CN114836572B

CN114836572B - MNP (MNP) marker locus of paraenterovirus, primer composition, kit and application of MNP marker locus

Info

Publication number: CN114836572B
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: 2023-06-16
Anticipated expiration: 2041-11-10
Also published as: CN114836572A

Abstract

The invention discloses a paramylon virus specific MNP (MNP) marker locus, a primer composition, a kit and application thereof, wherein the MNP marker locus refers to a genome region which is screened on a paramylon virus genome and is separated 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 locus can specifically identify the paraenterovirus and finely distinguish different strains; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that the sequence analysis can be performed on all the marker loci of multiple samples at one time, the method has the advantages of high throughput, multiple targets, high sensitivity and culture-free detection, can be applied to the identification and genetic variation detection of the paraenterovirus of large-scale samples, and has important significance for scientific research and epidemic prevention monitoring of the paraenterovirus.

Description

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

Technical Field

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

Background

Parechoviruses (Parechoviruses) are an RNA virus belonging to the genus Parechoviruses of the family MicroRNA virus. The paraenteric virus infection is very extensive, reports on the virus infection are all over the world, the transmission route is mainly fecal-oral transmission and droplet transmission, and the infected objects are mainly children. The virus mainly invades the digestive tract, respiratory tract, central nervous system and other parts, and most of adult infection has slight clinical symptoms, children infection has various clinical manifestations, and can be expressed as asymptomatic or slight respiratory and digestive system infection symptoms, and also can be expressed as serious infection such as myocarditis, pneumonia, meningitis and the like. The clinic manifestation of the paraenteric virus infection lacks specificity, is not easy to identify with bacteria and other virus infection, has no specific medicine at present, and mainly adopts symptomatic support therapy clinically. Therefore, the rapid and accurate detection of the paraenteric virus has important significance for diagnosing the etiology in time, realizing early detection and early treatment and reducing the disease deterioration. In addition, as a group organism, the parareovirus can generate variation in individuals in the group in interaction with a host and the environment, so that the detection or treatment method is disabled; for experimental studies, such undetectable variations can result in the same named strain being virtually different in different laboratories or different times in the same laboratory, resulting in irreproducible and incomparable experimental results. Therefore, developing a rapid, accurate and monitorable mutation detection and analysis method for parareovirus has important significance for clinical treatment, epidemic prevention detection and scientific research of parareovirus.

Classical methods for detecting parareoviruses, including isolation and culture, PCR techniques, whole genome and metagenome sequencing, etc., have one or more limitations in terms of duration, complexity of operation, detection throughput, accuracy and sensitivity of detecting variations, cost, etc. The targeted molecular marker detection technology integrating the ultra-multiplex PCR amplification and the high-throughput sequencing can enrich target microorganisms in a sample with low microorganism content in a targeted manner, avoids the limitation of massive data waste and background noise caused by the separation culture of the whole genome depending on pathogenic bacteria and metagenome sequencing, and has the advantages of small sample requirement, accurate diagnosis result, data quantity conservation, low-frequency variation detection and culture-free. 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 parareovirus and a detection technology thereof is a technical problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a specific MNP (MNP) marking site of a paraenteric virus, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on the paraenteric virus and have the effects of multiple targets, high flux, high sensitivity and fine typing.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect of the invention there is provided a paramylon virus specific MNP marker locus, the MNP marker locus being a genomic region screened on the genome of a paramylon virus that is distinct from other species and has a plurality of nucleotide polymorphisms within the species, comprising: marker loci of MNP-1, MNP-6 and MNP-15 of the reference genome by KY 556675.1; marker loci for MNP-2 and MNP-11 of the reference genome with GQ 183022.1; marker loci of MNP-3 to MNP-5, MNP-7, MNP-9 and MNP-12 to MNP-14 of the reference genome with NC_ 003976.2; marker loci for MNP-8 and MNP-10 of the reference genome with MK 904591.1.

In the above technical scheme, the marking sites of MNP-1 to MNP-15 are specifically shown in the specification table 1, and the starting and ending positions of the MNP marks marked in the table 1 are determined based on the reference sequences corresponding to the same row of MNPs in the table 1.

In a second aspect of the present invention, there is provided a multiplex PCR primer composition for detecting the MNP marker loci, the multiplex PCR primer composition comprising 15 pairs of primers, the specific primer sequences being shown in SEQ ID NO.1 to SEQ ID NO. 30.

In the above technical solution, the primers of each MNP marker locus include an upper primer and a lower primer, and are specifically shown in table 1 of the specification.

In a third aspect of the invention, there is provided a detection kit for detecting the MNP marker locus of a paramylon virus, 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 a parareovirus or said multiplex PCR primer composition or said detection kit for qualitative detection of a parareovirus for non-diagnostic purposes and for preparing a qualitative detection product of a parareovirus.

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

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

In a seventh aspect of the invention, there is provided the use of said MNP marker locus of a paramylon virus or said multiplex PCR primer composition or said detection kit in the accurate and finely divided detection of a paramylon virus.

In the application, the specific operation steps are as follows: firstly, obtaining virus total RNA of a sample to be tested; reverse transcription into cDNA using commercial reverse transcription kits; 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; and comparing the sequencing result with the reference sequence of the parareovirus to obtain the number of detection sequences and genotype data of the cDNA. And carrying out data quality control and data analysis on the sequencing data of the cDNA according to the number of the sequencing sequences of the paraenteric viruses and the number of the detected MNP sites obtained from the cDNA and the blank control, and obtaining the number of the detected MNP sites, the number of the sequencing sequences covering each MNP site and the genotype data of the MNP sites.

When the method is used for detecting the parareovirus, whether the sample to be detected contains the nucleic acid of the parareovirus or not is judged after quality control according to the number of sequencing sequences of the parareovirus detected in the sample to be detected and a blank control and the number of MNP sites detected. The quality control scheme and the judging method are characterized in that nucleic acid of the paraenteric virus with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the paraenteric virus are evaluated, and the quality control scheme and the judging method when the kit detects the paraenteric virus are formulated.

When used for parareovirus genetic variation detection, it includes inter-strain and intra-strain genetic variation detection. The detection of genetic variation among strains comprises the steps of obtaining genotype data of each strain to be compared at 15 MNP sites by using the kit and the method. By genotype comparison, whether the main genotypes of the strains to be compared are different at the 15 MNP sites is analyzed. If the strains to be compared have variation in the main genotype of at least one MNP site, then the two are judged to have genetic variation. Alternatively, 15 loci of strains to be compared can be amplified by single PCR, and then Sanger sequencing is performed on the amplified products to obtain sequences, and the genotypes of each MNP locus of the strains to be compared are aligned. If there are MNP sites of inconsistent major genotypes, there is variation between the strains to be compared. When detecting genetic variation inside the strain, determining whether the secondary genotype other than the primary genotype is detected at the MNP locus of the strain to be detected through a statistical model. If the strain to be tested has the subgenotype at least one MNP site, judging that the strain to be tested has genetic variation.

When the method is used for constructing a paraminovirus MNP fingerprint database, genotype data of the MNP locus of the paraminovirus identified from a sample is input into a database file to form the MNP fingerprint database of the paraminovirus; and each time different samples are identified, comparing the samples with an MNP fingerprint database of the paraenteric viruses, and identifying whether the paraenteric viruses in the samples and strains in the database have differences of main genotypes (with genotypes supported by more than 50% of sequencing fragments at one MNP site) at the MNP sites, wherein the paraenteric viruses with the main genotype differences at least 1 MNP site are new mutation types, and recording the new mutation types into a DNA fingerprint database.

When the method is used for the paraenteric virus typing, the paraenteric virus in a sample to be tested is identified, and the genotype of each MNP site is obtained. And identifying whether the parareovirus in the sample is an existing type or a new type by comparing with an MNP fingerprint database of the parareovirus, wherein the new type is recorded in the MNP fingerprint database. Therefore, the MNP fingerprint database can be continuously enriched by utilizing the primer combination.

The invention is initiated in the field of parareoviruses, and is not reported in related documents; MNP markers are developed mainly based on reference sequences, and MNP sites which are large-scale and are distinguished from other species, polymorphic in the interior of the species of the parareovirus and conserved in sequence at two sides can be mined according to the reported resequencing data of the species of the parareovirus; MNP site detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of the MNP site; and then a set of MNP locus with the largest polymorphism and high specificity and a primer combination with the best compatibility can be screened out according to the test result of the standard substance.

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

the invention provides a MNP (MNP) marking site of a paraenteric virus, a primer composition, a kit and application thereof. The provided 15 MNP loci of the paraenteric 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 requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection of the mycobacterium combination are met, and the requirements of the standard and sharable fingerprint data construction of the paraenteric virus are met; the need to accurately detect genetic variation between strains of parareovirus; the requirements of homozygosity and heterozygosity of the parareovirus are identified, and technical support is provided for scientific research, scientific monitoring and control of the parareovirus.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

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

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

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

Detailed Description

The advantages and various effects of the embodiments of the present invention will be more clearly apparent from the following detailed description and examples. Those skilled in the art will appreciate that these specific implementations and examples are provided to illustrate, but not limit, examples of the present invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. In case of conflict, the present specification will control.

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

The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:

the inventionNovel molecular markers-MNP markers specific for species suitable for detection of organisms in a population are clearly developed. MNP markers refer to polymorphic markers caused by multiple nucleotides in a region of the genome. MNP markers have the following advantages over SSR markers and SNP markers: (1) The alleles are abundant, and 2 are arranged on single MNP locus ⁿ Species alleles higher than SSR and SNP; (2) The species distinguishing capability is strong, the species identification can be realized by only a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting MNP labels based on the combination of super multiplex PCR and a second generation high throughput sequencing technology has the following advantages: (1) The output is a base sequence, a standardized database can be constructed for sharing without parallel experiments; (2) The method has high efficiency, breaks through the limitation of the number of sequencing samples by using the sample DNA bar code, and can type tens of thousands of MNP sites of hundreds of samples at one time; (3) High sensitivity, multiple targets are detected at one time by using multiple PCR, and high false negative and low sensitivity caused by single target amplification failure are avoided; (4) High accuracy, and sequencing the amplified product hundreds of times by using a second-generation high-throughput sequencer.

In view of the advantages and the characteristics, the MNP marking and the detection technology thereof can realize classification and tracing of the multi-allele types of the group organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, genetic variation detection and the like. At present, no report about MNP labeling exists in microorganisms, and corresponding technology is lacking. Thus, the present invention exploits the MNP marker locus of a parareovirus, which is a genomic region screened on the parareovirus genome that is distinct from other species and has multiple nucleotide polymorphisms within the species, comprising: marker loci of MNP-1, MNP-6 and MNP-15 of the reference genome by KY 556675.1; marker loci for MNP-2 and MNP-11 of the reference genome with GQ 183022.1; marker loci of MNP-3 to MNP-5, MNP-7, MNP-9 and MNP-12 to MNP-14 of the reference genome with NC_ 003976.2; marker loci for MNP-8 and MNP-10 of the reference genome with MK 904591.1.

Next, the present invention has developed a multiplex PCR primer composition for detecting the MNP marker locus of the paraenterovirus, characterized in that the multiplex PCR primer composition comprises 15 pairs of primers, the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO.1 to SEQ ID NO. 30. The primers do not collide with each other, and efficient amplification can be performed by multiplex PCR.

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

The kit of the invention can accurately and sensitively detect the parareovirus as low as 10 copies/reaction.

The MNP markers and the kit have high specificity in detecting the parareovirus in complex templates.

The MNP marker locus, primer composition, kit and use thereof of one of the parareoviruses of the present application will be described in detail below in conjunction with examples, comparative examples and experimental data.

Example 1 screening of Paracolone Virus MNP marker loci and design of multiplex PCR amplification primers

S1, screening of MNP (paraenterovirus) marker locus

Based on complete or partial sequences of genomes of 3794 different isolates of the parareovirus disclosed on the net, 15 MNP marking sites are obtained through sequence comparison. For species on which no genomic data is present on the net, genomic sequence information representing a minispecies of the microorganism species to be detected may also be obtained by high throughput sequencing, which may be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected markers, genomic sequences of at least 10 genetically representative isolates are generally used as reference. The 15 MNP marker loci screened are shown in table 1:

TABLE 1 MNP marker loci and detection primers starting position on the reference sequence

The step S1 specifically includes:

selecting a genome sequence of a representative strain of the parareovirus as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphic sites of each strain of the parareovirus;

on the reference genome, carrying out window translation by taking 100-300 bp as a window and taking 1bp as a step length, and screening to obtain a plurality of candidate MNP site areas, wherein the candidate MNP site areas contain more than or equal to 2 single nucleotide variation sites, and the single nucleotide polymorphism sites do not exist on sequences of 30bp at both ends;

screening a region with the discrimination DP more than or equal to 0.2 from the candidate polynucleotide polymorphism site region as an MNP marking site; wherein dp=d/t, t is the log of comparisons when all the minor species are compared pairwise in the candidate polynucleotide polymorphic site region, and d is the log of samples of differences in at least two single nucleic acid polymorphisms in the candidate polynucleotide polymorphic site region.

As an optional implementation mode, when screening is performed on the reference genome by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, so that the comprehensive screening is facilitated.

S2, design of multiplex PCR amplification primer

The multiplex PCR amplification primers of the MNP locus are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.

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

S3, evaluating detection efficiency of primer combination

The detection method of the MNP marker is that all MNP loci are amplified at one time through multiplex PCR, amplification products are sequenced through second-generation high-throughput sequencing, sequencing data are analyzed, and compatibility of the primer combination is evaluated according to the detected loci.

The method comprises the steps of using auxiliary reovirus RNA with known copy number provided by Hubei province disease control and prevention center, reversely transcribing the auxiliary reovirus RNA into cDNA through a commercial reverse transcription kit, adding the cDNA into human genome DNA to prepare a 1000-copy/reaction template, using the primer combination, screening the primer combination with uniform amplification and optimal compatibility according to detection conditions of MNP sites in 4 libraries, and finally screening the primer combination of 15 MNP sites in the table 1.

Threshold settings and Performance assessment for MNP site and primer identification of Paracolone Virus described in example 2

1. Detection of MNP markers

In this example, a known copy number of parareovirus RNA provided by the disease control and prevention center in Hubei 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 parareovirus mimetic samples. An equal volume of sterile water was set at the same time as a blank. A total of 4 samples, each of which was constructed as 3 replicate libraries per day, were tested continuously for 4 days, i.e., 12 sets of sequencing data were obtained per sample, as shown in table 2. And according to the number of sequencing fragments and the number of sites of the parareovirus MNP sites detected in the blank control and the parareovirus nucleic acid standard in 12 repeated experiments, evaluating the reproducibility, the accuracy and the 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.

TABLE 2 detection sensitivity and stability analysis of MNP labeling method of Paracolone virus

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

2. Reproducibility and accuracy assessment of detection of parareovirus by MNP (MNP) marker detection kit

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

TABLE 3 reproducibility and accuracy assessment of methods for detection of Paracolone viral MNP markers

As can be seen from Table 3, the number of MNP sites having a difference in the main genotypes was 0; according to the principle that the reproducible genotypes are considered to be accurate between 2 repeated experiments, the accuracy a=1- (1-r)/2=0.5+0.5r, and r represents the reproducibility, namely the ratio of the reproducible site number of the main genotype to the common site number. In the project reproducibility test, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.

3. Threshold value judgment for detecting parareovirus by MNP (MNP) marker detection kit

As shown in Table 2, sequences aligned to the parareovirus could be detected in 1 copy/reaction samples, covering at least 1 MNP site. The sequence of the parareovirus 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 4.5 megabases. The measurement and calculation basis is that the number of MNP loci detected by each sample is 15, and the length of one sequencing fragment is 300 bases, so that when the data size is more than 4.5 megabases, most samples can ensure that the number of sequencing fragments covering each locus reaches 1000 times by one experiment, and ensure the accurate analysis of the base sequence of each MNP locus.

2) Determining whether the contamination is acceptable based on the signal index S of the parareovirus in the test sample and the noise index P of the parareovirus in the blank, wherein:

the blank noise index p=nc/Nc, where Nc and Nc represent the number of sequenced fragments and total sequenced fragment number of the paramylon 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 and the total number of sequenced fragments, respectively, of the parareovirus in the test sample.

3) Calculating the detection rate of MNP marking sites in a test sample, wherein the detection rate refers to the ratio of the number of detected sites to the number of total designed sites.

TABLE 4 SNR of Paracolone Virus in samples to be tested

The results are shown in Table 4, where the average noise figure for the parareovirus in the control is 0.04%, the average signal figure in the 1 copy sample is 0.26%, and the average signal to noise ratio for the 1 copy sample and the control is 6.5, thus the present invention provides that contamination in the assay system can be judged to be acceptable when the signal to noise ratio is greater than 10 times.

At least 6 MNP sites were stably detected in the 10 copies of the 12 sets of data in response to the 10 copies, at an average signal-to-noise ratio of 67.2 for the 10 copies of the sample and the blank, accounting for 40.0% of the total sites. Therefore, under the condition of ensuring accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold of the parareovirus is 34, namely when the signal-to-noise ratio of the parareovirus in a sample is more than 34 and the site detection rate is more than or equal to 20%, the detection of the nucleic acid of the parareovirus in the sample is judged.

Therefore, the kit provided by the invention can accurately and sensitively detect the paraenteric virus with the copy of as low as 10 copies/reaction.

4. Specific evaluation for detecting parareovirus by MNP (MNP) marker detection method

The mixed template is prepared by artificially mixing nucleic acids of paraenteric viruses, mumps viruses, zika viruses, coronaviruses, influenza viruses, mycobacterium tuberculosis, acinetobacter strains, pertussis bautre bacteria, huo Shibao terus bacteria, chlamydia pneumoniae, mycoplasma pneumoniae, EB viruses, haemophilus influenzae, varicella zoster viruses, cytomegaloviruses, herpes simplex viruses, human bocaviruses, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and streptococcus pyogenes, and the method provided by the invention is adopted as a control to detect the epidemic mumps viruses in the mixed template, so that 3 repeated experiments are carried out. After sequence comparison and analysis according to the quality control scheme and the judgment threshold, 15 MNP sites of the paraenteric virus can be specifically detected in 3 repeated experiments, which shows that the MNP markers and the kit detect the high specificity of target microorganisms in complex templates.

Example 3 detection of genetic variation between Paracolone Virus strains

6 progeny strains of the paraenteric virus strain provided by the disease control of Hubei province are detected by using the kit and the MNP marking site detection method, samples are sequentially named as S1-S6, the average coverage of sequencing of each sample is 1416 times, and all 15 MNP marks can be detected by each strain (table 5). The results of pairwise comparison of fingerprints of 6 strains are shown in Table 5, and the differences between the major genotypes of partial loci of 1 part (S-2) and 5 parts of parareoviruses detected together in the same batch (Table 5) indicate that there are variations between strains.

TABLE 5 detection assay for 6 Paracolone viruses

As can be seen from Table 5, the application of the kit in identifying the genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of strains of the same named parareoviruses in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of the parareoviruses. In clinical terms, one can take into account the diagnostic regimen as to whether the site of the difference affects resistance.

Example 4 detection of genetic variation inside Paracolone Virus strains

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

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

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

Parameter e related to Table 6 _max (n=1) and e _max (n.gtoreq.2) refers to the highest proportion of the total sequence of the locus of the sequence of the wrong allele carrying n SNPs. e, e _max (n=1) and e _max (n.gtoreq.2) 1.03% and 0.0994%, respectively, are obtained from the frequency of all minor genotypes detected at 930 homozygous MNP sites.

TABLE 6-threshold for determining the hypo-isogenotypes at partial sequencing depth

According to the above parameters, nucleic acids of two strains having different genotypes were mixed in the following 8 ratios of 1/1000,3/1000,5/1000,7/1000,1/100,3/100,5/100,7/100, and artificial heterozygous samples were prepared, each sample was tested 3 times for repetition, and 24 sequencing data were obtained in total. By accurately comparing the genotypes of MNP loci of the two strains, loci with heterozygous genotypes are detected in 24 artificial heterozygous samples, and the applicability of the developed method for detecting the MNP marker of the parareovirus in detecting genetic variation inside a strain population is demonstrated.

Example 5 construction of Paracolone Virus DNA fingerprint database

Extracting RNA of all strains or samples for constructing a paraminovirus MNP fingerprint database by using a conventional CTAB method, a commercial kit and the like, and detecting the quality of the RNA by using agarose gel and an ultraviolet spectrophotometer. And (3) comparing the sequencing data of the 6 strains with the reference genotype in sequence, and obtaining the main genotype of each site of each strain to form the MNP fingerprint of each strain. And inputting the obtained MNP fingerprint of each strain into a database file to form a paraenteric virus MNP fingerprint database.

The constructed MNP fingerprint database is based on the gene sequence of the detected strain, is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared and being capable of being updated at any time. And comparing the MNP fingerprint of the strain obtained by each detection with an MNP fingerprint database constructed based on the existing genome data, and inputting the MNP fingerprint of the strain with the main genotype difference into the constructed MNP fingerprint database to achieve real-time updating and co-construction sharing of the database.

Example 6 use in Paraconavirus refinement

Firstly, constructing a reference sequence library of the parareovirus, wherein the reference sequence library consists of a published genome sequence of the parareovirus and a constructed fingerprint database of the parareovirus; obtaining MNP fingerprints of the parareoviruses in each sample to be detected by using the primer combination and the MNP marker locus detection method described in the embodiment 2; comparing the DNA fingerprint of each strain with a constructed reference sequence library, and screening to obtain a strain with the closest genetic distance to the sequence library; 100% identical to the genotype of the existing strain, the existing variant, the new variant with major genotype difference at least one MNP site, realizes the fine typing of the parareovirus.

The typing results of 6 strains of the paraenteric virus in the embodiment are shown in table 5, 6 strains can be divided into 2 types, and the strains of the reference sequence library have main genotype differences of more than 1 MNP locus, so that the strains are judged to be 2 new variant strains, and the fine typing of the paraenteric virus is 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.

Sequence listing

<110> Jiang Handa science

<120> MNP labeling site of Paracolone virus, 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 multiplex PCR primer composition for detecting parareovirus, which is characterized by comprising 15 pairs of primers, wherein the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 30.

2. A test kit for detecting a parareovirus, comprising the primer composition of claim 1.

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

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

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 strains of a parareovirus.

6. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2-3 for constructing a database of parareoviruses.

7. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 in the accurate and fine detection of parareovirus.