CN114107562A - MNP (MNP marker locus) of human parainfluenza virus, primer composition, kit and application of MNP marker locus - Google Patents

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

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CN114107562A
CN114107562A CN202111329187.6A CN202111329187A CN114107562A CN 114107562 A CN114107562 A CN 114107562A CN 202111329187 A CN202111329187 A CN 202111329187A CN 114107562 A CN114107562 A CN 114107562A
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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 an MNP marker site of human parainfluenza virus, a primer composition, a kit and application thereof, wherein the MNP marker site refers to a genome region which is screened on the genome of the human parainfluenza virus, is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker sites of MNP-1-MNP-15; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 30. The MNP marker site can specifically identify the human parainfluenza virus and monitor variation; the primers are not interfered with each other, and by integrating multiple amplification and sequencing technologies, sequence analysis can be performed on all marked sites of multiple samples at one time, so that the kit has the detection advantages of high throughput, multiple targets, high sensitivity, high accuracy and no culture, can be applied to identification and genetic variation detection of human parainfluenza viruses of large-scale samples, and has important significance on scientific research and epidemic prevention monitoring of the human parainfluenza viruses.

Description

MNP (MNP marker locus) of human parainfluenza virus, 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) marker site of human parainfluenza virus, 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, and is a common respiratory infection pathogen that is easily overlooked. Human parainfluenza viruses can cause not only upper respiratory tract infections such as colds, sore throats, etc., but also lower respiratory tract diseases such as pneumonia, bronchitis, bronchiolitis, etc., particularly in the elderly and in people with immunodeficiency. The main transmission route is droplet transmission through respiratory tract, or infection occurs through self-inoculation from hand to nose after the susceptible person is exposed to infectious secretion. At present, no effective vaccine is used for preventing human parainfluenza virus infection, the clinical manifestations of the vaccine are various, the etiological manifestations of the vaccine are similar to those of other common respiratory tract infections, and the clinical manifestations are difficult to identify. Therefore, the rapid and accurate detection of the human parainfluenza virus has important significance for diagnosing the cause of the disease in time, finding early treatment and reducing disease deterioration. In addition, human parainfluenza virus is taken as a group organism, and in the interaction with a host and the environment, the individual in the group can generate variation, so that the detection or treatment method is ineffective; for experimental studies, such inconspicuous variations can result in different laboratories or the same laboratory differing in the fact that identically named strains are different at different times, resulting in non-reproducible and incomparable experimental results. Therefore, the development of a rapid, accurate and mutation-monitoring human parainfluenza virus detection and analysis method is of great significance to clinical treatment, epidemic prevention detection and scientific research of human parainfluenza virus.
The classical human parainfluenza virus detection method comprises isolation culture, PCR technology, whole genome and metagenome sequencing and the like, and has one or more limitations in the aspects of time length, operation complexity, detection flux, accuracy and sensitivity of detection variation, cost and the like. The targeted molecular marker detection technology combining the ultra-multiplex PCR amplification and the high-throughput sequencing can be used for enriching target microorganisms in a sample with low microorganism content in a targeted manner, avoids a large amount of data waste and background noise caused by whole genome and metagenome sequencing, and has the advantages of less 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 (single nucleotide polymorphism) markers and SSR (simple sequence repeat) markers. SSR markers are generally accepted as the most polymorphic markers, but are few in microorganisms; the SNP markers are large in number, densely distributed and are binary markers, and the polymorphism of a single SNP marker is insufficient to capture the potential allelic diversity in a microbial population. Therefore, the development of novel molecular markers for human parainfluenza virus and techniques for detecting the same has become a technical problem to be solved.
Disclosure of Invention
The invention aims to provide an MNP (MNP marker locus) of human parainfluenza virus, a primer composition, a kit and application thereof, which can perform 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 purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a MNP marker site of human parainfluenza virus, said MNP marker site being a species-specific genomic region selected on the genome of human parainfluenza virus and having a plurality of nucleotide polymorphisms within a species, comprising: KF530250.1 is used as a marker locus of MNP-1, MNP-7-MNP-9 and MNP-14 of a reference genome; using AF533012.1 as a marker locus of MNP-2 and MNP-10-MNP-11 of a reference genome; marker loci of MNP-3 to MNP-6 and MNP-13 with JQ901980.1 as reference genome; KY674953.1 was used as a marker site for MNP-12 and MNP-15 of the reference genome.
In the above technical solution, the labeling sites of MNP-1 to MNP-15 are specifically shown in table 1 of the specification, and the starting and ending positions of the MNP label marked in table 1 are determined based on the reference sequence corresponding to the same row of MNP in table 1.
In a second aspect of the invention, a multiplex PCR primer composition for detecting the MNP marker locus is provided, and the multiplex PCR primer composition comprises 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.
In the above technical scheme, the primer of each MNP marker site includes an upper primer and a lower primer, which are specifically shown in table 1 of the specification.
In a third aspect of the invention, a detection kit for detecting the MNP marker site of human parainfluenza virus is provided, which comprises the primer composition.
Further, the kit also comprises a multiplex PCR premix.
In the fourth aspect of the invention, the MNP marker site of the human parainfluenza virus, the multiplex PCR primer composition or the detection kit is provided for the identification of the human parainfluenza virus with non-diagnostic purposes.
In the fifth aspect of the invention, the MNP marker site of the human parainfluenza virus, the multiplex PCR primer composition or the detection kit is provided for detecting the genetic variation inside and among human parainfluenza virus strains.
In the sixth aspect of the invention, the application of the MNP marker site of the human parainfluenza virus, the multiplex PCR primer composition or the detection kit in constructing a human parainfluenza virus database is provided.
In the seventh aspect of the invention, the MNP marker site of human parainfluenza virus or the multiplex PCR primer composition or the detection kit is provided for use in human parainfluenza virus typing detection.
In the above applications of identification of the human parainfluenza virus, genetic variation inside and among strains, database construction, and typing detection, the operation steps include: firstly, obtaining the virus total RNA of a sample to be detected, and carrying out reverse transcription to obtain cDNA by an industrialized kit; performing a first round of multiplex PCR amplification on the cDNA and a blank control by using the kit disclosed by the invention, wherein the cycle number is not higher than 25; after purifying the amplification product, adding a sample label based on the second round of PCR amplification and a second-generation sequencing adaptor; purifying and quantifying the second round amplification product; when detecting a plurality of strains, performing high-throughput sequencing by equivalently mixing the amplification products of the second round; and comparing the sequencing result with the reference sequence of the human parainfluenza virus to obtain the number and genotype data of the detected sequence of the cDNA. And performing data quality control and data analysis on the sequencing data of the total cDNA according to the quantity of the sequencing sequences of the human parainfluenza virus obtained from the cDNA and the blank control and the quantity of the detected MNP sites to obtain the quantity of the detected MNP sites, the quantity of the sequencing sequences covering each MNP site and the MNP site genotype data.
When the method is used for identifying the human parainfluenza virus, whether the nucleic acid of the human parainfluenza virus is contained in the sample to be detected or not is judged after quality control is carried out according to the sequencing sequence number of the human parainfluenza virus detected in the sample to be detected and blank control and the number of the detected MNP sites. The quality control scheme and the determination method are characterized in that RNA of human parainfluenza virus with known copy number is used as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the human parainfluenza virus are evaluated, and the quality control scheme and the determination method when the kit is used for detecting the human parainfluenza virus are established.
When the RNA is used for detecting the genetic variation of the human parainfluenza virus, the detection of the genetic variation among strains and in the strains is included. The detection of genetic variation among strains comprises the steps of obtaining genotype data of strains to be compared at 15 MNP sites by using the kit and the method. And analyzing whether the major genotypes of the strains to be compared on the 15 MNP sites have difference or not through genotype comparison. If the strains to be compared have variation in the major genotype of at least one MNP site, the strains are judged to have genetic variation. As an alternative, 15 sites of the strains to be compared can be amplified respectively through single PCR, and then the amplification products are subjected to Sanger sequencing, and after the sequences are obtained, the genotypes of all MNP sites of the strains to be compared are compared. If there are MNP sites that are not identical in major genotypes, there is variation between the strains to be compared. When detecting the genetic variation in the strain, judging whether a secondary genotype other than the main genotype is detected at the MNP site of the strain to be detected or not through a statistical model. And if the to-be-detected strain has a minor genotype at least one MNP site, judging that the genetic variation exists in the to-be-detected strain.
When the method is used for constructing the MNP fingerprint database of the human parainfluenza virus, the genotype data of the MNP locus of the human parainfluenza virus identified from a sample is recorded into a database file to form the MNP fingerprint database of the human parainfluenza virus; when different samples are identified, the identification is carried out by comparing the sample with the MNP fingerprint database of the human parainfluenza virus, whether the human parainfluenza virus in the sample has the difference of the major genotypes (more than 50 percent of genotypes supported by a sequencing fragment at one MNP site) at the MNP site with the strains in the database is identified, the human parainfluenza virus with the difference of the major genotypes at least 1 MNP site is a new variant type, and the new variant type is included in the MNP fingerprint database.
When the method is used for typing detection of the human parainfluenza virus, the human parainfluenza virus in a sample to be detected is identified to obtain the genotype of each MNP locus; collecting genome sequences of the human parainfluenza viruses disclosed on the Internet and a constructed MNP fingerprint database of the human parainfluenza viruses 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 the reference sequence library of the human parainfluenza virus. And identifying whether the human parainfluenza virus in the sample is the existing strain type or the new variant strain type according to the comparison result with the reference sequence library, thereby realizing the fine typing of the human parainfluenza virus.
RNARNARNA the invention belongs to the initiative in the field of human parainfluenza virus, and has no related literature report; MNP markers are mainly developed based on reference sequences, and MNP sites which are largely distinguished from other species, are polymorphic in human parainfluenza virus species and are conserved in sequences at two sides can be mined according to reported re-sequencing data of small species represented by human parainfluenza virus; MNP locus detection primers suitable for multiplex PCR amplification can be designed through conserved sequences on two sides of the MNP locus; and then according to the test result of the standard product, a primer combination and a detection kit with the maximum polymorphism, high specificity, the best compatibility and the set of MNP sites can be screened out.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the invention provides MNP (MNP marker locus) of human parainfluenza virus, a primer composition, a kit and application thereof, wherein the 15 MNP loci of the human parainfluenza virus and the primer composition thereof can be subjected to multiplex PCR (polymerase chain reaction) amplification, a second-generation sequencing platform is fused for sequencing an amplification product, the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on the human parainfluenza virus are met, and the requirements of standard and sharable fingerprint data construction of the human parainfluenza virus are met; the need to accurately detect genetic variation among human parainfluenza virus strains; the method identifies the homozygosis and heterozygosis requirements of the human parainfluenza virus, and provides technical support for scientific research, scientific monitoring and prevention and treatment of the human parainfluenza virus.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of the screening and primer design of the MNP marker site of human parainfluenza virus;
FIG. 3 is a flow chart of detection of MNP marker sites.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the embodiments of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that the present embodiments and examples are illustrative of the present invention and are not to be construed as limiting the present invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
the invention develops a novel molecular marker-MNP marker specific to species. MNP markers refer to polymorphic markers caused by multiple nucleotides over a region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages:(1) abundant alleles, 2 at a single MNP locusnSpecies alleles, higher than SSR and SNP, are suitable for identification of group organisms; (2) the species distinguishing capability is strong, species identification can be realized only by a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting the MNP label based on the combination of the super-multiplex PCR and the second-generation high-throughput sequencing technology has the following advantages: (1) the output is a base sequence, parallel experiments are not needed, and a standardized database can be constructed for sharing and sharing; (2) the efficiency is high, the sample DNA bar code is utilized, the limitation of the quantity of sequencing samples is broken through, and tens of thousands of MNP sites of hundreds of samples can be typed at one time; (3) the sensitivity is high, multiple targets are detected at one time by utilizing multiple PCR, and high false negative and low sensitivity caused by amplification failure of a single target are avoided; (4) high accuracy, using a second generation high throughput sequencer to sequence the amplification product hundreds of times.
In view of the advantages and the characteristics, the MNP marker and the detection technology MNP marking method thereof can realize the classification and the tracing of the multi-allelic genotypes of the population organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, detection of genetic variation and the like. At present, no report on MNP labeling exists in microorganisms, and corresponding technologies are lacked. The development, screening and application of the MNP marking method have better application foundation in plants.
Accordingly, the present invention has developed MNP marker sites for human parainfluenza virus, which are regions of the genome screened on the human parainfluenza virus genome that are different from other species and have a plurality of nucleotide polymorphisms within the species, including marker sites for MNP-1 to MNP-15 whose reference genome is the sequence identified in Table 1.
Then, the invention develops a multiplex PCR primer composition of the MNP marker locus of the human parainfluenza virus, the multiplex PCR primer composition comprises 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 30. The primers do not conflict with each other, and efficient amplification can be performed by multiplex PCR.
The multiplex PCR primer composition can be used as a detection kit for the MNP marker site of the human parainfluenza virus.
The kit provided by the invention can sensitively detect 10 copies/response of human parainfluenza virus.
In the reproducibility test of the invention, the logarithm of difference of the MNP marking main gene type among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100 percent, and the accuracy a is 100 percent.
The MNP markers and the kits of the invention detect high specificity of target microorganisms in complex templates.
The MNP labeling site, primer composition, kit and application of the human parainfluenza virus of the present application will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1 screening of human parainfluenza virus MNP marker sites and design of multiplex PCR amplification primers
S1 screening of human parainfluenza virus MNP marker sites
Based on the complete or partial genome sequences of 3794 different isolates of human parainfluenza virus disclosed in the network, 15 MNP marker sites are obtained by sequence alignment. For species without genomic data on the net, the genomic sequence information of the representative microspecies of the microbial species to be detected can also be obtained by high-throughput sequencing, wherein the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected marker, the genomic sequence of at least 10 genetically representative isolates is generally used as a reference. The 15 MNP marker sites screened are shown in table 1:
TABLE 1-starting position of the MNP marker site and detection primer on the reference sequence
Figure BDA0003348046640000061
The step S1 specifically includes:
selecting a genome sequence of a representative strain of the human parainfluenza virus as a reference genome, and performing sequence comparison on the genome sequence and the reference genome to obtain single nucleic acid polymorphic sites of each strain of the human parainfluenza virus;
on the reference genome, performing window translation by taking 100-300 bp as a window and 1bp as a step length, and screening to obtain a plurality of candidate MNP site regions, wherein the candidate MNP site regions contain more than or equal to 2 single nucleotide variation sites, and the single nucleotide polymorphism sites do not exist on sequences of 30bp at two ends;
screening a region with the division DP of more than or equal to 0.2 in the candidate polynucleotide polymorphic site region as an MNP marker site; wherein, DP ═ d/t, t is the comparison logarithm of all the minor species in the region of the candidate polynucleotide polymorphic site when compared pairwise, d is the sample logarithm of at least two single nucleic acid polymorphisms that differ in the region of the candidate polynucleotide polymorphic site.
As an optional implementation mode, when the reference genome is screened by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, which is beneficial to comprehensive screening.
S2 design of multiplex PCR amplification primers
And designing the multiplex PCR amplification primers of the MNP sites through primer design software, wherein the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely all the designed primers can be normally amplified in one amplification reaction.
S3 evaluation of detection efficiency of primer combination
The RNA quality control of human parainfluenza virus (cargo number: VIP (VC)) 105 with known copy number purchased is used, after being reverse transcribed into cDNA by a commercial reverse transcription kit, the cDNA is added into human genome DNA to prepare a 1000 copy/reaction template, and the detection is carried out by the MNP mark detection method by using the primer combination. 4 repeated sequencing libraries are constructed, primer combinations with even amplification and optimal compatibility are screened according to the detection condition of MNP sites in the 4 libraries, and finally primer compositions of 15 MNP sites disclosed in the invention table 1 are screened.
Example 2 identification of human parainfluenza Virus by MNP sites and primers threshold settings and Performance evaluation
1. Detection of MNP markers
In this example, human parainfluenza virus RNA quality control samples (cargo number: VIP (VC)) 105 with known copy numbers were reverse-transcribed into cDNA using a commercial reverse transcription kit, and added to human genomic DNA to prepare human parainfluenza virus mock samples of 1 copy/reaction, 10 copies/reaction, and 100 copies/reaction. An equal volume of sterile water was also set as a blank. A total of 4 samples were obtained, each sample was constructed into 3 duplicate libraries each day, and the assay was continued for 4 days, i.e. 12 sets of sequencing data were obtained for each sample, as shown in table 2. According to the sequencing fragment number and the site number of the MNP site of the human parainfluenza virus detected in the blank control and the human parainfluenza virus simulation sample in 12 times of repeated experiments, the repeatability, the accuracy and the sensitivity of the detection method are evaluated, and the threshold values of the pollution of a quality control system and the detection of the target pathogen are set. The flow of detection of MNP markers is shown in figure 3.
1. Sensitivity and stability evaluation of MNP (MNP) marker detection kit for detecting human parainfluenza virus
As shown in Table 2, the kit can stably detect more than 1-3 MNP sites in a sample with 1 copy/reaction, stably detect more than 6 MNP sites in a sample with 10 copies/reaction, and detect 1 MNP site at most in a sample with 0 copy/reaction, can obviously distinguish the samples with 10 copies/reaction and 0 copies/reaction, and has technical stability and detection sensitivity as low as 10 copies/reaction.
TABLE 2 detection sensitivity, stability analysis of MNP labeling method for human parainfluenza Virus
Figure BDA0003348046640000081
2. Evaluation of reproducibility and accuracy of MNP (MNP) marker detection kit for detecting human parainfluenza virus
And evaluating the reproducibility and accuracy of the MNP marker detection method for detecting the human parainfluenza virus based on whether the genotype of the co-detected site can be reproduced in two times of repetition. Specifically, two-by-two comparisons were made for each of 12 sets of data for 100 copies/reaction samples, and the results are shown in Table 3.
TABLE 3 evaluation of reproducibility and accuracy of human parainfluenza virus MNP marker detection methods
Figure BDA0003348046640000082
As can be seen from Table 3, the number of MNP sites differing in major genotypes was 0; the accuracy rate a is 1- (1-r)/2 is 0.5+0.5r, and r represents the reproducibility rate, i.e., the ratio of the number of sites where the major genotype is reproducible to the number of common sites, which is considered to be the principle of accuracy among 2 repeated experiments. In the reproducibility test of the invention, the logarithm of difference of the MNP marking main gene type among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100 percent, and the accuracy a is 100 percent.
3. Threshold determination for detecting human parainfluenza virus by MNP (protein) marker detection kit
In 1 copy/reaction sample, the sequence aligned to human parainfluenza virus can be detected, at least 1 MNP site is covered. The sequences of human parainfluenza virus were also detected in the partial blank control, as shown in Table 2. Due to the extreme sensitivity of MNP marker detection methods, contamination of the data during detection is likely to result in the generation of false positives. Therefore, the following quality control schemes are prepared in this example.
The quality control scheme is as follows:
1) the amount of sequencing data was greater than 4.5 megabases. The measuring and calculating basis is that the number of MNP sites detected by each sample is 15, and the length of a sequencing fragment is 300 bases, so that when the data volume is more than 4.5 million bases, the number of the sequencing fragments covering each site can be ensured to reach 1000 times by one-time experiment of most samples, and the accurate analysis of the base sequence of each MNP site is ensured.
2) Determining whether the contamination is acceptable according to the signal index S of the human parainfluenza virus in the test sample and the noise index P of the human parainfluenza virus in the blank control, wherein:
blank control noise index P ═ Nc/Nc, where Nc and Nc represent the number of sequenced fragments and the total number of sequenced fragments of human parainfluenza virus in the blank control, respectively.
3) And the signal index S of the test sample is Nt/Nt, wherein Nt and Nt respectively represent the number of sequencing fragments of the human parainfluenza virus and the total number of sequencing fragments in the test sample.
And calculating the detection rate of the MNP marker locus in the test sample, which is the ratio of the number of the detected locus to the number of the total design locus.
TABLE 4-Signal to noise ratio of human parainfluenza Virus in samples tested
Figure BDA0003348046640000091
As shown in Table 4, the average value of the noise index of human parainfluenza virus in the blank was 0.03%, while the average value of the signal index in the sample of 1 copy was 0.27%, and the average value of the signal-to-noise ratio of the sample of 1 copy and the blank was 9.3, and thus, the present invention provides that when the signal-to-noise ratio is more than 10 times, contamination in the detection system can be judged to be acceptable.
The average of the signal-to-noise ratios of the 10 copies of the sample and blank was 81.1, and at least 6 MNP sites were stably detected in the 10 copies/reaction of 12 sets of data, accounting for 40.0% of the total sites. Therefore, the present standard stipulates that the signal-to-noise ratio determination threshold for human parainfluenza virus is 40 with accuracy ensured, that is, when the signal-to-noise ratio for human parainfluenza virus in a sample is greater than 40 and the site detection rate is 30% or more, it is determined that nucleic acid of human parainfluenza virus is detected in the sample.
Therefore, the kit provided by the invention can sensitively detect 10 copies/response of human parainfluenza virus.
4. Specific evaluation of MNP marker detection method for detecting human parainfluenza virus
Human parainfluenza virus, coronavirus, human rhinovirus, measles virus, enterovirus, influenza virus, mycobacterium tuberculosis, staphylococcus aureus, acinetobacter strain, bordetella pertussis, bordetella holtzeri, 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, streptococcus pyogenes are artificially mixed together to prepare a mixed template, and after reverse transcription into cDNA, the blank template is used as a control, the method provided by the invention is adopted to detect the human parainfluenza virus in the mixed template, and 3 repeated experiments are carried out. After sequence comparison and analysis according to the quality control scheme and the judgment threshold value, 15 MNP sites of the human parainfluenza virus can be specifically detected in 3 repeated experiments, which shows that the MNP marker and the kit detect the high specificity of the target microorganism in a complex template.
Example 3 detection of genetic variation between human parainfluenza Virus strains
6 copies of 1 human parainfluenza virus strain provided by Hubei province disease prevention and control center are detected by using the kit, samples are sequentially named as S1-S6, the sequencing average coverage multiple of each sample reaches 3110 times, and all 15 MNP markers can be detected from each strain (Table 5). The fingerprints of 6 strains are compared pairwise, and the results are shown in table 5, wherein 1 part of human parainfluenza viruses and 5 parts of human parainfluenza viruses detected together in the same batch have differences in major genotypes of 10 MNP sites (table 5), and virus variation exists among strains.
TABLE 5-6 detection assay for human parainfluenza virus
Figure BDA0003348046640000101
Figure BDA0003348046640000111
The kit can be used for ensuring the genetic consistency of the same named human parainfluenza virus strains in different laboratories by detecting the MNP marker and identifying the genetic variation among the strains, thereby ensuring the comparability of the research result, and having important significance for the scientific research of the human parainfluenza virus. In clinical settings, diagnostic protocols can be considered as to whether differential sites affect drug resistance.
Example 4 detection of genetic variation within human parainfluenza Virus strains
As a population organism, some individuals within the human parainfluenza virus population are mutated so that the population is no longer homozygous, forming a heterogeneous heterozygous population, affecting, inter alia, the stability and consistency of the phenotype of the test microorganism. Such variants exhibit an allelic profile outside the major genotype of the locus when tested for molecular markers in a population. When the variant individuals have not accumulated, they account for a very small fraction of the population and are characterized by a low frequency of alleles. Low frequency alleles tend to be confused with technical errors, making prior art techniques difficult to distinguish. The present invention detects highly polymorphic MNP markers. The technical error rate of MNP labeling is significantly lower than that of SNP labeling, based on the probability of multiple errors occurring simultaneously being lower than the probability of one error occurring.
The authenticity assessment of the sub-allelic genotypes of this example was performed as follows: allelic types with strand bias (ratio of the number of sequencing sequences overlaid on the RNA duplex) were first excluded according to the following rule: the strand preference is greater than 10-fold, or the difference from the strand preference of the dominant allele is greater than 5-fold.
Genotypes without strand preference were judged for authenticity based on the number and proportion of sequences sequenced in table 6. Inv function calculation under a 99.9999% probability guarantee, emax(n-1) and emax(n.gtoreq.2) 1.03% and 0.0994%, respectively, the number of sequenced sequences of the sub-allelic gene in each locus is a critical value, and only when the number of sequenced sequences of the sub-allelic gene exceeds the critical value, the true sub-allelic gene is determined. When multiple candidate sub-alleles are present, multiple corrections are made to the P-value for each candidate allele, FDR<0.5% of the candidate alleles were judged to be true sub-allelic genotypes.
TABLE 6 critical value for determination of sub-allelic genotypes at partial sequencing depth
Figure BDA0003348046640000112
Figure BDA0003348046640000121
Table 6 relates to the parameter emax(n-1) and emax(n.gtoreq.2) means that the highest proportion of the number of sequencing sequences carrying the wrong allele of n SNPs to the total number of sequencing sequences at that site. e.g. of the typemax(n-1) and emax(n.gtoreq.2) 1.03% and 0.0994%, respectively, were obtained from the frequency of all the minor alleles detected at 930 homozygous MNP sites.
Nucleic acids of two strains having differences in genotype were mixed in the following 8 ratios of 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100, 7/100 according to the above parameters to prepare artificial heterozygous samples, and each sample was examined for 3 replicates to obtain a total of 24 sequencing data. By accurately comparing the gene types of the MNP sites of the two strains, the sites with heterozygous gene types are detected in 24 artificial heterozygous samples, thereby demonstrating the applicability of the developed MNP marker detection method of the human parainfluenza virus in detecting the genetic variation in the strain population.
Example 5 construction of a human parainfluenza Virus MNP fingerprint database
Extracting all strains or RNA of a sample for constructing a human parainfluenza virus MNP fingerprint database by using a conventional CTAB method, a commercial kit and other methods, and detecting the quality of the RNA by using agarose gel and an ultraviolet spectrophotometer. And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain a main genotype of each site of each strain, and forming the MNP fingerprint of each strain. The MNP fingerprint of each strain is compared with an MNP fingerprint database constructed on the basis of the existing genome data, and the MNP fingerprints of the strains with the main genotypes having differences are recorded into the constructed MNP fingerprint database. The constructed MNP fingerprint database is based on the gene sequences of detected strains, is compatible with all high-throughput sequencing data, and has the characteristics of complete co-construction and sharing and update at any time.
Example 6 application in human parainfluenza Virus typing
The kit is used for detecting the human parainfluenza virus in the sample to be detected, and the MNP fingerprint of the human parainfluenza virus in each sample is obtained; 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 the MNP fingerprint of the human parainfluenza virus of each sample with a constructed reference sequence library, identifying the MNP fingerprint as a very similar strain when the MNP fingerprint is identical to the established reference sequence, identifying the MNP fingerprint as a new variant strain when the MNP fingerprint has main genotype difference at more than one MNP site, 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 the other 5 human parainfluenza virus samples have different major genotypes at 10 MNP sites of the 15 MNP sites, and are classified into 2 types, which are similar to the human parainfluenza virus sequences of type I and III, respectively, and may be mislabeling events during the experiment. Therefore, the resolution of the method for human parainfluenza virus reaches the level of single base, and the fine typing of the human parainfluenza virus in the sample can be realized.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the embodiments of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to encompass such modifications and variations.
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Claims (8)

1. A MNP marker site of human parainfluenza virus, wherein said MNP marker site is a region of the genome screened on the genome of human parainfluenza virus that is distinct from other species and has a plurality of nucleotide polymorphisms within a species, comprising: KF530250.1 is used as a marker locus of MNP-1, MNP-7-MNP-9 and MNP-14 of a reference genome; using AF533012.1 as a marker locus of MNP-2 and MNP-10-MNP-11 of a reference genome; marker loci of MNP-3 to MNP-6 and MNP-13 with JQ901980.1 as reference genome; KY674953.1 was used as a marker site for MNP-12 and MNP-15 of the reference genome.
2. The multiplex PCR primer composition for detecting the MNP marker site of the human parainfluenza virus as claimed in claim 1, wherein the multiplex PCR primer composition comprises 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID No. 1-SEQ ID No. 30.
3. An assay kit for detecting the MNP marker site of human parainfluenza virus of claim 1, comprising the primer composition of claim 2.
4. The test kit of claim 3, wherein the kit further comprises a multiplex PCR premix.
5. Use of the MNP marker site of human parainfluenza virus of claim 1 or the primer composition of claim 2 or the detection kit of any one of claims 3-4 for the identification of human parainfluenza virus for non-diagnostic purposes.
6. Use of the MNP marker site of human parainfluenza virus of claim 1 or the primer composition of claim 2 or the detection kit of any one of claims 3-4 for detecting genetic variations within and among human parainfluenza virus strains.
7. Use of the MNP marker site of human parainfluenza virus of claim 1 or the primer composition of claim 2 or the detection kit of any one of claims 3-4 for constructing a database of human parainfluenza viruses.
8. Use of the MNP marker site of human parainfluenza virus of claim 1 or the primer composition of claim 2 or the detection kit of any one of claims 3-4 for human parainfluenza virus typing detection.
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