CN114836572A

CN114836572A - MNP (MNP protein) marker site of paraenterovirus, primer composition, kit and application of MNP marker site

Info

Publication number: CN114836572A
Application number: CN202111327682.3A
Authority: CN
Inventors: 周俊飞; 方治伟; 李甜甜; 高利芬; 肖华锋; 陈利红; 李论; 彭海; 万人静
Original assignee: Jianghan University
Current assignee: Jianghan University
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2022-08-02
Anticipated expiration: 2041-11-10
Also published as: CN114836572B

Abstract

The invention discloses a specific MNP marker locus of a parareovirus, a primer composition, a kit and application thereof, wherein the MNP marker locus refers to a genome region which is screened from a parareovirus genome and is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker loci of MNP-1-MNP-15; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 30. The MNP marker site can specifically identify the paraenterovirus and finely distinguish different strains; the primers are not interfered with each other, and by integrating multiple amplification and sequencing technologies, sequence analysis can be performed on all marked sites of multiple samples at one time, so that the kit has the detection advantages of high throughput, multiple targets, high sensitivity and no culture, can be applied to identification and genetic variation detection of the parareoviruses of large-scale samples, and has important significance on scientific research and epidemic prevention monitoring of the parareoviruses.

Description

MNP (MNP protein) marker site of paraenterovirus, primer composition, kit and application of MNP marker site

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marker site of a paraenterovirus, a primer composition, a kit and application thereof.

Background

Paraenterovirus (Parechovirus) is a RNA virus belonging to the genus Paraenterovirus of the family picornaviridae. Paraenterovirus infection is very wide, reports on the virus infection are reported all over the world, the transmission routes mainly comprise feces-oral route transmission and droplet transmission, and the infected objects mainly comprise children. The virus mainly invades the alimentary canal, the respiratory tract, the central nervous system and other parts, the adult infection mostly only has slight clinical symptoms, the child infection has various clinical manifestations, and the child infection can be manifested as asymptomatic or slight infection symptoms of the respiratory and digestive systems, and can also be manifested as serious infection such as myocarditis, pneumonia, meningitis and the like. The clinical manifestation of the paraenteric virus infection lacks specificity, is not easy to be identified with the bacterial infection and other virus infections, has no specific medicine at present, and is mainly based on symptomatic support therapy clinically. Therefore, the rapid and accurate detection of the paraenterovirus has important significance for diagnosing the cause of the disease in time, finding early treatment and reducing the disease deterioration. In addition, the paraenterovirus serves as a group organism, and in the interaction with a host and the environment, individuals in the group can generate variation, so that the detection or treatment method is ineffective; 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 parareovirus detection and analysis method capable of monitoring variation is of great significance to clinical treatment, epidemic prevention detection and scientific research of parareovirus.

The classical detection method of the paraenterovirus comprises separation culture, PCR technology, whole genome and metagenome sequencing and the like, and has one or more limitations in the aspects of time length, operation complexity, detection flux, 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 the limitation that the whole genome depends on the massive data waste and background noise caused by the separation culture of pathogenic bacteria and the metagenome sequencing, and has the advantages of less sample requirement, accurate diagnosis result, data amount saving, low-frequency variation detection and no culture. The molecular markers detected by the existing targeted detection technology mainly comprise SNP (single nucleotide polymorphism) markers and SSR (simple sequence repeat) markers. SSR markers are generally accepted as the most polymorphic markers, but are few in microorganisms; the SNP markers are large in number, densely distributed and are binary markers, and the polymorphism of a single SNP marker is insufficient to capture the potential allelic diversity in a microbial population.

Therefore, the development of a novel highly polymorphic molecular marker for a paramyxovirus and a technique for detecting the same has become a technical problem to be solved.

Disclosure of Invention

The invention aims to provide a specific MNP marker locus of an paraenterovirus, a primer composition, a kit and application thereof, which can be used for qualitatively identifying and detecting variation of the paraenterovirus 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 paramyxovirus-specific MNP marker site that is a region of the genome that is selected on the paramyxovirus genome to be distinguished from other species and that has a plurality of nucleotide polymorphisms within a species, comprising: KY556675.1 is taken as a marker locus of MNP-1, MNP-6 and MNP-15 of a reference genome; GQ183022.1 is taken as a marker locus of MNP-2 and MNP-11 of a reference genome; marker loci of MNP-3-MNP-5, MNP-7, MNP-9, MNP-12-MNP-14 with NC-003976.2 as reference genome; marker sites for MNP-8 and MNP-10 of the reference genome MK 904591.1.

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

In a second aspect of the invention, a multiplex PCR primer composition for detecting said MNP marker site is provided, said multiplex PCR primer composition comprising 15 pairs of primers, the specific primer sequence being shown in SEQ ID No.1 to SEQ ID No. 30.

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

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

Further, the kit also comprises a multiplex PCR premix.

In the fourth aspect of the invention, the application of the MNP marker site of the parareovirus, the multiple PCR primer composition or the detection kit in qualitative detection of the parareovirus for non-diagnosis purposes and preparation of a qualitative detection product of the parareovirus is provided.

In the fifth aspect of the invention, the MNP marker site of the paraenteric virus, the multiple PCR primer composition or the detection kit is provided for the application of detecting the genetic variation inside the paraenteric virus strain and among strains.

In a sixth aspect of the invention, the invention provides the application of the MNP marker site of the parareovirus, the multiplex PCR primer composition or the detection kit in the construction of a parareovirus database.

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

In the above application, the specific operation steps are as follows: firstly, acquiring the virus total RNA of a sample to be detected; reverse transcription into cDNA by using a commercial reverse transcription kit; 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; and (3) comparing the sequencing result with the reference sequence of the paraenterovirus to obtain the number and genotype data of the detected sequence of the cDNA. And performing data quality control and data analysis on the sequencing data of the cDNA according to the quantity of the sequencing sequences of the paraenteroviruses obtained from the cDNA and the blank control and the quantity of the detected MNP sites to obtain the quantity of the detected MNP sites, the quantity of the sequencing sequences covering each MNP site and the genotype data of the MNP sites.

When the method is used for identifying the parareovirus, whether the nucleic acid of the parareovirus is contained in the sample to be detected or not is judged after quality control is carried out according to the sequencing sequence number of the detected parareovirus and the number of detected MNP sites in the sample to be detected and blank control. The quality control scheme and the determination method are characterized in that nucleic acid of the parareovirus with known copy number is used as a detection sample, the sensitivity, the accuracy and the specificity of the kit for detecting the parareovirus are evaluated, and the quality control scheme and the determination method when the kit is used for detecting the parareovirus are established.

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

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

When the method is used for the parareovirus typing, the parareovirus in a sample to be detected is identified, and the genotype of each MNP locus is obtained. And comparing the sample with the MNP fingerprint database of the parareovirus to identify whether the parareovirus in the sample is of the existing type or the new type, and recording the new type into the MNP fingerprint database. Therefore, the MNP fingerprint database can be continuously enriched by utilizing the primer combination.

The invention belongs to the initiative in the field of paraenteric virus, and has no related literature report; MNP marks are mainly developed based on reference sequences, and MNP sites which are largely distinguished from other species, are polymorphic in the species of the parareovirus and are conserved in sequences at two sides can be excavated according to reported re-sequencing data of representative small species of the parareovirus; MNP locus detection primers suitable for multiplex PCR amplification can be designed through conserved sequences on two sides of the MNP locus; and then according to the test result of the standard product, a primer combination and a detection kit with the maximum polymorphism, high specificity, the best compatibility and the set of MNP sites can be screened out.

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

the invention provides an MNP marker locus of a paraenterovirus, a primer composition, a kit and application thereof. The provided 15 MNP sites of the paraenteric virus and the primer combination thereof can carry out multiple PCR amplification, fuse a second-generation sequencing platform to carry out sequencing on an amplification product, meet the requirements of carrying out high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on the combined mycobacterium and meet the requirements of standard and sharable fingerprint data construction of the paraenteric virus; the need to accurately detect genetic variation between strains of paraenteric viruses; the requirements of homozygous and heterozygous Parareovirus are identified, and a technical support is provided for scientific research, scientific monitoring and prevention and treatment of the Parareovirus.

Drawings

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

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

FIG. 2 is a flow chart of the screening and primer design of the MNP marker site of the paraenterovirus;

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

Detailed Description

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

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. If there is a conflict, the present specification will control.

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

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

the invention develops a novel species-specific molecular marker-MNP marker which is suitable for detecting group organisms. 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; (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 based on the combination of the super-multiplex PCR and the second-generation high-throughput sequencing technology has the following advantages: (1) the output is a base sequence, parallel experiments are not needed, and a standardized database can be constructed for sharing and sharing; (2) the efficiency is high, the sample DNA bar code is utilized, the limitation of the quantity of sequencing samples is broken through, and tens of thousands of MNP sites of hundreds of samples can be typed at one time; (3) the sensitivity is high, multiple targets are detected at one time by utilizing multiple PCR, and high false negative and low sensitivity caused by amplification failure of a single target are avoided; (4) high accuracy, using a second generation high throughput sequencer to sequence the amplification product hundreds of times.

In view of the advantages and the characteristics, the MNP marker and the detection technology MNP marking method thereof can realize the classification and the tracing of the multi-allelic genotypes of the population organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, detection of genetic variation and the like. At present, no report on MNP labeling exists in microorganisms, and corresponding technologies are lacked. Accordingly, the present invention develops a MNP marker site for a parareovirus, which is a region of the genome screened on the parareovirus genome that is distinct from other species and has multiple nucleotide polymorphisms within a species, including: KY556675.1 is taken as a marker locus of MNP-1, MNP-6 and MNP-15 of a reference genome; GQ183022.1 is taken as a marker locus of MNP-2 and MNP-11 of a reference genome; marker loci of MNP-3-MNP-5, MNP-7, MNP-9, MNP-12-MNP-14 with NC-003976.2 as reference genome; the marker sites of MNP-8 and MNP-10 of the reference genome are MK 904591.1.

Then, the invention develops a multiple PCR primer composition for detecting the MNP marker locus of the paraenterovirus, which is characterized by comprising 15 pairs of primers, wherein the nucleotide sequences of the 15 pairs of primers are shown in SEQ ID NO. 1-SEQ ID NO. 30. The primers do not conflict with each other, and efficient amplification can be performed by multiplex PCR.

The multiplex PCR primer composition can be used as a detection kit for detecting the MNP marker locus of the paraenterovirus.

The kit can accurately and sensitively detect the paraenterovirus with the copy/reaction as low as 10.

The MNP marker and the kit have high specificity in detecting the paraenterovirus in the complex template.

The MNP marker site, the primer composition, the kit and the application of the paracolovirus are explained in detail in the following by combining the embodiment, the comparative example and the experimental data.

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

S1 screening of MNP (minor enterovirus) marker sites

Based on the complete or partial genome sequences of 3794 different isolates of the paraenterovirus disclosed on the network, 15 MNP marker loci are obtained by sequence alignment. For species without genomic data on the net, the genomic sequence information of the representative microspecies of the microbial species to be detected can also be obtained by high-throughput sequencing, wherein the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected marker, the genomic sequence of at least 10 genetically representative isolates is generally used as a reference. The 15 MNP marker sites screened are shown in table 1:

TABLE 1-starting position of the MNP marker site and detection primer on the reference sequence

The step S1 specifically includes:

selecting a genome sequence of a representative strain of the paraenteric virus as a reference genome, and performing sequence comparison on the genome sequence and the reference genome to obtain single nucleic acid polymorphic sites of each strain of the paraenteric 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.

In this embodiment, the primers used to identify the MNP marker sites are shown in table 1.

S3 evaluation of detection efficiency of primer combination

The detection method of the MNP marker comprises the steps of amplifying all MNP sites at one time through multiplex PCR, sequencing amplification products through second-generation high-throughput sequencing, analyzing sequencing data, and evaluating the compatibility of the primer combination according to the detected sites.

Paraenterovirus RNA with known copy number provided by Hubei province disease control and prevention center is added into human genome DNA after being reverse transcribed into cDNA by a commercial reverse transcription kit to prepare a 1000-copy/reaction template, the primer combination is used for screening the primer combination with even amplification and optimal compatibility according to the detection condition of MNP sites in 4 libraries, and finally, the primer combination of 15 MNP sites disclosed in the invention in the table 1 is screened out.

Thresholding and Performance evaluation of the MNP sites and primers described in example 2 to identify Paraenteroviruses

1. Detection of MNP markers

In this example, the RNA of the parainfluenza virus with a known copy number provided by the disease control and prevention center in north Hu province was reverse-transcribed into cDNA using a commercial reverse transcription kit, and then added to human genomic DNA to prepare 1 copy/reaction, 10 copy/reaction, and 100 copy/reaction parainfluenza virus mock samples. An equal volume of sterile water was also set as a blank. A total of 4 samples were obtained, each sample was constructed into 3 duplicate libraries each day, and the assay was continued for 4 days, i.e. 12 sets of sequencing data were obtained for each sample, as shown in table 2. According to sequencing fragment number and site number of an paratenovirus MNP site detected in a blank control and a paratenovirus nucleic acid standard substance in 12 repeated experiments, the repeatability, the accuracy and the sensitivity of the detection method are evaluated, and a threshold value for detecting the pollution of a quality control system and a target pathogen is set. The flow of detection of MNP markers is shown in figure 3.

TABLE 2 detection sensitivity, stability analysis of MNP labeling method for Paraenterovirus

As shown in Table 2, the kit can stably detect more than 6 MNP sites in a sample of 10 copies/reaction, and can detect 1 MNP site at most in a sample of 0 copies/reaction, the kit can obviously distinguish the samples of 10 copies/reaction and 0 copies/reaction, and has technical stability and detection sensitivity as low as 10 copies/reaction.

2. Evaluation of reproducibility and accuracy of MNP (MNP) labeled detection kit for detecting paraenteric virus

And evaluating the reproducibility and accuracy of the MNP marker detection method for detecting the paraenteric virus based on whether the genotype of the co-detected site can be reproduced in the two times of repetition. Specifically, 12 sets of data for 100 copies/reaction samples were compared in pairs, respectively, and the results are shown in Table 3.

TABLE 3 evaluation of reproducibility and accuracy of the Parareovirus MNP marker detection method

As can be seen from Table 3, the number of MNP sites with differences in major genotypes is 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 project, the logarithm of difference of the MNP labeling main genotypes 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 paraenterovirus by MNP (MNP) marker detection kit

As shown in table 2, sequences aligned to the paraenterovirus could be detected in 1 copy/reaction sample, covering at least 1 MNP site. In the partial blank control, the sequence of the paraenterovirus was also detected. 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 4.5 megabases. The measuring and calculating basis is that the number of MNP sites detected by each sample is 15, and the length of a sequencing fragment is 300 bases, so that when the data volume is more than 4.5 million bases, the number of the sequencing fragments covering each site can be ensured to reach 1000 times by one-time experiment of most samples, and the accurate analysis of the base sequence of each MNP site is ensured.

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

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

The signal index of the test sample is Nt/Nt, wherein Nt and Nt represent the number of sequencing fragments of the parareovirus and the total number of 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.

TABLE 4 SNR of Parareovirus in samples tested

As shown in Table 4, the average noise index of the Paraenterovirus in the blank was 0.04%, while the average signal index in the 1 copy sample was 0.26%, and the average signal-to-noise ratio of the 1 copy sample and the blank was 6.5, so that the present invention provides that when the signal-to-noise ratio is more than 10 times, contamination in the detection system can be judged to be acceptable.

The mean of the signal-to-noise ratios of the 10 copies of the sample and blank 67.2 consistently detected at least 6 MNP sites in 10 copies/reaction of the 12 sets of data, accounting for 40.0% of the total sites. Therefore, the standard provides that the threshold value for determining the signal-to-noise ratio of the parareovirus is 34 under the condition of ensuring the accuracy, namely, the nucleic acid of the parareovirus in the sample is determined to be detected when the signal-to-noise ratio of the parareovirus in the sample is greater than 34 and the site detection rate is greater than or equal to 20%.

Therefore, the kit provided by the invention can accurately and sensitively detect the paraenterovirus with the copy/response as low as 10.

4. Specific evaluation of MNP (MNP) marker detection method for detecting paraenterovirus

Artificially mixing nucleic acids of paraenteric orphan virus, mumps virus, Zika virus, coronavirus, influenza virus, mycobacterium tuberculosis, acinetobacter strain, Bordetella pertussis, Bordetella holtzeri, Chlamydia pneumoniae, mycoplasma pneumoniae, EB virus, haemophilus influenzae, varicella zoster virus, cytomegalovirus, herpes simplex virus, human bocavirus, Klebsiella pneumoniae, Legionella, Moraxella catarrhalis, Pseudomonas aeruginosa, Rickettsia, Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes together to prepare a mixed template, and detecting the mumps virus in the mixed template by using a blank template as a control for 3 repeated experiments. After sequence comparison and analysis according to the quality control scheme and the judgment threshold value, 15 MNP sites of the paraenterovirus can be specifically detected in 3 repeated experiments, and the fact that the MNP marker and the kit detect high specificity of target microorganisms in a complex template is shown.

Example 3 detection of genetic variation between strains of Parareovirus

The kit and the MNP marker locus detection method are utilized to detect 6 sub-passage strains of the parareovirus strains provided by Hubei province disease control, samples are sequentially named as S1-S6, the average coverage multiple of sequencing of each sample reaches 1416 times, and all 15 MNP markers can be detected from each strain (Table 5). The fingerprints of 6 strains are compared in pairs, and the results are shown in table 5, wherein the major genotype differences of partial sites exist in 1 part (S-2) of paraenteroviruses and 5 parts of paraenteroviruses detected together in the same batch (table 5), which indicates that the variation among strains exists.

TABLE 5-6 detection assays for Paraenteroviruses

As can be seen from table 5, the application of the kit of the present invention to identify genetic variation among strains by detecting MNP markers can be used to ensure genetic consistency of identically named strains of parareovirus in different laboratories, thereby ensuring comparability of the research results, which is of great significance to scientific research of parareovirus. In clinical settings, diagnostic protocols can be considered as to whether differential sites affect drug resistance.

Example 4 detection of genetic variation within Paraenterovirus strains

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

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

Genotypes without strand preference were judged for authenticity based on the number and proportion of sequences sequenced in table 6. Inv function calculation with 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

Nucleic acids of two strains with genotype difference were mixed according to the above parameters according to the following 8 ratios of 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100 and 7/100 to prepare artificial heterozygous samples, and each sample was tested for 3 replicates to obtain a total of 24 sequencing data. By accurately comparing the gene types of the MNP sites of the two strains, the sites with heterozygous gene types are detected in 24 artificial heterozygous samples, thereby demonstrating the applicability of the developed MNP marker detection method for the mycoplasma pneumoniae in detecting the genetic variation in the strain population.

Example 5 construction of Paraenterovirus DNA fingerprint database

Extracting all strains or RNA of a sample for constructing an MNP fingerprint database of the parareovirus by using a conventional CTAB method, a commercial kit and other methods, and detecting the quality of the RNA by using agarose gel and an ultraviolet spectrophotometer. And (3) after sequence comparison is carried out on the sequencing data of the 6 strains and a reference genotype, obtaining a main genotype of each site of each strain, and forming an MNP fingerprint of each strain. And (4) recording the obtained MNP fingerprint of each strain into a database file to form an MNP fingerprint database of the parareovirus.

The constructed MNP fingerprint database is based on the gene sequences of detected strains, is compatible with all high-throughput sequencing data, and has the characteristics of complete co-construction and sharing and update at any time. The MNP fingerprint of the strains obtained by each detection is compared with an MNP fingerprint database constructed based on the existing genome data, and the MNP fingerprint of the strains with the difference in main genotypes is recorded into the constructed MNP fingerprint database, so that the real-time updating and co-construction sharing of the database are realized.

Example 6 application in Fine typing of Parareovirus

Firstly, constructing a reference sequence library of the parareovirus, wherein the reference sequence library consists of a disclosed genome sequence of the parareovirus and a constructed fingerprint database of the parareovirus; obtaining the MNP fingerprint of the parareovirus in each sample to be detected by utilizing the primer combination and the MNP marker locus detection method in the embodiment 2; comparing the DNA fingerprint of each strain with a constructed reference sequence library, and screening to obtain the strain with the genetic distance closest to that in the sequence library; the gene type of the strain is 100 percent same as that of the existing strain, is an existing variant, and the variant with main gene type difference at least one MNP site is a new variant, so that the fine typing of the paraenterovirus is realized.

The results of typing 6 strains of parareovirus in this example are shown in table 5, where 6 strains can be classified into 2 types, and the strains of the reference sequence library have a major genotype difference with more than 1 MNP site, and are determined as 2 new variants, thereby realizing fine typing of parareovirus.

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.

Sequence listing

<110> university of Jianghan

<120> MNP (MNP marker locus) of paraenterovirus, primer composition, kit and application thereof

<160> 30

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agtaagtttg taagacgtct gatga 25

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aaccaatccc aaagggtctg ttaaa 25

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

caagacgctt aaagcatggt gtaa 24

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acgtcacaca aacttactag aggat 25

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaagccacgt ctcgaagcat a 21

<210> 6

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccgcctcccg caattgttt 19

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttcctggctc cacctacaag 20

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aatcatcata aatttctttc ccatgc 26

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctgagaatat gcaatttgcc caatc 25

<210> 10

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggacatagaa acatcattaa attctctcca 30

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tctttaacta atctccccca tgtgt 25

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgaaagaggt gtccaaacat agacc 25

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tggtgcattg tctagatttt tctgc 25

<210> 14

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tttgattcca aagtagctgt ccgg 24

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

taaggcccac gaaggatgc 19

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

aaataaaagg aaaccaggga tcccc 25

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gcctacacac aggatgaagc t 21

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atcaatcctc agatgtccca aggag 25

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

aaccaagcaa acagtccatg aaatt 25

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gtgtttttgt gaatttcgct cttcc 25

<210> 21

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gccgaggttt agcagacc 18

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

agtgtcgctt gttacctaca g 21

<210> 23

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cgagctgcag cagttcactt t 21

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ggactacacc attcaaggga ctg 23

<210> 25

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

agtcttatct tgtatgtgtc ctgca 25

<210> 26

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cgggtgtgga atgcactgtc 20

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gctttggtca acttgctgat agtc 24

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ggcagtatat ttaatggtgg gtcga 25

<210> 29

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cacaaatttg acccaacacc catc 24

<210> 30

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ccatgaagct gtgtccaatc ttata 25

Claims

1. A MNP marker site of a parareovirus, wherein said MNP marker site is a region of the genome screened on the parareovirus genome that is distinct from other species and has a plurality of nucleotide polymorphisms within a species, comprising: KY556675.1 is taken as a marker locus of MNP-1, MNP-6 and MNP-15 of a reference genome; GQ183022.1 is taken as a marker locus of MNP-2 and MNP-11 of a reference genome; marker loci of MNP-3-MNP-5, MNP-7, MNP-9, MNP-12-MNP-14 with NC-003976.2 as reference genome; the marker sites of MNP-8 and MNP-10 of the reference genome are MK 904591.1.

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

3. A test kit for detecting the MNP marker site of the paraenteric virus of claim 1, comprising the primer composition of 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 a parareovirus 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 qualitative detection of parareovirus for non-diagnostic purposes and for the preparation of a product for the qualitative detection of parareovirus.

6. Use of the MNP marker site of the parareovirus of claim 1 or the primer composition of claim 2 or the detection kit of any one of claims 3 to 4 for detecting genetic variations within and between strains of the parareovirus.

7. Use of the MNP marker site of the parareovirus of claim 1 or the primer composition of claim 2 or the detection kit of any one of claims 3 to 4 for constructing a database of parareoviruses.

8. Use of the MNP marker site of the parareovirus of claim 1 or the primer composition of claim 2 or the detection kit of any one of claims 3 to 4 for the fine typing detection of the parareovirus.