CN115029479B - MNP (MNP) marking site of Zika virus, primer composition, kit and application of MNP marking site - Google Patents

Abstract

The invention discloses MNP (MNP) marking sites of Zika virus, a primer composition, a kit and application thereof, wherein the MNP marking sites refer to genome regions which are screened on the genome of the Zika virus and are separated from other species and have a plurality of nucleotide polymorphisms in the species, and the marking sites comprise MNP-1-MNP-15; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 30. The MNP marker locus can specifically identify Zika virus 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 identification and genetic variation detection of the Zika virus of large-scale samples, and has important significance on scientific research and epidemic prevention monitoring of the Zika virus.

Description

A kind of MNP marker site, primer composition, kit and application of Zika virus

technical field

The embodiment of the present invention relates to the field of biotechnology, in particular to a Zika virus MNP marker site, primer composition, kit and application thereof.

Background technique

Zika virus belongs to the Flaviviridae family and belongs to the Flavivirus genus. It is a single-stranded positive-sense RNA virus and is an arbovirus transmitted by mosquitoes. In 1947, the Zika virus was first discovered in rhesus monkeys in the Zika jungle of Uganda, hence the name. At present, a total of 84 countries around the world have reported cases of Zika virus infection, and the virus is gradually spreading in Southeast Asia and other regions. Symptoms of Zika virus infection are similar to those of dengue fever, including fever, rash, joint pain, muscle pain, headache and conjunctivitis. The World Health Organization (WHO) believes that neonatal microcephaly and Guillain-Barré syndrome may be related to Zika virus infection. Diagnosis of Zika virus is difficult due to cross-reactivity with other flaviviruses such as dengue virus, West Nile virus and yellow fever virus, and there are currently no vaccines, preventive drugs or specific treatments for Zika virus medicine. Therefore, rapid and accurate detection of Zika virus is of great significance for timely diagnosis of the cause, early detection and early treatment, reduction of disease progression and prevention of pathogen transmission. In addition, as Zika virus is a group organism, in the interaction with the host and the environment, individuals in the group will mutate, leading to the failure of detection or treatment methods; for experimental research, this undetectable variation will lead to different The strains with the same name in the same laboratory or in different periods in the same laboratory are actually not the same, resulting in irreproducible and incomparable experimental results. Therefore, the development of rapid, accurate, and variable-monitoring Zika virus detection and analysis methods is of great significance for the clinical treatment, epidemic prevention detection and scientific research of Zika virus.

Classic Zika virus detection methods, including isolation and culture, PCR technology, whole genome and metagenomic sequencing, etc., have one or more limitations in terms of time length, operational complexity, detection throughput, accuracy and sensitivity of detection of mutations, cost, etc. . The targeted molecular marker detection technology that combines ultra-multiplex PCR amplification and high-throughput sequencing can enrich target microorganisms in samples with low microbial content, avoiding the isolation and culture of pathogenic bacteria and metagenomic sequencing of the whole genome. Due to the limitation of a large amount of data waste and background noise, it has the advantages of less sample requirements, accurate diagnostic results, saving data, detecting low-frequency mutations, and exempting culture. The molecular markers detected by existing targeted detection technologies mainly include SNP and SSR markers. SSR markers are recognized as the most polymorphic markers, but their number is small in microorganisms; SNP markers are huge in number, densely distributed, and are dimorphic markers, and the polymorphism of a single SNP marker is not enough to capture potential alleles in microbial populations genetic diversity.

Therefore, the development of new molecular markers with high polymorphism of Zika virus and its detection technology has become a technical problem to be solved urgently.

Contents of the invention

The purpose of the present invention is to provide a Zika virus-specific MNP marker site, primer composition, kit and application thereof, which can carry out qualitative identification and variation detection of Zika virus, and have multi-target, high-throughput, and high-sensitivity and fine-tuning effects.

In order to achieve the above object, the present invention adopts the following technical solutions:

In the first aspect of the present invention, a Zika virus-specific MNP marker site is provided, and the MNP marker site refers to a Zika virus genome screened on the Zika virus genome that is different from other species and has multiple cores within the species. Genomic regions of nucleotide polymorphisms, including: marker sites of MNP-1, MNP-3, MNP-9, MNP-11 and MNP-13 with MH675624.1 as the reference genome; MT505349.1 as the reference genome The marker sites of MNP-2, MNP-4, MNP-7, MNP-12 and MNP-14~MNP-15; MNP-5~MNP-6, MNP-8 and MNP with KU955594.1 as the reference genome -10 marker sites.

In the above technical solution, the marking sites of MNP-1 to MNP-15 are specifically shown in Table 1 of the instruction manual. The starting and ending positions of the MNP marking marked in Table 1 are based on the references corresponding to the same row of MNP in Table 1. The sequence is determined.

In a second aspect of the present invention, a multiplex PCR primer composition for detecting the MNP marker site is provided, the multiplex PCR primer composition includes 15 pairs of primers, and the specific primer sequence is as SEQ ID NO.1 ~shown in SEQ ID NO.30.

In the above technical solution, the primers for each MNP marker site include an upper primer and a lower primer, as shown in Table 1 of the specification.

In the third aspect of the present invention, a detection kit for detecting the Zika virus MNP marker site is provided, the kit includes the primer composition.

Further, the kit also includes a multiplex PCR master mix.

In the fourth aspect of the present invention, the MNP marker site of the Zika virus or the multiple PCR primer composition or the detection kit is provided for the qualitative detection and preparation of Zika virus for non-diagnostic purposes. Applications in virus qualitative detection products.

In a fifth aspect of the present invention, the MNP marker site of the Zika virus or the multiplex PCR primer composition or the detection kit is provided for detecting Zika virus strains and strains. Application in Variation.

In the sixth aspect of the present invention, the application of the MNP marker site of Zika virus or the multiplex PCR primer composition or the detection kit in constructing Zika virus database is provided.

In the seventh aspect of the present invention, the application of the MNP marker site of Zika virus or the multiplex PCR primer composition or the detection kit in fine typing detection of Zika virus is provided.

In the application described above, the specific operation steps are: firstly, obtain the viral total RNA of the sample to be tested; utilize a commercialized reverse transcription kit to reverse transcribe into cDNA; Carry out the first round of multiplex PCR amplification, the number of cycles is not higher than 25; after the amplification products are purified, add sample tags and next-generation sequencing adapters based on the second round of PCR amplification; Quantification after purification; when multiple strains are detected, high-throughput sequencing is performed by mixing equal amounts of the second-round amplification products; the sequencing results are compared to the reference sequence of the Zika virus, and the cDNA is obtained Detect sequence number and genotype data. According to the number of Zika virus sequencing sequences and the number of detected MNP sites obtained in the cDNA and the blank control, data quality control and data analysis were performed on the sequencing data of the cDNA to obtain the number of detected MNP sites, The number of sequencing sequences covering each of the MNP sites and the genotype data of the MNP sites.

When used for Zika virus identification, according to the number of sequenced sequences of Zika virus detected in the sample to be tested and the blank control and the number of detected MNP sites, after quality control, it is determined whether the sample to be tested contains Zika The nucleic acid of the virus. Wherein, the quality control scheme and determination method are to use the RNA of Zika virus with known copy number as the detection sample, evaluate the sensitivity, accuracy and specificity of the kit for detecting Zika virus, and formulate the kit Quality control scheme and determination method for Zika virus detection.

When used for the detection of Zika virus genetic variation, it includes the detection of genetic variation between strains and within strains. The detection of genetic variation between strains includes using the kit and method to obtain the genotype data of each of the 15 MNP sites of the strains to be compared. Through genotype comparison, analyze whether there are differences in the main genotypes of the strains to be compared at the 15 MNP sites. If there is a variation in the main genotype of at least one MNP locus of the strain to be compared, it is determined that there is a genetic variation between the two. As an alternative, the 15 loci of the strain to be compared can also be amplified by single-plex PCR, and then the amplified products are subjected to Sanger sequencing. After the sequence is obtained, each MNP locus of the strain to be compared is genotype comparison. If there are MNP sites with inconsistent main genotypes, there is variation among the strains to be compared. When detecting genetic variation within a strain, a statistical model is used to determine whether a secondary genotype other than the main genotype is detected at the MNP site of the strain to be tested. If the strain to be tested has a subgenotype at at least one MNP site, it is determined that there is genetic variation within the strain to be tested.

When used to construct the Zika virus MNP fingerprint database, the genotype data of the MNP site of the Zika virus identified from the sample is entered into the database file to form the MNP fingerprint database of the Zika virus; During the sample, by comparing with the MNP fingerprint database of the Zika virus, identify whether the Zika virus in the sample and the strain in the database have a main genotype at the MNP site (with more than 50 genotypes at one MNP site). % genotypes supported by sequencing fragments), Zika virus with major genotype differences at at least one MNP locus is a new variant type, which is included in the MNP fingerprint database.

When used for Zika virus typing, the Zika virus in the sample to be tested is identified to obtain the genotype of each MNP site. By comparing with the MNP fingerprint database of the Zika virus, it is identified whether the Zika virus in the sample is an existing type or a new type, and the new type is included in the MNP fingerprint database. Therefore, using the primer combination, the MNP fingerprint database can be continuously enriched.

The present invention is the first in the field of Zika virus, and there are no related literature reports; MNP markers are mainly developed based on reference sequences. MNP sites with polymorphic and conserved sequences on both sides of the Zika virus species; MNP site detection primers suitable for multiplex PCR amplification can be designed through the conserved sequences on both sides of the MNP site; then according to the test results of the standard, A set of MNP sites with the largest polymorphism and high specificity, a primer combination with the best compatibility and a detection kit 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 a Zika virus MNP marker site, primer composition, kit and application thereof. The provided 15 MNP sites of Zika virus and their primer combinations can be used for multiplex PCR amplification, combined with the next-generation sequencing platform to sequence the amplified products, meeting the high-throughput, high-efficiency, The demand for high-accuracy and high-sensitivity detection meets the requirements for Zika virus standard and shareable fingerprint data construction; the demand for accurate detection of genetic variation among Zika virus strains; the demand for identification of Zika virus homozygosity and heterozygosity , to provide technical support for scientific research, scientific monitoring and prevention and control of Zika virus.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings that need to be used in the description of the embodiments. Obviously, the drawings in the following description are some implementations of the embodiments of the present invention. For example, those skilled in the art can also obtain other drawings based on these drawings without creative work.

Figure 1 is a schematic diagram of MNP marker polymorphism;

Fig. 2 is the screening and primer design flowchart of Zika virus MNP marker site;

Fig. 3 is a flowchart of the detection of MNP marker sites.

Detailed ways

The following will specifically describe the embodiments of the present invention in combination with specific implementations and examples, and the advantages and various effects of the embodiments of the present invention will be presented more clearly. Those skilled in the art should understand that these specific implementations and examples are for illustrating the embodiments of the present invention, rather than limiting the embodiments of the present invention.

Throughout the specification, unless otherwise specified, terms used herein should be understood as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of the present invention belong. In case of conflict, this specification shall take precedence.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the examples of the present invention can be purchased from the market or prepared by existing methods.

The technical solution of the embodiment of the present application is to solve the above-mentioned technical problems, and the general idea is as follows:

The invention develops a new species-specific molecular marker-MNP marker, which is suitable for detecting population organisms. MNP markers refer to polymorphic markers caused by multiple nucleotides in an upper region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) rich alleles, 2 ⁿ alleles on a single MNP locus, higher than SSR and SNP markers; (2) strong species discrimination ability, Only a small amount of MNP markers are needed to achieve species identification, reducing the detection error rate. The MNP marker method based on ultra-multiplex PCR combined with next-generation high-throughput sequencing technology to detect MNP markers has the following advantages: (1) The output is base sequence, without parallel experiments, and a standardized database can be built for sharing; (2) High Efficiency, using sample RNA barcodes, breaking through the limitation of the number of sequencing samples, and can type tens of thousands of MNP sites in hundreds of thousands of samples at one time; (3) High sensitivity, using multiplex PCR to detect multiple targets at one time, avoiding Failure to amplify a single target leads to high false negatives and low sensitivity; (4) high accuracy, using a second-generation high-throughput sequencer to sequence the amplified product hundreds of times.

In view of the above advantages and characteristics, MNP marker and its detection technology MNP marker method can realize the classification and traceability of multi-allelic genotypes in populations, and has application potential in the identification of pathogenic microorganisms, fingerprint database construction, and genetic variation detection. At present, in microorganisms, there is no report on MNP labeling, and there is a lack of corresponding technology. Therefore, the present invention has developed the MNP marker site of Zika virus, and the MNP marker site is a genomic region screened on the Zika virus genome that is distinguished from other species and has multiple nucleotide polymorphisms within the species , including: marker sites of MNP-1, MNP-3, MNP-9, MNP-11 and MNP-13 with MH675624.1 as the reference genome; MNP-2 and MNP-4 with MT505349.1 as the reference genome , MNP-7, MNP-12 and MNP-14~MNP-15 marker sites; MNP-5~MNP-6, MNP-8 and MNP-10 marker sites with KU955594.1 as the reference genome.

Next, the present invention has developed a multiplex PCR primer composition for detecting the Zika virus MNP marker site, characterized in that the multiplex PCR primer composition includes 15 pairs of primers, and the nucleotides of the 15 pairs of primers The sequence is shown as SEQ ID NO.1-SEQ ID NO.30. The primers do not conflict with each other and can be efficiently amplified by multiplex PCR.

The multiplex PCR primer composition can be used as a detection kit for detecting the Zika virus MNP marker site.

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

The MNP marker of the present invention and the kit have high specificity for detecting Zika virus in complex templates.

A Zika virus MNP marker site, primer composition, kit and application of the present application will be described in detail below in combination with examples, comparative examples and experimental data.

Embodiment 1, the screening of Zika virus MNP marker site and the design of multiple PCR amplification primers

S1. Screening of Zika virus MNP marker sites

Based on the complete or partial genome sequences of 3794 Zika virus isolates published online, 15 MNP marker sites were obtained through sequence alignment. For species that do not have genomic data on the Internet, the genome sequence information of representative races of the microbial species to be detected can also be obtained through high-throughput sequencing, where high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure the polymorphism of the selected markers, the genome sequences of at least 10 genetically representative isolates are generally used as references. The 15 MNP marker sites screened are shown in Table 1:

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

The step S1 specifically includes:

Select the genome sequence of a representative strain of the Zika virus as a reference genome, compare the genome sequence with the reference genome, and obtain the single nucleic acid polymorphism sites of each strain of the Zika virus;

On the reference genome, use 100-300 bp as a window, and use 1 bp as a step to perform window translation, and screen to obtain a plurality of candidate MNP site regions, wherein the candidate MNP site regions contain ≥ 2 of the mononuclei Nucleotide variation sites, and the single nucleic acid polymorphism sites do not exist on the 30bp sequences at both ends;

In the candidate polynucleotide polymorphic site region, the region with a discrimination degree DP≥0.2 is selected as the MNP marker site; wherein, DP=d/t, t is at the candidate polynucleotide polymorphic site d is the logarithm of comparisons of at least two single nucleic acid polymorphism differences in the region of candidate polynucleotide polymorphism sites.

As an optional implementation, other step lengths can also be used when screening on the reference genome with a window of 100-300 bp. In this embodiment, the step size is 1 bp, which is conducive to comprehensive screening.

S2, the design of multiple PCR amplification primers

The multiplex PCR amplification primers of the MNP site are designed by primer design software. The primer design follows that the primers do not interfere with each other. All primers can be combined into a primer pool for multiplex PCR amplification, that is, all designed primers can be used in one amplification reaction. Amplified normally.

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

S3. Evaluation of detection efficiency of primer combinations

The detection method of the MNP marker is to amplify all MNP sites at one time through multiplex PCR, sequence the amplified products through second-generation high-throughput sequencing, analyze the sequencing data, and evaluate the detected sites according to the detected sites. Compatibility of the primer combinations described above.

In this example, the Zika virus RNA with a known copy number provided by the Hubei Provincial Center for Disease Control and Prevention was reverse-transcribed into cDNA by a commercial reverse transcription kit, and then added to human genomic RNA to prepare 1000 copies/ For the template of the reaction, use the primer combination to screen the primer combination with uniform amplification and optimal compatibility according to the detection of the MNP sites in the 4 libraries, and finally screen out the 15 primer combinations described in Table 1 of the present invention. Primer compositions for MNP loci.

Threshold setting and performance evaluation of Zika virus identified by MNP sites and primers described in Example 2

1. Detection of MNP markers

In this example, the Zika virus RNA with a known copy number provided by the Hubei Provincial Center for Disease Control and Prevention was reverse-transcribed into cDNA by a commercial reverse transcription kit, and then added to human genomic RNA to prepare 1 copy/reaction , 10 copies/reaction and 100 copies/reaction of Zika virus mock samples. At the same time, an equal volume of sterile water was set as a blank control. A total of 4 samples were constructed, and 3 replicate libraries were constructed per day for each sample, and were tested continuously for 4 days, that is, 12 sets of sequencing data were obtained for each sample, as shown in Table 2. According to the number of sequenced fragments and the number of sites of Zika virus MNP sites detected in the blank control and Zika virus nucleic acid standards in 12 repeated experiments, the reproducibility, accuracy and sensitivity of the detection method were evaluated, and the formula was formulated. Thresholds for quality control system contamination and detection of target pathogens. The detection process of MNP markers is shown in Figure 3.

Table 2 - Detection sensitivity and stability analysis of Zika virus MNP labeling method

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

2. Evaluation of the reproducibility and accuracy of the MNP marker detection kit for detecting Zika virus

Based on whether the genotypes of the common detection loci are reproducible in two replicates, the reproducibility and accuracy of the MNP marker detection method for detecting Zika virus were evaluated. Specifically, 12 sets of data of samples with 100 copies/reaction were compared in pairs, and the results are shown in Table 3.

Table 3-Reproducibility and accuracy evaluation of Zika virus MNP marker detection method

It can be seen from Table 3 that the number of MNP loci with differences in the main genotype is 0; according to the principle that the reproducible genotype between two repeated experiments is considered accurate, the accuracy rate a=1-(1-r)/2 =0.5+0.5r, r represents the recurrence rate, that is, the ratio of the number of reproducible loci of the main genotype to the number of common loci. In the reproducibility test of this project, the logarithm of the difference of the main genotype of the MNP marker between different libraries and different database construction batches of each sample is 0, the reproducibility rate r=100%, and the accuracy rate a=100%. Therefore, the kit of the present invention can accurately detect Zika virus as low as 10 copies/reaction.

3. Threshold determination of Zika virus detected by MNP marker detection kit

As shown in Table 2, sequences aligned to Zika virus can be detected in 1 copy/reaction sample, covering at least 1 MNP site. In some blank controls, the sequence of Zika virus was also detected. Due to the extreme sensitivity of the MNP marker detection method, data contamination during the detection process can easily lead to false positives. Therefore, in this example, a quality control plan is formulated, as follows:

1) The amount of sequencing data is greater than 4.5 million bases. The calculation is based on the fact that the number of MNP sites detected in each sample is 15, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 4.5 million bases, most samples can be guaranteed to cover every site in one experiment. The number of sequencing fragments at each point reaches 1000 times, ensuring accurate analysis of the base sequence of each MNP site.

2) According to the signal index S of the Zika virus in the test sample and the noise index P of the Zika virus in the blank control, determine whether the pollution is acceptable, wherein:

Blank control noise index P=nc/Nc, wherein nc and Nc respectively represent the number of sequenced fragments and the total number of sequenced fragments of Zika virus in the blank control.

The signal index of the test sample S=nt/Nt, wherein nt and Nt respectively represent the number of sequenced fragments and the total number of sequenced fragments of Zika virus in the test sample.

3) Calculate the detection rate of MNP marker sites in the test sample, which refers to the ratio of the number of detected sites to the total number of designed sites.

Table 4 - Signal-to-noise ratio of Zika virus in samples to be tested

The results are shown in Table 4, the mean value of the noise index of Zika virus in the blank control is 0.04%, and the mean value of the signal index in the sample of 1 copy is 0.33%, the signal of the sample of 1 copy and the blank control The average value of the noise ratio is 8.25. Therefore, the present invention stipulates 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.

The average signal-to-noise ratio of the samples with 10 copies and the blank control is 80.75, and at least 6 MNP sites can be stably detected in 12 sets of data with 10 copies/reaction, accounting for 40.0% of the total sites. Therefore, in the case of ensuring accuracy, this standard stipulates that the signal-to-noise ratio threshold of Zika virus is 40, that is, when the signal-to-noise ratio of Zika virus in the sample is greater than 40, and the detection rate of the locus is greater than or equal to 30%. , it was determined that the nucleic acid of Zika virus was detected in the sample.

Therefore, the kit provided by the present invention can accurately and sensitively detect Zika virus as low as 10 copies/reaction.

4. Specificity evaluation of MNP marker detection method for detection of Zika virus

Man-made Zika virus and Mycobacterium tuberculosis, Acinetobacter strains, Bordetella pertussis, Bordetella hallii, Chlamydia pneumoniae, Mycoplasma pneumoniae, Epstein-Barr virus, Haemophilus influenzae, varicella zoster virus, Cytomegalovirus, herpes simplex virus, human bocavirus, Klebsiella pneumoniae, Legionella, Moraxella catarrhalis, Pseudomonas aeruginosa, Rickettsia, Staphylococcus aureus, Streptococcus pneumoniae 1. The nucleic acid of Streptococcus pyogenes was mixed together to prepare a mixed template, and a blank template was used as a control, and the Zika virus in the mixed template was detected by the method provided by the present invention, and three repeated experiments were carried out. After sequence alignment and analysis according to the quality control scheme and decision threshold, 15 MNP markers of the Zika virus can be specifically detected in three repeated experiments, indicating that the MNP markers and all The above kit can detect target microorganisms with high specificity in complex templates.

Example 3, Detection of Genetic Variation Between Zika Virus Strains

Using the kit and MNP marker site detection method to detect 6 progeny strains of the Zika virus strain provided by Hubei Provincial Disease Control and Prevention, the samples were named S1-S6 in sequence, and the average sequencing coverage of each sample Up to 1416 times, each strain can detect all 15 MNP markers (Table 5). The fingerprints of the 6 strains were compared in pairs, and the results are shown in Table 5. There is 1 copy (S-2) and 5 copies of Zika virus detected together with the same batch. There are 5 major genes marked by MNP Type differences (Table 5), indicating the existence of inter-strain variation.

Table 5-6 Detection analysis of Zika virus

As can be seen from Table 5, the application of the kit of the present invention to identify genetic variation between strains by detecting MNP markers can be used to ensure the genetic consistency of the same named Zika virus strain in different laboratories, thereby ensuring the comparability of research results, This is of great significance for the scientific research of Zika virus. In clinical practice, the diagnostic scheme can be considered according to whether the difference site affects drug resistance.

Example 4. Detection of genetic variation within Zika virus strains

As a group organism, some individuals within the Zika virus group mutate, making the group no longer homozygous and forming a heterogeneous heterozygous group, which affects the stability and consistency of the microbial phenotype, especially in the test. This variant appears as an allelic type outside the main genotype of the locus when the population is tested for molecular markers. When the variant individual has not yet accumulated, it only accounts for a very small part of the population, showing a low frequency allele type. Low-frequency allele types are often mixed with technical errors, making them difficult to distinguish with existing techniques. The present invention detects highly polymorphic MNP markers. The technical error rate of MNP markers is significantly lower than that of SNP markers, based on the fact that multiple errors are less likely to occur simultaneously than one error.

The authenticity evaluation of the secondary allelic type in this embodiment is carried out as follows: first, the allelic type with strand preference (ratio of the number of sequenced sequences covered on the RNA double strand) is excluded according to the following rules: strand preference is greater than 10 times, or more than 5 times different from the strand preference of the main allele type.

The authenticity of genotypes without strand preference was determined based on the number and proportion of sequenced sequences in Table 6. Table 6 lists the calculations based on the BINOM.INV function under the probability guarantee of α=99.9999%, when e _max (n=1) and e _max (n≥2) are 1.03% and 0.0994%, respectively, in each site The critical value of the number of sequencing sequences of the minor allele type, only when the number of sequencing sequences of the minor allele type exceeds the critical value, it is determined as the real minor allele type. When there are multiple candidate minor alleles, the P value of each candidate allele type is multiple-corrected, and the candidate alleles with FDR<0.5% are judged to be true minor allele types.

The parameters e _max (n=1) and e _max (n≥2) involved in Table 6 refer to the highest ratio of the number of sequenced sequences carrying the wrong alleles of n SNPs to the total number of sequenced sequences at this site. e _max (n=1) and e _max (n≥2) of 1.03% and 0.0994%, respectively, were obtained from the frequencies of all minor alleles detected at 930 homozygous MNP loci.

Table 6-Critical value for judging minor allelic genotypes under partial sequencing depth

According to the above parameters, the nucleic acids of the two strains with different genotypes are divided into the following 8 ratios: 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100, 7 /100 mixed to prepare artificial heterozygous samples, each sample was detected 3 times, and a total of 24 sequencing data were obtained. Through accurate comparison with the genotypes of the MNP sites of the two strains, the sites with heterozygous genotypes were detected in 24 artificial heterozygous samples, which shows that the MNP of the Zika virus developed Applicability of marker detection methods to detect genetic variation within strain populations.

Embodiment 5, the construction of Zika virus MNP fingerprint database

The RNA of all strains or samples used to construct the Zika virus MNP fingerprint database was extracted by conventional CTAB method and commercial kits, and the quality of RNA was detected by agarose gel and ultraviolet spectrophotometer. After comparing the sequencing data of the above six strains with the reference genotype, the main genotype of each locus of each strain was obtained to form the MNP fingerprint of each strain. The obtained MNP fingerprint of each virus strain is entered into the database file to form the Zika virus RNA fingerprint database.

The constructed MNP fingerprint database is based on the gene sequences of the detected strains, so it is compatible with all high-throughput sequencing data, and has the characteristics of being completely co-constructed, shared, and updateable at any time. Compare the MNP fingerprints of the strains obtained for each detection with the MNP fingerprint database constructed based on existing genomic data, and enter the MNP fingerprints of strains with differences in main genotypes into the constructed MNP fingerprint database to reach the database. Real-time updates and co-construction and sharing.

Embodiment 6, the application in Zika virus fine typing

First construct the reference sequence library of Zika virus, which is composed of the genome sequence of Zika virus that has been disclosed and the fingerprint database of Zika virus that has been constructed; Utilize the primer combination and MNP marker site detection method described in Example 2 to obtain The MNP fingerprint of Zika virus in each sample to be tested; the RNA fingerprint of each strain is compared with the reference sequence library constructed, and the strain with the closest genetic distance to the sequence library is obtained by screening; and the existing virus If the genotypes of the strains are 100% identical, it is an existing variant, and if there is a main genotype difference at at least one MNP site, it is a new variant, so as to realize the fine typing of Zika virus. The typing results of the 6 Zika virus strains in this example are shown in Table 5. The 6 strains can be divided into 2 types, and the strains in the reference sequence library all have more than one main MNP locus. The difference in genotype was determined to be two new mutant strains, and the fine typing of Zika virus was realized.

Finally, it should also be noted that the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, but also Other elements not expressly listed, or inherent to the process, method, article, or apparatus are also included.

Having described preferred embodiments of embodiments of the present invention, additional changes and modifications can be made to these embodiments by those skilled in the art once the basic inventive concept is appreciated. Therefore, the appended claims are intended to be interpreted to cover the preferred embodiment and all changes and modifications which fall within the scope of the embodiments of the present invention.

Apparently, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. In this way, if the 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 equivalent technologies thereof, the embodiments of the present invention are also intended to include these modifications and variations.

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<210> 9

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 9

aacatagttc gtctcaagag tggag 25

<210> 10

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 10

ccagtccccc accatagaga 20

<210> 11

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 11

gaggctggag ctctgatcac 20

<210> 12

<211> 26

<212>DNA

<213> Artificial Sequence

<400> 12

tctcgtcact gtatagataa gggaag 26

<210> 13

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 13

taccggataa tgctatcagt gcat 24

<210> 14

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 14

caagcttcca aaacctccca ag 22

<210> 15

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 15

cgctcccttc tcactctaca ag 22

<210> 16

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 16

ccaggcaatg gcaacggc 18

<210> 17

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 17

gaagaccatg cacactggct 20

<210> 18

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 18

gtcttccttt gctccgtcct aa 22

<210> 19

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 19

ctggagctct agaggctgag at 22

<210> 20

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 20

agctgggacc ttggtgaatg 20

<210> 21

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 21

agaaccaccc atataggaca tgg 23

<210> 22

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 22

tgtcggtcat ggctattcct 20

<210> 23

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 23

gggggacgtc aagcagga 18

<210> 24

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 24

ttgaatattc caggcagagt ctgaa 25

<210> 25

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 25

tgataacatc accttggcaa tcct 24

<210> 26

<211> 25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

aatggtaagt tcttcttcac actgc 25

<210> 27

<211> 19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ggtgctcggt ggacttctc 19

<210> 28

<211> 18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cctcttccca agcctgct 18

<210> 29

<211> 23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ggtcatagat tccaggagat gcc 23

<210> 30

<211> 25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

atgtactcat ctccaggtt gttgg 25

Claims (7)

1. The multiplex PCR primer composition for detecting the Zika virus 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 zika virus, said kit 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 the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 for qualitative detection of a non-diagnostic purpose of a zika virus and for preparing a product for qualitative detection of a zika virus.

5. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 for detecting genetic variation within and among strains of a zika virus.

6. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 in constructing a database of a card virus.

7. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 in the detection of the split type of the Zika virus.

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