CN114107562B - MNP (human parainfluenza virus) marker locus, primer composition, kit and application of MNP marker locus - Google Patents

MNP (human parainfluenza virus) marker locus, primer composition, kit and application of MNP marker locus Download PDF

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CN114107562B
CN114107562B CN202111329187.6A CN202111329187A CN114107562B CN 114107562 B CN114107562 B CN 114107562B CN 202111329187 A CN202111329187 A CN 202111329187A CN 114107562 B CN114107562 B CN 114107562B
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肖华锋
李论
彭海
周俊飞
高利芬
李甜甜
陈利红
方治伟
万人静
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Abstract

The invention discloses a MNP (MNP) marker locus of human parainfluenza virus, a primer composition, a kit and application thereof, wherein the MNP marker locus refers to a genome region which is screened on a human parainfluenza 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 human parainfluenza viruses and monitor variation; 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 human parainfluenza viruses of large-scale samples, and the method has important significance on scientific research and epidemic prevention monitoring of the human parainfluenza viruses.

Description

MNP (human parainfluenza virus) marker locus, 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 (human parainfluenza virus) marking site, a primer composition, a kit and application thereof.
Background
Human parainfluenza virus (human_parainfluenza_virus) is a single-stranded enveloped RNA virus of the paramyxoviridae family, divided into 4 subtypes, which are common and easily ignored pathogens of respiratory tract infections. The human parainfluenza virus can cause upper respiratory tract infection such as cold and sore throat, and lower respiratory tract diseases such as pneumonia, bronchitis, bronchiolitis, and the like, especially in the elderly and people with immunodeficiency. The main transmission route is the transmission of droplets through the respiratory tract, or the infection occurs by a "self-inoculation" mode from hand to nose after a susceptible person contacts with infectious secretions. At present, no effective vaccine is available for preventing human parainfluenza virus infection, and the clinical manifestations are various, the etiology manifestations are similar to other common respiratory tract infections, and the clinical manifestations are not easy to identify. Therefore, the rapid and accurate detection of human parainfluenza virus has important significance for timely diagnosis of etiology, early detection and early treatment, and reduction of disease deterioration. In addition, human parainfluenza virus is used as a group organism, and individuals in the group can be mutated in interaction with a host and the environment, so that a 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 method for detecting and analyzing human parainfluenza virus has important significance for clinical treatment, epidemic prevention detection and scientific research of human parainfluenza virus.
Classical human parainfluenza virus detection methods, including isolation 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 detection variation, 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 a large amount of data waste and background noise caused by sequencing of a whole genome and a metagenome, and has the advantages of small sample requirement, accurate diagnosis result, data quantity saving and low-frequency variation detection.
The molecular markers detected by the existing targeted detection technology mainly comprise SNP 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 of human parainfluenza virus and a detection technology thereof become a technical problem to be solved.
Disclosure of Invention
The invention aims to provide a MNP (MNP) marking site of human parainfluenza virus, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on the human parainfluenza virus and have the effects of high flux, high accuracy, high specificity, high sensitivity and accurate 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 MNP marker locus for a human parainfluenza virus, the MNP marker locus being specific for a species selected on the genome of the human parainfluenza virus and having a plurality of nucleotide polymorphisms within the species, comprising: marking sites of MNP-1, MNP-7-MNP-9 and MNP-14 of the reference genome by KF 530250.1; marker loci of MNP-2 and MNP-10-MNP-11 of the reference genome with AF 533012.1; marking loci of MNP-3-MNP-6 and MNP-13 of a reference genome by using JQ 901980.1; marker loci for MNP-12 and MNP-15 of the reference genome with KY 674953.1.
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 nucleotide sequences of the 15 pairs of primers being shown as 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 the human parainfluenza 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 human parainfluenza virus or said multiplex PCR primer composition or said detection kit for the identification of human parainfluenza virus for non-diagnostic purposes.
In a fifth aspect of the invention, there is provided the use of said MNP marker locus of human parainfluenza virus or said multiplex PCR primer composition or said detection kit for detecting genetic variation within and between human parainfluenza virus strains.
In a sixth aspect of the invention, there is provided the use of said MNP marker locus of human parainfluenza virus or said multiplex PCR primer composition or said detection kit for the construction of a human parainfluenza virus database.
In a seventh aspect of the invention, there is provided the use of said MNP marker locus of human parainfluenza virus or said multiplex PCR primer composition or said detection kit in the genotyping detection of human parainfluenza virus.
In the above application of human parainfluenza virus identification, intra-strain and inter-strain genetic variation, database construction and typing detection, the operation steps comprise: firstly, obtaining virus total RNA of a sample to be detected, and carrying out reverse transcription into cDNA by using a commercialized 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 result is compared with the reference sequence of the human parainfluenza virus, and the number of detection sequences and genotype data of the cDNA are obtained. And carrying out data quality control and data analysis on the sequencing data of the total cDNA according to the number of human parainfluenza virus sequencing sequences obtained from the cDNA and the blank control and the number of detected MNP sites, and obtaining the number of detected MNP sites, the number of sequencing sequences covering each MNP site and the MNP site genotype data.
When the method is used for identifying the human parainfluenza virus, whether the sample to be detected contains nucleic acid of the human parainfluenza virus or not is judged after quality control according to the number of sequencing sequences of the human parainfluenza virus detected in the sample to be detected and the blank control and the number of MNP sites detected. The quality control scheme and the judging method are characterized in that RNA of human parainfluenza viruses with known copy numbers is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the human parainfluenza viruses are evaluated, and the quality control scheme and the judging method when the kit detects the human parainfluenza viruses are formulated.
RNA, when used in human parainfluenza virus genetic variation detection, 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 human parainfluenza virus MNP fingerprint database, genotype data of the MNP locus of the human parainfluenza virus identified from a sample is input into a database file to form the MNP fingerprint database of the human parainfluenza virus; each time a different sample is identified, comparing the sample with the MNP fingerprint database of the human parainfluenza virus, identifying whether the human parainfluenza virus in the sample has a difference of main genotype (the genotype supported by more than 50% of sequencing fragments at one MNP site) with strains in the database at the MNP sites, wherein the human parainfluenza virus with the main genotype difference at least 1 MNP site is a new mutation type, and recording the new mutation type in the MNP fingerprint database.
When the method is used for human parainfluenza virus typing detection, the human parainfluenza virus in a sample to be detected is identified, and the genotype of each MNP locus is obtained; collecting genome sequences of human parainfluenza viruses disclosed on the internet and a constructed human parainfluenza virus MNP fingerprint database to construct a human parainfluenza virus reference sequence library; and comparing the genotype of the human parainfluenza virus in the sample to be detected with a reference sequence library of the human parainfluenza virus. And identifying whether the human parainfluenza virus in the sample is an existing strain type or a new variant strain type according to the comparison result with the reference sequence library, and realizing the fine typing of the human parainfluenza virus.
RNARNARNA the invention is initiated in the field of human parainfluenza viruses 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 human parainfluenza virus species and conserved in sequence at two sides can be mined according to the reported resequencing data of the human parainfluenza virus representative race; 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 MNP (MNP) marking sites of human parainfluenza virus, a primer composition, a kit and application thereof, wherein the provided 15 MNP sites of human parainfluenza virus and the primer composition thereof can be used for multiplex PCR (polymerase chain reaction) amplification, and a second generation sequencing platform is fused for sequencing an amplification product, so that the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on the human parainfluenza virus are met, and the requirements of human parainfluenza virus standard and sharable fingerprint data construction are met; the need to accurately detect genetic variation between human parainfluenza virus strains; the requirements of homozygosity and heterozygosity of human parainfluenza viruses are identified, and technical support is provided for scientific research, scientific monitoring and prevention and control of human parainfluenza viruses.
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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 for MNP marker loci of human parainfluenza viruses;
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 invention develops a novel molecular marker-MNP marker specific to species. 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 The seed alleles, which are higher than SSR and SNP, are suitable for the identification of the 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 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, and a standardized database can be constructed for carrying out without parallel experimentsSharing and sharing; (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.
Thus, the present invention developed MNP marker loci for human parainfluenza virus that are genomic regions screened on the human parainfluenza virus genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including MNP-1-MNP-15 marker loci that reference the sequences identified in Table 1.
Next, the present invention developed a multiplex PCR primer composition for the MNP marker loci of human parainfluenza viruses, comprising 15 pairs of primers, the nucleotide sequences of the 15 pairs of primers being 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 the MNP marker locus of the human parainfluenza virus.
The kit provided by the invention can sensitively detect 10 copies/reaction of human parainfluenza virus.
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 detect high specificity of target microorganisms in complex templates.
The MNP marker loci, primer compositions, kits and uses thereof of one of the human parainfluenza viruses of the present application will be described in detail below in conjunction with examples, comparative examples and experimental data.
Example 1 screening of human parainfluenza MNP marker loci and design of multiplex PCR amplification primers
S1, screening of MNP (human parainfluenza virus) marker loci
Based on complete or partial sequences of genomes of different isolates of the 3794 human parainfluenza viruses disclosed on the net, 15 MNP (MNP) marker loci 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 human parainfluenza virus 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 human parainfluenza virus;
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.
S3, evaluating detection efficiency of primer combination
The RNA quality control product (goods number: VIP (VC) 105) of the purchased human parainfluenza virus with known copy number is used, is subjected to reverse transcription into cDNA by a commercial reverse transcription kit, is added into human genome DNA to prepare a 1000-copy/reaction template, and is detected by the MNP labeling detection method by using the primer combination. 4 repeated sequencing libraries were constructed, and the primer combinations of the 15 MNP sites described in Table 1 of the present invention were finally selected by screening for primer combinations of uniform amplification and optimal compatibility according to the detection conditions of MNP sites in the 4 libraries.
Example 2 MNP site and primer identification threshold settings and Performance assessment of human parainfluenza Virus
1. Detection of MNP markers
In this example, a human parainfluenza virus RNA quality control (cargo number: VIP (VC) 105) with a known copy number was reverse transcribed into cDNA using a commercial reverse transcription kit, and then added to human genomic DNA to prepare 1-, 10-, and 100-copy/reaction human parainfluenza virus simulated 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 human parainfluenza virus MNP sites detected in the blank control and human parainfluenza virus simulation samples in 12 repeated experiments, evaluating the reproducibility, accuracy and sensitivity of the detection method, and formulating thresholds for quality control system pollution and target pathogen detection. The detection flow of MNP markers is shown in fig. 3.
1. Sensitivity and stability assessment of MNP marker detection kit for detecting human parainfluenza virus
As shown in Table 2, the kit can stably detect 1-3 MNP sites in a 1-copy/reaction sample, 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.
TABLE 2 detection sensitivity and stability analysis of MNP markers of human parainfluenza virus
2. Reproducibility and accuracy assessment of MNP marker detection kit for detecting human parainfluenza virus
Based on whether the genotype of the co-detected site is reproducible in the two replicates, the reproducibility and accuracy of detection of human parainfluenza virus by the MNP marker detection method is evaluated. Specifically, the data of 12 sets of 100 copies/reaction samples were compared in pairs, and the results are shown in Table 3.
TABLE 3 reproducibility and accuracy assessment of human parainfluenza MNP marker detection method
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 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%.
3. Threshold value judgment for detecting human parainfluenza virus by MNP (MNP) mark detection kit
Sequences aligned to human parainfluenza virus can be detected in 1 copy/reaction samples, covering at least 1 MNP site. Human parainfluenza virus sequences were also detected in partial blank, as shown in Table 2. 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 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 human parainfluenza virus in the test sample and the noise index P of human parainfluenza virus in the blank, wherein:
the placebo noise figure p=nc/Nc, where Nc and Nc represent the number of sequenced fragments and total sequenced fragment number, respectively, of human parainfluenza virus in the placebo.
3) The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments of human parainfluenza virus and the total number of sequenced fragments, respectively, in the test sample.
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 human parainfluenza Virus in samples to be tested
The results are shown in Table 4, where the average noise figure of human parainfluenza virus in the blank is 0.03%, the average signal figure in the 1-copy sample is 0.27%, and the average signal to noise ratio of the 1-copy sample and the blank is 9.3, thus the present invention provides that when the signal to noise ratio is greater than 10 times, it can be determined that the contamination in the test system is acceptable.
The average signal to noise ratio of the 10 copies of the sample and the blank was 81.1, and at least 6 MNP sites were stably detected in the 10 copies/reaction 12 sets of data, accounting for 40.0% of the total sites. Therefore, under the condition of ensuring the accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold value of the human parainfluenza virus is 40, namely when the signal-to-noise ratio of the human parainfluenza virus in the sample is more than 40 and the site detection rate is more than or equal to 30 percent, the nucleic acid of the human parainfluenza virus is judged to be detected in the sample.
Therefore, the kit provided by the invention can sensitively detect 10 copies/reaction of human parainfluenza virus.
4. MNP marker detection method for detecting specificity evaluation of human parainfluenza virus
Human parainfluenza virus, coronavirus, human rhinovirus, measles virus, enterovirus, influenza virus, mycobacterium tuberculosis, staphylococcus aureus, acinetobacter strain, pertussis baud bacteria, huo Shibao teryle, chlamydia pneumoniae, mycoplasma pneumoniae, haemophilus influenzae, EB virus, varicella zoster virus, cytomegalovirus, herpes simplex virus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and streptococcus pyogenes are artificially mixed together to prepare a mixed template, the mixed template is used as a reference, and the human parainfluenza virus in the mixed template is detected by using the method provided by the invention and subjected to 3 repeated experiments. After sequence comparison and analysis according to the quality control scheme and the judgment threshold, 15 MNP sites of the human parainfluenza 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 human parainfluenza Virus strains
The kit is used for detecting 6 copies of 1 human parainfluenza virus strain provided by the disease prevention control center of Hubei province, samples are sequentially named as S1-S6, the average coverage of sequencing of each sample is 3110 times, and all 15 MNP markers can be detected for each strain (table 5). The fingerprints of 6 strains were aligned pairwise, and the results are shown in Table 5, where 1 human parainfluenza virus detected together with the same lot had a difference in the major genotypes at 10 MNP sites (Table 5), and there was an inter-strain variation.
TABLE 5-6 detection and analysis of human Paraffin Virus
The application of the kit for identifying the genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of the same named human parainfluenza virus strains in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of human parainfluenza viruses. 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 human parainfluenza Virus strains
As a group organism, the human parainfluenza virus group has variation of partial individuals inside, 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 RNA 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.
TABLE 6-threshold for determining the hypo-isogenotypes at partial sequencing depth
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.
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 MNP marker detection method for human parainfluenza viruses in detecting genetic variation inside a strain population is demonstrated.
Example 5 construction of human parainfluenza Virus MNP fingerprint database
Extracting RNA of all strains or samples for constructing a human parainfluenza virus 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 sequence of the sequencing data of the 6 strains to obtain the main genotype of each site of each strain, thereby forming the MNP fingerprint of each strain. The MNP fingerprint of each strain is compared with an MNP fingerprint database constructed based on the existing genome data, and the MNP fingerprint of the strain with the main genotype difference is input into the constructed 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.
Example 6 use in human parainfluenza Virus typing
Detecting human parainfluenza viruses in samples to be detected by using the kit, and obtaining MNP fingerprints of the human parainfluenza viruses in each sample; constructing a reference sequence library consisting of the genome sequence of the disclosed human parainfluenza virus and the MNP fingerprint database of the constructed human parainfluenza virus; comparing MNP fingerprint of human parainfluenza virus of each sample with a constructed reference sequence library, identifying the MNP fingerprint as a very similar strain with the same reference sequence, identifying the MNP fingerprint as a new variant strain with main genotype difference at more than one MNP locus, and realizing the fine typing of the human parainfluenza virus.
Detection of 6 human parainfluenza virus samples as shown in table 5, the detected 6 human parainfluenza virus S2 and other 5 human parainfluenza virus S2 were different in major genotypes of 10 MNP sites at the 15 MNP sites, and were classified into 2 types, which are similar to type I and type III human parainfluenza virus sequences, respectively, and may be error-marked events during the experiment. Therefore, the resolution of the method for human parainfluenza virus reaches the level of single base, and the method can realize the fine typing of the human parainfluenza virus in the 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.
Sequence listing
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Claims (7)

1. A multiplex PCR primer composition for detecting human parainfluenza virus, 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 human parainfluenza virus, 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 the identification of human parainfluenza virus for non-diagnostic purposes.
5. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for the detection of genetic variation within and between strains of human parainfluenza virus for non-diagnostic purposes.
6. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2-3 for the construction of a human parainfluenza virus database.
7. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2-3 for the genotyping detection of human parainfluenza virus for non-diagnostic purposes.
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