles virus and the primer combination thereof can carry out multiplex PCR amplification, fuse a second-generation sequencing platform to carry out sequencing on an amplification product, meet the detection requirements of high throughput, high efficiency, high accuracy, high sensitivity and no culture on the measles virus, and meet the requirements of accurately detecting the genetic variation among measles virus strains and in a strain group; the requirement of construction of sharable fingerprint data meeting the measles virus standard provides technical support for scientific research and monitoring of measles virus.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of the screening and primer design of the MNP marker sites of measles virus;
FIG. 3 is a flow chart of detection of MNP marker sites.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the embodiments of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that the present embodiments and examples are illustrative of the present invention and are not to be construed as limiting the present invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
screening MNP markers suitable for detecting the population organisms as detection targets. MNP markers refer to polymorphic markers caused by multiple nucleotides over a region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) abundant alleles, 2 at a single MNP locus n Species alleles, higher than SSR and SNP, are suitable for detection of microorganisms, a typical population of organisms; (2) the species distinguishing capability is strong, species identification can be realized only by a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting the MNP label is fused with 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 efficiency is high, the sample DNA bar code is utilized, the limitation of the quantity of sequencing samples is broken through, and tens of thousands of MNP sites of hundreds of samples can be typed at one time; (3) the sensitivity is high, multiple targets are detected at one time by utilizing multiple PCR, and high false negative and low sensitivity caused by amplification failure of a single target are avoided; (4) high accuracy, sequencing the amplified product by using a second generation high-throughput sequencerThe treatment is carried out for hundreds of times.
In view of the advantages and the characteristics, the MNP marker and the detection technology MNP marking method thereof can realize the classification and the tracing of the multi-allelic genotypes of the population organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, detection of genetic variation and the like. At present, no report on MNP labeling exists in microorganisms, and corresponding technologies are lacked. The development, screening and application of the MNP marking method have better application foundation in plants.
Accordingly, the present invention has developed MNP marker sites for measles virus, which are regions of the genome screened on the genome of measles virus that are distinct from other species and have multiple nucleotide polymorphisms within the species, including marker sites for MNP-1 to MNP-5 on the AF266288.2 genome.
Next, the present invention has developed a multiplex PCR primer composition for detecting the MNP marker site of measles virus, comprising 5 pairs of primers, wherein the nucleotide sequences of the 5 pairs of primers are shown in SEQ ID NO. 1-SEQ ID NO. 10. The primers are not conflicted with each other, and can be efficiently amplified through multiple PCR;
the multiplex PCR primer composition can be used for a detection kit for detecting the MNP marker locus of the measles virus.
The kit provided by the invention can sensitively detect the measles virus with 10 copies/response.
In the reproducibility test of the invention, the logarithm of difference of the MNP labeling main gene type among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100%, and the accuracy a is 100%.
The MNP marker and the kit of the present invention have high specificity in detecting a target microorganism in a complex template.
The MNP marker site, primer composition, kit and use of a measles virus according to the present invention will be described in detail with reference to examples, comparative examples and experimental data.
Example 1 screening of the MNP marker sites of measles Virus and design of the multiplex PCR amplification primers
S1 screening for MNP marker sites of measles virus
Based on the complete or partial sequence of the genome of the different isolates of the 3794 measles viruses published on the web, 5 MNP marker sites were obtained by sequence alignment. For species without genomic data on the net, the genomic sequence information of the representative microspecies of the microbial species to be detected can also be obtained by high-throughput sequencing, wherein the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected marker, the genomic sequence of at least 10 genetically representative isolates is generally used as a reference. The 5 MNP marker sites screened are shown in table 1:
TABLE 1-starting position of the MNP marker site and detection primer on the reference sequence
The step S1 specifically includes:
selecting a genome sequence of one representative subtype of the measles virus as a reference genome, and performing sequence alignment on the genome sequence and the reference genome to obtain single nucleic acid polymorphic sites of each strain of the measles virus;
on the reference genome, performing window translation by taking 100-300 bp as a window and 1bp as a step length, and screening to obtain a plurality of candidate MNP site regions, wherein the candidate MNP site regions contain more than or equal to 2 single nucleotide variation sites, and the single nucleotide polymorphism sites do not exist on sequences of 30bp at two ends;
screening a region with the division DP of more than or equal to 0.2 in the candidate polynucleotide polymorphic site region as an MNP marker site; wherein, DP ═ d/t, t is the comparison logarithm of all the minor species in the region of the candidate polynucleotide polymorphic site when compared pairwise, d is the sample logarithm of at least two single nucleic acid polymorphisms that differ in the region of the candidate polynucleotide polymorphic site.
As an optional implementation mode, when the reference genome is screened by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, which is beneficial to comprehensive screening.
S2 design of multiplex PCR amplification primers
And designing the multiplex PCR amplification primers of the MNP sites through primer design software, wherein the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely all the designed primers can be normally amplified in one amplification reaction.
S3 evaluation of detection efficiency of primer combination
Measles virus RNA with known copy number provided by Hubei province disease prevention control center is reversely transcribed into cDNA by a commercial reverse transcription kit, and then added into human genome DNA to prepare a 1000-copy/reaction simulation template, detection is carried out by the MNP mark detection method, 4 repeated sequencing libraries are constructed, primer combinations with uniform amplification and optimal compatibility are screened according to the detection condition of MNP sites in the 4 libraries, and finally, primer compositions of 5 MNP sites disclosed in the table 1 of the invention are screened out.
Example 2 detection of measles Virus by MNP sites and primers
Measles virus RNA with known copy number provided by disease control and prevention control center in Hubei province is added into human genome DNA after being reverse transcribed into cDNA by commercial reverse transcription kit to prepare measles virus simulant samples of 1 copy/reaction, 10 copies/reaction and 100 copies/reaction. An equal volume of sterile water was also set as a blank. A total of 4 samples were obtained, each sample was constructed into 3 duplicate libraries each day, and the assay was continued for 4 days, i.e. 12 sets of sequencing data were obtained for each sample, as shown in table 2. The flow of detection of MNP markers is shown in figure 3. According to the sequencing fragment number and the site number of the MNP site of the measles virus detected in the blank control and the measles virus nucleic acid standard in 12 repeated experiments, the repeatability, the accuracy and the sensitivity of the detection method are evaluated, and the threshold values of quality control system pollution and target pathogen detection are set.
The flow of detection of MNP markers is shown in figure 3.
1. Sensitivity and stability evaluation of MNP (MNP) marker detection kit for detecting measles virus
As shown in Table 2, the kit can stably detect more than 1-3 MNP sites in a sample with 1 copy/reaction, stably detect more than 6 MNP sites in a sample with 10 copies/reaction and detect 2 MNP sites at most in a sample with 0 copies/reaction, can obviously distinguish the samples with 10 copies/reaction and 0 copies/reaction, and has technical stability and detection sensitivity as low as 10 copies/reaction.
TABLE 2 analysis of detection sensitivity and stability of MNP labeling method for measles virus
2. Evaluation of reproducibility and accuracy of MNP (MNP) marker detection kit for detecting measles virus
The reproducibility and accuracy of the MNP marker detection method for detecting measles virus was evaluated based on whether the genotype of the co-detected site was reproducible in two replicates. Specifically, two-by-two comparisons were made for each of 12 sets of data for 100 copies/reaction samples, and the results are shown in Table 3.
TABLE 3 evaluation of reproducibility and accuracy of the MNP marker detection method for measles virus
As can be seen from Table 3, the number of MNP sites differing in major genotypes was 0; the accuracy rate a is 1- (1-r)/2 is 0.5+0.5r, and r represents the reproducibility rate, i.e., the ratio of the number of sites where the major genotype is reproducible to the number of common sites, which is considered to be the principle of accuracy among 2 repeated experiments. In the reproducibility test of the invention, the logarithm of difference of the MNP labeling main gene type among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100%, and the accuracy a is 100%.
3. Threshold judgment for detecting measles virus by MNP (MNP) marker detection kit
The sequence aligned to measles virus could be detected in 1 copy/reaction sample, covering at least 2 MNP sites. The sequence of measles virus was also detected in the partial blank control. Due to the extreme sensitivity of MNP marker detection methods, contamination of the data during detection is likely to result in the generation of false positives. Therefore, in this example, a quality control scheme is established as follows:
1) the amount of sequencing data was greater than 1.5 megabases. The measuring and calculating basis is that the number of MNP sites detected by each sample is 5, and the length of a sequencing fragment is 300 bases, so that when the data volume is more than 1.5 million bases, the number of the sequencing fragments covering each site can be ensured to reach 1000 times by one-time experiment of most samples, and the accurate analysis of the base sequence of each MNP site is ensured.
2) Determining whether the contamination is acceptable according to the signal index S of the measles virus in the test sample and the noise index P of the measles virus in the blank, wherein:
the blank control noise index P is Nc/Nc, wherein Nc and Nc represent the number of measles virus sequencing fragments and the total sequencing fragments in the blank control.
The signal index S of the test sample is Nt/Nt, where Nt and Nt represent the number of the sequencing fragments of measles virus and the total number of the sequencing fragments in the test sample, respectively.
3) And calculating the detection rate of the MNP marker locus in the test sample, which is the ratio of the number of the detected locus to the number of the total design locus. The results are shown in Table 4;
TABLE 4 SNR of measles Virus in samples tested
As can be seen from Table 4, the mean noise index of measles virus in the blank was 0.04%, while the mean signal index in the 1 copy sample was 0.30%, and the mean signal-to-noise ratio of the 1 copy sample and the blank was 7.5, so that the present invention provides that when the signal-to-noise ratio is more than 10 times, contamination in the test system can be judged to be acceptable. The average signal-to-noise ratio of the 10 copies of the sample and blank was 70.0, and at least 6 MNP sites were stably detected in 12 sets of 10 copies/reaction, accounting for 40.0% of the total sites. Therefore, the present standard stipulates that the threshold for determination of the signal-to-noise ratio of measles virus is 35, i.e., that nucleic acid of measles virus is detected in a sample when the signal-to-noise ratio of measles virus in the sample is greater than 35 and the site detection rate is 30% or more, while ensuring accuracy.
Therefore, the kit provided by the invention can sensitively detect the measles virus with 10 copies/response.
4. Specific evaluation of MNP (MNP) marker detection kit for detecting measles virus
The method is characterized in that nucleic acids of measles virus, human rhinovirus, coronavirus, influenza virus, mycobacterium tuberculosis, staphylococcus aureus, acinetobacter strain, human parainfluenza virus, bordetella holtzeri, chlamydia pneumoniae, mycoplasma pneumoniae, haemophilus influenzae, EB virus, varicella zoster virus, cytomegalovirus, herpes simplex virus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and streptococcus pyogenes are mixed together to prepare a mixed template, the blank template is used as a control, the method provided by the invention is adopted to detect the measles virus in the mixed template, and 3 repeated experiments are carried out. 5 MNP sites of the measles virus can be specifically detected in 3 repeated experiments, and after the measles virus is analyzed 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 the MNP marker and the kit detect the high specificity of the target microorganism in the complex template.
Example 3 detection of genetic variation between measles Virus strains
The kit and the MNP marker locus detection method are used for detecting 6 measles virus strains which are collected, samples are named as S1-S6 in sequence, the sequencing average coverage multiple of each MNP locus reaches 4220 times, and each strain can detect all 5 MNP markers (Table 5). The fingerprints of the 6 strains were aligned pairwise and the results are shown in table 5, with 1 (S-2) and 5 measles viruses tested together in the same batch differing in major genotypes at all sites (table 5), possibly among other species mislabeled.
TABLE 5-6 detection assays for measles Virus
As can be seen from Table 5, the kit can be used for ensuring the genetic consistency of the measles virus strains named identically in different laboratories by detecting the MNP marker and identifying the genetic variation among the strains, thereby ensuring the comparability of the research results, which has important significance for the scientific research of the measles virus. In clinical settings, diagnostic protocols can be considered as to whether differential sites affect drug resistance.
Example 4 detection of genetic variation of measles Virus
Detection of genetic variation of measles virus, including both inter-and intra-strain variation. Because measles virus is parasitic in the host, the genetic variation of measles virus between and within the host is examined. The variation between hosts is detected by comparing major genotypes, the obtained measles virus fingerprints are pairwise compared, the major genotypes are identified with 100% reproducibility and accuracy based on an MNP marking method, and the major genotype difference of one site of two strains can be detected.
Whereas the variation within the host of measles virus is difficult to detect. As a population organism, measles virus has a mutation in the host or in the population, and when the population is subjected to molecular marker detection, it appears as an allelic form outside the major genotype of the locus. When the variant individuals have not accumulated, they account for a very small fraction of the population and are characterized by a low frequency of alleles. Low frequency alleles tend to be confused with technical errors, making prior art techniques difficult to distinguish. The present invention detects highly polymorphic MNP markers. The technical error rate of MNP markers is significantly lower than that of SNP markers, based on the fact that the probability of multiple errors occurring simultaneously is lower than that of one error. The invention distinguishes real sub-allelic genotypes and wrong genotypes caused by technical errors through a statistical model. Specifically, the method comprises the following steps:
the authenticity assessment of the sub-allelic genotypes of this example was performed as follows: allelic types with strand bias (ratio of the number of sequencing sequences overlaid on a DNA double strand) were first excluded according to the following rule: the strand preference is greater than 10-fold, or the difference from the strand preference of the dominant allele is greater than 5-fold.
Genotypes without strand preference were judged for authenticity based on the number and ratio of sequenced sequences in table 6. Inv function calculation under a 99.9999% probability guarantee, e max (n-1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, the number of sequenced sequences of the sub-allelic gene in each locus is a critical value, and only when the number of sequenced sequences of the sub-allelic gene exceeds the critical value, the true sub-allelic gene is determined. When multiple candidate sub-alleles are present, multiple corrections are made to the P-value for each candidate allele, FDR<0.5% of the candidate alleles were judged to be true sub-allelic genotypes.
Table 6 relates to the parameter e max (n-1) and e max (n.gtoreq.2) means that the highest proportion of the number of sequencing sequences carrying the wrong allele of n SNPs to the total number of sequencing sequences at that site. e.g. of the type max (n-1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, were obtained from the frequency of all the minor alleles detected at 930 homozygous MNP sites.
TABLE 6 critical value for determination of sub-allelic genotypes at partial sequencing depth
The RNA of measles virus of different variants was mixed according to the above parameters in the following 8 ratios 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100, 7/100 to prepare artificial heterozygous samples, each of which was tested for 3 replicates to obtain a total of 24 sequencing data. By accurately comparing the genotypes of the MNP loci of the two variants of the measles virus, the heterozygous genotype loci can be detected in 24 artificial heterozygous samples, thereby demonstrating the applicability of the developed MNP marker detection method of the measles virus in detecting the genetic variation of strains.
Example 5 construction of MNP fingerprint database for measles Virus
Extracting RNA of all strains or samples for constructing a measles virus MNP fingerprint database by using a conventional CTAB method, a commercial kit and other methods, and detecting the quality of the RNA by adopting agarose gel electrophoresis and an ultraviolet spectrophotometer.
And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain a major genotype of each site of each strain, forming an MNP fingerprint of each strain, and recording the MNP fingerprint into a database file to form a measles virus MNP fingerprint database. The constructed MNP fingerprint database is based on the gene sequences of the detected strains and is therefore compatible with all high throughput sequencing data. The MNP fingerprint spectrum of the strains obtained by each detection is compared with the established MNP fingerprint database, and the MNP fingerprint database established by the MNP fingerprint spectrum of the strains with different main genotypes realizes the co-establishment sharing and the random updating of the database.
Example 6 application in Fine typing of measles Virus
The measles virus in a sample to be detected is detected by using the kit, and the MNP fingerprint of the measles virus in each sample is obtained; constructing a reference sequence library consisting of the disclosed genome sequence of measles virus and the MNP fingerprint database of the constructed measles virus; the MNP fingerprint of the measles virus of each sample is compared with a constructed reference sequence library, the measles virus is identified as a very similar strain when being identical with the existing reference sequence, and is identified as a new variant strain when the main genotype difference exists at more than one MNP site, thereby realizing the fine typing of the measles virus.
The test on 6 measles virus samples as shown in table 5, the 6 measles viruses tested were 1 and 5 other major genotypes at the 5 MNP sites, and were also different from the strains of the reference sequence library, either as new variants or as mislabels during the experimental process or as contamination events with other strains. Therefore, the method achieves the resolution of measles virus to the single base level, and can realize the fine classification of measles virus in a sample.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the embodiments of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to encompass such modifications and variations.
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Claims (8)
1. A MNP marker site for measles virus, which is a region of the genome screened for on the genome of measles virus that is distinct from other species and has a plurality of nucleotide polymorphisms within a species, including marker sites for MNP-1 to MNP-5 on the AF266288.2 genome.
2. A multiplex PCR primer composition for the detection of the MNP marker sites of measles virus according to claim 1, comprising 5 primers, wherein the nucleotide sequences of said 5 primers are represented by SEQ ID No.1 to SEQ ID No. 10.
3. A test kit for the detection of the MNP marker sites of measles virus according to claim 1, comprising the primer composition according to claim 2.
4. The test kit of claim 3, wherein the kit further comprises a multiplex PCR premix.
5. Use of the MNP marker site of measles virus according to claim 1 or of the primer composition according to claim 2 or of the detection kit according to any one of claims 3 to 4 for the qualitative detection of measles virus for non-diagnostic purposes.
6. Use of the MNP marker site of measles virus according to claim 1 or the primer composition according to claim 2 or the detection kit according to any of claims 3 to 4 for the detection of genetic variations within and between measles virus strains.
7. Use of the MNP marker site of measles virus according to claim 1 or the primer composition according to claim 2 or the detection kit according to any one of claims 3 to 4 for the construction of measles virus databases.
8. Use of the MNP marker site of measles virus according to claim 1 or of the primer composition according to claim 2 or of the detection kit according to any one of claims 3 to 4 for the detection of the fine typing of measles virus.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634610A (en) * | 2012-05-07 | 2012-08-15 | 镇江和创生物科技有限公司 | Primer probe combination for specific detection of measles virus and rubella virus and kit |
| CN103409554A (en) * | 2013-07-02 | 2013-11-27 | 江苏硕世生物科技有限公司 | Nucleic acid detection kit for rapidly detecting measles virus/rubella virus |
| CN107937502A (en) * | 2017-12-07 | 2018-04-20 | 江汉大学 | A kind of method for screening the high polymorphic molecular marker site of microorganism |
| CN108085415A (en) * | 2017-12-18 | 2018-05-29 | 北京卓诚惠生生物科技股份有限公司 | Detect the primed probe group and method of dengue fever, datum hole Kenya and measles virus |
| CN110982940A (en) * | 2019-12-30 | 2020-04-10 | 广州市疾病预防控制中心 | Composition and kit for detecting nucleic acid of measles virus, rubella virus and mumps virus based on melting curve |
| CN111411169A (en) * | 2019-01-08 | 2020-07-14 | 苏秀兰 | Nucleic acid sequence, kit and method for detecting measles virus by isothermal amplification and application |
| CN113549711A (en) * | 2021-08-12 | 2021-10-26 | 上海伯杰医疗科技有限公司 | Primer combination for detecting human bocavirus whole genome and detection method thereof |
-
2021
- 2021-11-10 CN CN202111327708.4A patent/CN114836573B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634610A (en) * | 2012-05-07 | 2012-08-15 | 镇江和创生物科技有限公司 | Primer probe combination for specific detection of measles virus and rubella virus and kit |
| CN103409554A (en) * | 2013-07-02 | 2013-11-27 | 江苏硕世生物科技有限公司 | Nucleic acid detection kit for rapidly detecting measles virus/rubella virus |
| CN107937502A (en) * | 2017-12-07 | 2018-04-20 | 江汉大学 | A kind of method for screening the high polymorphic molecular marker site of microorganism |
| CN108085415A (en) * | 2017-12-18 | 2018-05-29 | 北京卓诚惠生生物科技股份有限公司 | Detect the primed probe group and method of dengue fever, datum hole Kenya and measles virus |
| CN111411169A (en) * | 2019-01-08 | 2020-07-14 | 苏秀兰 | Nucleic acid sequence, kit and method for detecting measles virus by isothermal amplification and application |
| CN110982940A (en) * | 2019-12-30 | 2020-04-10 | 广州市疾病预防控制中心 | Composition and kit for detecting nucleic acid of measles virus, rubella virus and mumps virus based on melting curve |
| CN113549711A (en) * | 2021-08-12 | 2021-10-26 | 上海伯杰医疗科技有限公司 | Primer combination for detecting human bocavirus whole genome and detection method thereof |
Non-Patent Citations (3)
| Title |
|---|
| CHEN M等: ".Endemic and imported measles virus-associated outbreaks among adults", EMERGING INFECTIOUS DISEASES, vol. 21, no. 3, pages 477 - 479 * |
| 周剑惠,王爽,陈超,许强,姬奕昕,林琳,田鑫,曹凤瑞,刘胜贺,李振,许文波: "限制性片段长度多态性分析方法应用于中国麻疹野病毒基因型别的鉴定", 中国计划免疫, no. 01, pages 5 - 8 * |
| 黄泓滟;周淑洁;靳玉惠;柯文鸿;黄保军;: "合肥市71例麻疹病例麻疹病毒基因型分析", 现代预防医学, no. 08, pages 1486 - 1490 * |
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