CN115044705B - MNP (MNP) marker locus of human coronavirus HCoV-NL63, primer composition, kit and application of MNP marker locus - Google Patents

Abstract

The invention discloses a MNP (MNP) marking site of human coronavirus HCoV-NL63, a primer composition, a kit and application thereof, wherein the MNP marking site comprises 15 marks of a reference genome NC_005831.2, and the nucleotide sequence of the primer is shown as SEQ ID NO.1-SEQ ID NO. 30. The MNP marker locus can specifically identify the HCoV-NL63 of the human coronavirus; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that the sequence analysis can be performed on all the marker loci of multiple samples at one time, the detection advantages of high flux, multiple targets, high sensitivity, high precision and culture-free are achieved, the method can be applied to the identification and genetic variation detection of the human coronavirus HCoV-NL63 of a large-scale sample, and the method has important significance to scientific research and epidemic prevention monitoring of the human coronavirus HCoV-NL63.

Description

MNP (MNP) marker locus of human coronavirus HCoV-NL63, 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 an MNP (MNP) marking site of human coronavirus HCoV-NL63, a primer composition, a kit and application thereof.

Background

Coronaviruses are classified systematically into the genus Coronavirus (Coronavir), which is a RNA virus with a linear single positive strand genome, and are a broad class of viruses that are widely found in nature. Viruses are enveloped and the envelope is observed by electron microscopy to have spinous processes shaped like coronaries, so that such viruses are named coronaviruses. Coronaviruses are expelled from the body through respiratory secretions, are transmitted through oral fluids, sneezes, contact, and are transmitted through air droplets. Coronavirus infections are distributed in many areas of the world and cause pandemics many times. The 2019 outbreak of novel coronavirus-infected pathogenic bacteria novel coronaviruses (2019-nCoV or SARS-CoV-2, eliciting novel coronavirus infection COVID-19) are currently known as the 7 th coronaviruses that can infect humans, the remaining 6 are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (eliciting severe acute respiratory syndrome) and MERS-CoV (eliciting middle east respiratory syndrome), respectively. HCoV-NL63 infection usually causes less symptoms, but is susceptible to infection with other respiratory viruses, primarily through respiratory droplets and human-to-human contact. It is usually prevalent in the late winter season, with a latency period ranging from 2-5 days, and mainly infects infants and adults with low immune function or underlying diseases, and symptoms include fever, cough, pneumonia, and the like.

Existing human coronavirus detection technologies mainly rely on detection of pathogens and antibodies for serotype identification, indirect or direct immunofluorescence methods and molecular detection technologies for detecting genetic material, including PCR, nucleotide hybridization and sequencing technologies. These techniques have advantages, but also have one or more limitations in terms of duration, complexity of operation, throughput of detection, accuracy and sensitivity of detecting variations, cost, etc. For example, virus separation and identification operations are complex and time-consuming; serotype identification, indirect or direct immunofluorescence methods are prone to cross-reactions, resulting in inaccurate detection and inability to monitor variation; the PCR detection technique detects only 1 to 2 markers of one virus at a time, is inefficient, and is prone to detection failure due to virus variation. Metagenomic sequencing is another technique for detecting human coronaviruses, but it often involves large amounts of host sequencing data, especially requiring ultra-deep sequencing for detection of low viral load samples, resulting in high costs. Therefore, the development of a rapid, accurate and one-time high-throughput detection and analysis method for detecting and typing human coronaviruses with various human coronaviruses has important significance for detection and epidemic prevention of the human coronaviruses.

Therefore, the development of a rapid, accurate and one-time high-throughput detection and analysis method for detecting and typing human coronaviruses with various human coronaviruses has important significance for detection and epidemic prevention of the human coronaviruses.

Disclosure of Invention

The invention aims to provide MNP (MNP) marking sites, primer compositions, kits and application of the MNP marking sites, the primer compositions and the kits for the human coronavirus HCoV-NL63, which can identify and variant the human coronavirus HCoV-NL63 and have the effects of multiple targets, high flux, high sensitivity and fine typing.

In a first aspect of the invention, there is provided a MNP marker locus of human coronavirus HCoV-NL63, said MNP marker locus referring to a species-specific genomic region screened on the human coronavirus HCoV-NL63 genome and having a plurality of nucleotide polymorphisms within the species, comprising 15 markers on the human coronavirus HCoV-NL63 reference genome NC_ 005831.2.

Description table 1 further illustrates that the start and end positions of the MNP markers noted in table 1 are determined based on the reference sequences corresponding to the same row of MNPs in 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, each MNP-marked primer comprising an upper primer and a lower primer, the specific primer nucleotide sequences being shown in SEQ ID NO.1 to SEQ ID NO. 30. Table 1 of the specification further illustrates this.

In a third aspect of the invention, a detection kit for detecting the marker locus of human coronavirus HCoV-NL63 MNP is provided, said kit comprising said primer composition.

Further, the kit further comprises a multiplex PCR premix.

And the application of the marking locus, the primer composition and the kit in qualitative detection of the HCoV-NL63 of the human coronavirus of the non-eruption purpose and in preparation of a product for qualitative detection of the HCoV-NL63 of the human coronavirus.

In a fourth aspect of the invention, the application of the MNP labeling site of the human coronavirus HCoV-NL63 or the multiplex PCR primer composition or the detection kit in the identification of the human coronavirus HCoV-NL63, the construction of a DNA fingerprint database and the detection of genetic variation is provided.

In the above application, firstly, the total viral RNA of the sample to be tested is obtained; cDNA synthesis is carried out on the total RNA by using a commercial kit; carrying out a first round of multiplex PCR amplification on the cDNA and the blank control by using the kit, wherein the cycle number is not higher than 25; purifying the amplified product, and then adding a sample tag and a second generation sequencing joint based on the second-round PCR amplification; quantifying after purifying the second round of amplification products; detecting a plurality of strains by mixing the amplification products of the second round in equal amounts and then performing high-throughput sequencing; the sequencing results were aligned to the reference sequence of the human coronavirus HCoV-NL63 to obtain the number of detection sequences in the cDNA and genotype data. And performing data quality control and data analysis on the sequencing data of the cDNA according to the number of sequencing sequences of the human coronavirus obtained from the cDNA and the blank control and the number of detected MNP labels, and obtaining the number of MNP labels of the human coronavirus HCoV-NL63 detected in the sample, the number of sequencing sequences covering each MNP label and the MNP label genotype data.

When used for the identification of human coronavirus HCoV-NL63, the quality control is performed to determine whether the sample to be tested contains nucleic acid of human coronavirus HCoV-NL63 based on the number of sequencing sequences of human coronavirus HCoV-NL63 detected in the sample to be tested and the blank control and the number of MNP sites detected. The quality control scheme and the judging method are characterized in that DNA of the human coronavirus HCoV-NL63 with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the human coronavirus HCoV-NL63 are evaluated, and the quality control scheme and the judging method when the kit detects the human coronavirus HCoV-NL63 are formulated.

When used for the detection of genetic variation of human coronavirus HCoV-NL63, it includes the detection of genetic variation of strains between individuals and within individuals of a case of HCoV-NL63 infection. The genetic variation detection of the strains among individuals comprises the step of utilizing the kit and the method to obtain genotype data of 15 MNP marks of the strains infected by the individuals to be compared. By genotype comparison, whether there was a difference in the major genotypes across the 15 MNP markers was analyzed. If the compared strains have a variation in the major genotype of at least one MNP marker, then it is determined that there is a genetic variation in both. Alternatively, the 15 markers of the strains to be compared may be amplified by single PCR, respectively, and the amplified products may be subjected to Sanger sequencing to obtain sequences, and the genotypes of each MNP marker of the strains to be compared may be aligned. If MNP markers of non-identical major genotypes are present, variations between the strains to be compared are present. When detecting whether genetic variation exists in the interior of a strain infected by an individual, determining whether the strain detected in the interior of the individual detects a secondary genotype other than the primary genotype at the MNP marker by a statistical model. If there is a subgenotype in at least one MNP marker, it is determined that there is a genetic variation within the tested strain.

When the MNP fingerprint database of the human coronavirus HCoV-NL63 is constructed, genotype data of the MNP marker of the human coronavirus HCoV-NL63 identified from a sample is input into a database file to form the MNP fingerprint database of the human coronavirus HCoV-NL63; each time a different sample is identified, by comparing with the MNP fingerprint database of the human coronavirus HCoV-NL63, it is identified whether the human coronavirus HCoV-NL63 in the sample differs from the strains in the database in that the MNP markers have a major genotype (with a genotype supported by more than 50% of the sequenced fragments in one MNP marker), and the human coronavirus HCoV-NL63 having a major genotype difference in at least 1 MNP marker is the new variant type, which is recorded in the MNP fingerprint database.

In the application, when the kit is used for the typing detection of the human coronavirus HCoV-NL63, the human coronavirus HCoV-NL63 in a sample to be detected is identified, and the genotype of each MNP locus is obtained; the genome sequence of the human coronavirus HCoV-NL63 disclosed on the collection network and the constructed DNA fingerprint database of the human coronavirus HCoV-NL63 form a human coronavirus HCoV-NL63 reference sequence library; comparing the genotype of the human coronavirus HCoV-NL63 in the sample to be tested with a reference sequence library of the human coronavirus HCoV-NL63, and screening strains which are genetically identical or closest to each other to obtain the genotyping of the human coronavirus HCoV-NL63 in the sample to be tested. And identifying whether the human coronavirus HCoV-NL63 in the sample is an existing type or a new variant according to the comparison result with the reference sequence library, and realizing the fine typing of the human coronavirus HCoV-NL63.

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

the invention provides MNP marking sites, primer compositions, kits and applications of the human coronavirus HCoV-NL63, 15 MNP marks of the human coronavirus HCoV-NL63 and primer combinations thereof, which can be used for multiplex PCR amplification, and a second generation sequencing platform is fused for sequencing amplification products, so that the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection of the human coronavirus HCoV-NL63 are met, and the requirements of shared fingerprint data construction of the human coronavirus HCoV-NL63 standard are met; meets the requirement of accurately detecting the genetic variation of strains among individuals and inside individuals of human coronavirus HCoV-NL63 infection cases; the invention is initiated in the field of human coronavirus HCoV-NL63, and is not reported in related documents.

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 the screening and primer design for the HCoV-NL63 MNP marker of human coronavirus;

fig. 3 is a flow chart of detection of MNP markers.

Detailed Description

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

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

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

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

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

In view of the advantages and the characteristics, the MNP marking and the detection technology thereof can realize classification and tracing of the multi-allele types of the group organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, genetic variation detection and the like. At present, no report about MNP labeling exists in microorganisms, and corresponding technology is lacking. The development, screening and application of MNP labeling method has better application foundation in plants.

Accordingly, the present invention provides a MNP marker locus for human coronavirus HCoV-NL63, characterized in that said MNP marker locus is a genomic region screened on the human coronavirus HCoV-NL63 genome that is distinct from other species and has multiple nucleotide polymorphisms within the species, comprising: 15 MNP-marker loci of MNP-1 to MNP-15 of the reference genome with NC_ 006213.1.

Next, the present invention developed a multiplex PCR primer composition for the human coronavirus HCoV-NL63 MNP marker loci, comprising 15 pairs of primers, the specific primer nucleotide sequences being shown in SEQ ID NO.1-SEQ ID NO. 30. The primers do not collide with each other, and efficient amplification can be performed through multiplex PCR;

the multiplex PCR primer composition can be used for a detection kit for detecting the HCoV-NL63 MNP marker locus of the human coronavirus. The kit can stably and sensitively detect HCoV-NL63 in a range as low as 10 copies/reaction.

In the reproducibility test of the invention, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.

The MNP markers and the kits of the invention have high specificity in detecting target microorganisms in complex templates.

The MNP-labeling site, primer composition, kit and use thereof of one of the human coronaviruses HCoV-NL63 of the present application will be described in detail below in connection with examples, comparative examples and experimental data.

Example 1 selection of human coronavirus HCoV-NL63 MNP marker locus and design of multiplex PCR amplification primers

Screening of S1, human coronavirus HCoV-NL63 MNP marker loci

15 HCoV-NL 63-specific MNP markers were obtained by sequence alignment based on the 913 of the network-published HCoV-NL63, 547 of the HCoV-229E, 518 of the HCoV-HKU1, 1426 of the HCoV-OC43, 1438 of the MERS-CoV, 40855 of the SARS-CoV-2 and 44326 genomes of the SARS-CoV as reference sequences and comparison with the NCBI database. For species on which no genomic data is present, genomic sequence information of the microorganism species representative of the isolate 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 isolates are generally used as reference.

The 15 MNP markers screened are shown in Table 1 and SEQ ID NO.1-SEQ ID NO. 30:

MNP labeling sites and initial positions of detection primers on the reference sequence as described in Table 1

The step S1 specifically includes:

selecting one or more genome sequences of representative strains of the human coronavirus HCoV-NL63 as a reference genome, and comparing the genome sequences with the reference genome to obtain single nucleotide polymorphism markers of each strain of the human coronavirus HCoV-NL63;

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

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

As an optional implementation mode, when screening is performed on the reference genome by taking 100-300bp 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 MNP marked multiplex PCR amplification primers are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.

S3, evaluating detection efficiency of primer combination

The 1000 copies/reaction template was prepared by reverse transcription of human coronavirus HCoV-NL63 RNA of known copy number into cDNA using commercial kit and addition to 2 ng/reaction human genomic DNA. Detection was performed by multiplex PCR in combination with the detection method of second generation sequencing using the designed primer combinations, constructing 4 duplicate detection libraries. According to the test results of the 4 repeated detection libraries, the 15 MNP labels with the best compatibility and the detection primer composition thereof provided by the invention are finally obtained, and are specifically shown in the table 1 and SEQ ID NO.1-SEQ ID NO. 30.

Example 2 characterization of MNP marker loci and primers evaluation and threshold settings of human coronavirus HCoV-NL63

1. Detection of MNP markers

In this example, a sample of human coronavirus HCoV-NL63 having a known copy number was reverse transcribed into cDNA using a commercial kit, and then added to human genomic DNA to prepare 1, 10 and 100 copies/reaction of human coronavirus HCoV-NL 63-simulated samples, with an equal volume of sterile water as a blank. The present example measures a total of 4 samples, each of which was constructed as 3 replicate libraries per day, and was tested continuously for 4 days, i.e., 12 sets of sequencing data were obtained per sample, as shown in Table 2. The reproducibility, accuracy and sensitivity of the detection method are evaluated according to the number of sequencing fragments and the number of markers of MNP markers of human coronavirus HCoV-NL63 detected in a blank control and a human coronavirus HCoV-NL63 nucleotide standard in 12 repeated experiments, and thresholds and judgment standards for quality control system pollution and target pathogen detection are formulated. The detection flow of MNP markers is shown in fig. 3.

TABLE 2 detection sensitivity and stability analysis of MNP labeling method of human coronavirus HCoV-NL63

As shown in Table 2, 2-3 MNP sites were detected in the 1-copy/reaction 12-set data, and 1 site was also detected in part in the blank; in the 12 sets of data of 10 copies/reaction, at least 8 MNP markers could be stably detected, and the number of MNP sites detected in the blank control was far higher, indicating that the kit could stably and sensitively detect HCoV-NL63 as low as 10 copies/reaction.

2. Reproducibility and accuracy assessment of detection of human coronavirus HCoV-NL63 by MNP marker detection kit

Based on whether the genotype of the marker is reproducible or not in the two replicates, the reproducibility and accuracy of the detection of human coronavirus HCoV-NL63 by the MNP marker detection method is evaluated. Specifically, the data of 12 sets of 100 copies/reaction sample shown in Table 2 were compared in pairs, and as a result, as shown in Table 3, the number of MNP markers having a difference in the main genotype 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, r represents the reproducibility, i.e. the ratio of the number of reproducible markers of the main genotype to the number of common markers. 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%.

TABLE 3 reproducibility and accuracy assessment of human coronavirus HCoV-NL63 MNP marker detection method

3. Threshold determination of detection of human coronavirus HCoV-NL63 by MNP marker detection kit

As shown in Table 2, the sequence aligned to human coronavirus HCoV-NL63 could be detected in 1 copy/reaction samples, covering at least 2 MNP markers. The sequence of human coronavirus HCoV-NL63 was also detected in a partial blank. Because of the extreme sensitivity of MNP marker detection methods, contamination of the data in the detection is prone to false positives. The following quality control scheme is formulated in this example.

The quality control scheme is 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 labels 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 label reaches 1000 times by one experiment, and the accurate analysis of the base sequence of each MNP label is ensured.

2) Determining whether the contamination is acceptable based on the signal index S of human coronavirus HCoV-NL63 in the test sample and the noise index P of human coronavirus HCoV-NL63 in the blank, wherein:

the noise figure p=nc/Nc for the control, where Nc and Nc represent the number of sequenced fragments and the total number of sequenced fragments of human coronavirus HCoV-NL63, respectively, in the control.

The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments and the total number of sequenced fragments of human coronavirus HCoV-NL63, respectively, in the test sample.

3) The detection rate of MNP markers in the test sample is calculated and refers to the ratio of the number of detected markers to the total number of designed markers.

As shown in Table 4, the noise figure average of human coronavirus HCoV-NL63 in the blank is 0.04%, the signal figure average in 1 copy sample is 0.22%, and the signal to noise ratio of 1 copy sample and blank is 5.5, therefore the present invention provides that when the signal to noise ratio is greater than 10 times, it can be judged that the contamination in the detection system is acceptable.

As shown in Table 4, the average signal-to-noise ratio of the 10 copies of the sample and the blank was 60, and at least 8 MNP markers were stably detected in the 10 copies/12 sets of data, accounting for 53.3% of the total markers. Thus, the present standard specifies that the signal-to-noise ratio decision threshold for human coronavirus HCoV-NL63 is 10, while ensuring accuracy, i.e. when the signal-to-noise ratio for human coronavirus HCoV-NL63 in a sample is greater than 10 and the label detection rate is greater than or equal to 26.5%, the nucleotides of human coronavirus HCoV-NL63 are decided to be detected in the sample. Based on the set judgment threshold, the kit provided by the invention can accurately and sensitively detect the human coronavirus HCoV-NL63 with the copy/response as low as 10.

TABLE 4 Signal to noise ratio of human coronavirus HCoV-NL63 in test sample

4. MNP marker detection method for detecting specificity evaluation of human coronavirus HCoV-NL63

The human coronavirus HCoV-229E and the RNA of human coronavirus HCoV-NL63, HCoV-HKU1, HCoV-NL63SARS-CoV-2, and human parainfluenza virus, metapneumovirus, human rhinovirus, mumps virus, measles virus, respiratory syncytial virus, influenza A virus, influenza B virus, influenza C virus, avian influenza virus and Zika virus were artificially mixed together to prepare a mixed template, and the kit provided by the invention was used for detecting pathogens in the mixed template by using a blank template as a control, and 3 repeated experiments were performed. As a result, in 3 replicates, the sequenced sequences obtained in the mixed template were aligned specifically only to the MNP sites of HCoV-NL63, and the number of MNP sites detected was 14,14 and 15, respectively. After the judgment is carried out according to the quality control scheme and the threshold value, only the human coronavirus in the mixed template can be specifically detected in 3 repeated experiments, which shows that the MNP mark and the kit detect the high specificity of the target microorganism in the complex template.

Example 3 detection of genetic variation of strains among individuals with human coronavirus HCoV-NL63 infection

6 copies of the collected HCoV-NL63 strain of the human coronavirus are detected by using the kit and the MNP marker locus detection method, samples are sequentially named as S1-S6, the average coverage of sequencing of each MNP marker is 3000 times, and all 15 MNP markers can be detected by each strain (Table 5). The results of pairwise alignment of the fingerprints of 6 strains are shown in Table 5, and 1 part (S-2) of human coronavirus HCoV-NL63 and 5 parts of human coronavirus HCoV-NL63 detected in the same batch all have a partially marked major genotype difference (Table 5) and have inter-strain variation.

The application of the kit for identifying the genetic variation among strains by detecting MNP markers can be used for monitoring the genetic variation of viruses and can be used for ensuring the genetic consistency of the same named human coronavirus HCoV-NL63 strains in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for epidemic prevention monitoring, accurate treatment and scientific research of the human coronavirus HCoV-NL63.

TABLE 5 detection analysis of 6 copy samples of 1 coronavirus HCoV-NL63 strain

EXAMPLE 4 detection of genetic variation of strains within human coronavirus HCoV-NL63 infection case

After infection of humans with coronaviruses, as a population organism, the individuals within the internal part of human coronaviruses HCoV-NL63 undergo a variation, resulting in a complex infection. When the variant individuals do not accumulate, the individuals occupy a very small part of the population, and when the population is subjected to molecular marker detection, the individuals show minor genotypes outside the low-frequency major genotypes. Low frequency sub-isogenotypes are often mixed with technical errors, resulting in indistinguishable prior art. The invention detects MNP marks with high polymorphism, and the technical error rate of the MNP marks is obviously lower than that of the prior marks, such as SNP marks, based on that the probability of simultaneous occurrence of a plurality of errors is lower than that of one error. The key to detection of complex infections is to determine the authenticity of the hypo-genotype of the MNP site detected in the infected individual.

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) means the highest ratio of the number of sequences of the wrong allele carrying n SNPs to the total number of sequences of the markerExamples are shown. 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 markers.

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

According to the above parameters, the nucleotides of two strains having the genotypes different from each other as shown in Table 5 were mixed in the following 8 ratios of 1/1000,3/1000,5/1000,7/1000,1/100,3/100,5/100,7/100 to prepare artificial heterozygous samples, each sample was tested 3 times for repetition, and 24 total sequencing data were obtained. Through the accurate comparison with the MNP marker genotypes of the two strains, the marker with the heterozygous genotype is detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marker detection method of the human coronavirus HCoV-NL63 in detecting low-frequency genetic variation inside a strain population is demonstrated.

EXAMPLE 5 construction of human coronavirus HCoV-NL63DNA fingerprint database

The DNA of all strains or samples used for constructing a human coronavirus HCoV-NL63DNA fingerprint database is extracted by using a conventional CTAB method, a commercial kit and other methods, and the quality of the DNA is detected by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm and 230nm is more than 2.0, the ratio of the absorbance values of 260nm and 280nm is between 1.6 and 1.8, the DNA electrophoresis main band is obvious, no obvious degradation and RNA residues exist, the genome DNA reaches the relevant quality requirements, and the subsequent experiments can be carried out.

And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain the main genotype of each mark of each strain, forming MNP fingerprint of each strain, and recording the fingerprint with at least 1 main genotype difference with other strains into a database file after pairwise comparison to form a human coronavirus HCoV-NL63DNA fingerprint database. The MNP fingerprint of the strain obtained by detection in each new sample is compared with the constructed MNP fingerprint database, and the MNP fingerprint database constructed by the MNP fingerprint of the strain with the main genotype difference is realized, so that the co-construction sharing and the random updating of the database are realized. The constructed MNP fingerprint database is based on the gene sequence of the detected strain and is therefore compatible with all high throughput sequencing data.

Example 6 use in human coronavirus HCoV-NL63 fine-particle type

And detecting the 6 human coronavirus HCoV-NL63 strains by using the primer combination and MNP labeling site detection method, so as to obtain MNP fingerprint of each strain. The DNA fingerprint of each strain is compared with the constructed fingerprint database in pairs, and the DNA fingerprint of each strain is defined as the existing variant, and the DNA fingerprint of each strain is defined as the new variant when at least one MNP mark has main genotype difference, so that the fine typing of the human coronavirus HCoV-NL63 is realized. Detection of 6 samples of human coronavirus HCoV-NL63 As shown in Table 5, 1 of 6 human coronavirus HCoV-NL63 detected was different from the other 5 major genotypes of the 5 MNP markers, and may have been mutated to different variants. Therefore, the resolution of the method for the human coronavirus HCoV-NL63 reaches the level of single base, and the method can realize the fine typing of the human coronavirus HCoV-NL63 in a sample.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiment and all such alterations and modifications as fall within the scope of the embodiments of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, the embodiments of the present invention are intended to include such modifications and alterations insofar as they come within the scope of the embodiments of the invention as claimed and the equivalents thereof.

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<400> 5

cacacacttt cttgtcgttg tca 23

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

acaaactatg actaccagga atact 25

<210> 7

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

aggtttatgt ccatttacgt aactgg 26

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

agtggccata aacaccataa aacag 25

<210> 9

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

atattcaact tgtgacattt gtaaaagt 28

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

acgtccatta acaaacttaa cacgt 25

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tgttgttaca cttaaagata gtgatgt 27

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

acaagcaaaa tcaagaaaag tttca 25

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

agggtgatgg tggtgtttta gg 22

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

agtctaactc gattgtgttg caac 24

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ccctgttaaa cctgctagta aacag 25

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

ataaacacag ccttactcca acttg 25

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

tttaactaag ggaccacatg agttt 25

<210> 18

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

acaacagcat ctgtcttaac aacat 25

<210> 19

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

aggtgacttt attcaaggtc ctttt 25

<210> 20

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

acaacaacag tggctttatg tttaca 26

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

ttcacacctt gtaaactgtg tttct 25

<210> 22

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

aatgtaatct ttagataatg actgaagttg 30

<210> 23

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

atcgcaaacg taatcagaaa ccttt 25

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

gaagagtctc gtgagttgtt acgag 25

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

ttgttagctt ccattcaaca tcttc 25

<210> 26

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

accaaaaata accttaccac tttgca 26

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

ggttctgtgt tgcgtattaa ggttt 25

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

attaacacca aaaacaccag atgca 25

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

ccaaatggta ctgttatgac aagca 25

<210> 30

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

acgacacaca attgaaaatt ctgga 25

Claims (7)

1. A multiplex PCR primer composition for detecting human coronavirus HCoV-NL63, which is characterized in that the multiplex PCR primer composition comprises 15 pairs of primers, and the specific primer nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO. 30.

2. A detection kit for detecting human coronavirus HCoV-NL63, characterized in that the kit comprises the primer composition of claim 1.

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

4. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 for the preparation of a qualitative detection product of human coronavirus HCoV-NL63.

5. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 for detecting inter-and intra-individual variations in human coronavirus HCoV-NL63 infected cases.

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 human coronaviruses HCoV-NL63.

7. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 in the detection of human coronavirus HCoV-NL63 fine type.

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