WO2023077490A1 - 一种甲、乙和丙型流感病毒的mnp标记组合、引物对组合、试剂盒及其应用 - Google Patents
一种甲、乙和丙型流感病毒的mnp标记组合、引物对组合、试剂盒及其应用 Download PDFInfo
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Definitions
- the embodiment of the present invention relates to the field of biotechnology, in particular to a combination of MNP markers of influenza A, B and C viruses, a combination of primer pairs, a kit and applications thereof.
- Influenza virus is referred to as influenza virus for short. It is divided into three types: A (A), B (B), and C (C). It can cause infection and disease in various animals such as humans, poultry, pigs, horses, and bats. Pathogens of bird flu, swine flu, equine flu and other human and animal diseases. Influenza viruses are mainly spread through droplets in the air, contact between a susceptible person and an infected person, or contact with contaminated items. Among them, influenza A virus is prone to mutation, and its genetic material contains eight single-stranded negative-strand RNAs such as H and N. The H and N strands are variable regions, so the existing subtypes are mainly classified based on H and N.
- H can be divided into 18 subtypes (H1 ⁇ H18), and N has 11 subtypes (N1 ⁇ N11).
- H1N1, H2N2, and H3N2 mainly infect humans, and the natural hosts of many other subtypes are various birds and animals, and the H5, H7, and H9 subtype strains are the most harmful to poultry.
- influenza viruses infecting humans influenza A virus has a strong variability, followed by influenza B, and the antigenicity of influenza C virus is very stable.
- the high variability of influenza A virus makes it difficult for people to deal with influenza. People cannot accurately predict the subtype of the virus that will be popular, so they cannot carry out targeted preventive vaccination, which seriously threatens human health.
- Existing influenza virus detection techniques include virus isolation and identification, serotype identification, indirect or direct immunofluorescence, and molecular detection techniques for detecting genetic material, including PCR, nucleotide hybridization, and sequencing techniques. These technologies have their own advantages, but there are also one or more limitations in terms of time length, operational complexity, detection throughput, accuracy and sensitivity of detecting variants, and cost. For example, virus isolation and identification are complicated and time-consuming; serotype identification, indirect or direct immunofluorescence methods are prone to cross-reactions, resulting in inaccurate detection and inability to monitor mutations; PCR detection technology is mainly for a single subtype of a virus. It is inefficient to use two markers for detection, and it is easy to cause detection failure due to virus mutation.
- Metagenome sequencing technology is another technology for detecting influenza viruses, but it often includes a large amount of host sequencing data. When testing samples with low viral loads, ultra-deep sequencing is especially required, resulting in high costs. Therefore, the development of rapid, accurate, one-time high-throughput influenza virus detection and analysis methods for the detection of three types of influenza A, B and C is of great importance for the detection of influenza virus, scientific research, fingerprint database construction and epidemic strain detection. significance.
- the invention develops a new type of molecular marker MNP marker, and integrates super multiplex PCR amplification and high-throughput sequencing technology to analyze and detect MNP markers, and can type tens of thousands of MNP markers in hundreds of thousands of samples at one time, realizing the detection of A , high-throughput, sensitive detection and precise typing of influenza B and C viruses, with the advantages of less sample requirements, simultaneous coverage of 3 viruses, accurate diagnostic results, saving data volume, and subtype detection.
- MNP markers refer to polymorphic markers caused by multiple nucleotides in an upper region of the genome. Compared with traditional SSR markers and SNP markers, MNP markers have the following advantages: (1) rich in alleles, there are 2 n alleles on a single MNP marker, 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 labeling method based on super 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 DNA barcodes, breaking through the limitation of the number of sequencing samples, and can type tens of thousands of MNP markers in hundreds of samples at one time; (3) High sensitivity, using multiplex PCR to detect multiple targets at a time, avoiding single Target amplification failures lead 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.
- MNP marker and its detection technology MNP marker method has application potential in the simultaneous identification and typing of multiple viruses, fingerprint database construction, and genetic variation detection.
- MNP marker method has application potential in the simultaneous identification and typing of multiple viruses, fingerprint database construction, and genetic variation detection.
- detection of influenza A, B and C there is no report on MNP markers, and there is a lack of corresponding technology.
- the combination of markers and primers developed in this invention will also be used to formulate national standards for pathogen detection (plan number 20201830-T-469), which will be released by the end of 2021.
- the purpose of the embodiment of the present invention is to provide a MNP marker combination, primer pair combination, kit and application of A, B and C influenza viruses, which can identify and mutate three types of influenza viruses A, B and C at one time Detection, with multi-target, high throughput, high sensitivity and fine typing effects.
- a combination of MNP markers of influenza A, B and C viruses is provided.
- the MNP markers refer to the conserved and differentiated genes that are screened on the genomes of influenza A, B and C viruses respectively. Genomic regions of other species with multiple nucleotide polymorphisms within the species, the MNP marker combination includes 265 markers from MNP-1 to MNP-265 on the reference sequences of influenza A, B and C viruses.
- the multiple PCR primer pair combination includes 265 pairs of primers, and the specific primer sequences are as SEQ ID NO.266- Shown in SEQ ID NO.795, wherein ID NO.266-SEQ ID NO.530 is the upper primer, ID NO.531-SEQ ID NO.795.
- each MNP-labeled primer includes an upper primer and a lower primer, as shown in Table 1 of the specification.
- a detection kit for detecting the combination of MNP markers of influenza A, B and C viruses includes the combination of primer pairs.
- kit also includes a multiplex PCR master mix.
- the MNP marker combination of the A, B and C influenza viruses or the multiple PCR primer pair combination or the detection kit in the A, B and C influenza viruses The application of identification, genetic variation detection, construction of DNA fingerprint database and fine typing.
- the viral total RNA of the sample to be tested Utilize the commercial kit to carry out cDNA synthesis to the total RNA; Utilize the kit of the present invention to carry out the first round of multiplexing of the cDNA and the blank control PCR amplification, the number of cycles is not more than 25; after the amplification product is purified, add sample tags and next-generation sequencing adapters based on the second round of PCR amplification; quantify the second round of amplification products after purification; detection For multiple strains, high-throughput sequencing is performed by mixing the second-round amplification products in equal amounts; the sequencing results are compared to the reference sequences of influenza A, B, and C viruses, and obtained in the cDNA The number of detected sequences and genotype data.
- data quality control and data analysis are performed on the sequencing data of the cDNA, obtained in the The number of MNP markers of influenza A, B and C viruses detected in the sample, the number of sequencing sequences covering each of the MNP markers and the genotype data of the MNP markers.
- the quality control scheme and determination method are based on the detection samples of influenza A, B and C influenza virus nucleotide standards with known copy numbers, and evaluate the ability of the kit to detect influenza A, B and C influenza viruses. Sensitivity, accuracy and specificity, formulate the quality control plan and determination method for the detection of influenza A, B and C by the kit. And in turn as a basis, the influenza virus in the measured sample is detected.
- the detection of genetic variation between strains includes using the above kit and method to obtain the genotype data of 265 part or all of the MNP markers of each strain to be compared. Through genotype comparison, it was analyzed whether there were differences in the main genotypes of the strains to be compared on the 265 commonly detected MNP markers. If there is a variation in at least one main genotype of the MNP marker in the strain to be compared, it is determined that there is a genetic variation between the two.
- the strains it is also possible to amplify part or all of the 265 MNP markers of the strains to be compared by single-plex PCR according to the purpose of the research, and then perform Sanger sequencing on the amplified products.
- the genotypes of common MNP markers detected by the strains were compared for comparison. If there are MNP markers with inconsistent main genotypes, there is variation among the strains to be compared.
- a statistical model is used to determine whether a subgenotype other than the main genotype is detected in the MNP marker of the strain to be tested. If the strain to be tested has subgenotypes in at least one MNP marker, it is determined that there is genetic variation within the strain to be tested.
- A, B and C type influenza virus DNA fingerprint database When being used for constructing A, B and C type influenza virus DNA fingerprint database, will identify from the genotype data of the described MNP mark of A, B and C type influenza virus in the sample, input database file, form A, B respectively and the DNA fingerprint database of influenza virus C; when different samples are identified each time, by comparing with the DNA fingerprint database of influenza A, B and C viruses, whether the influenza A, B and C viruses in the sample are identified There are major genotype differences (genotypes supported by more than 50% sequencing fragments at one MNP marker) in the MNP markers and strains in the database, and there are major genotype differences in at least one MNP marker A, B and Influenza C virus is a new variant type, included in the DNA fingerprint database.
- the present invention When used for type A, B and C influenza virus typing, it is to identify the A, B and C influenza virus in the sample to be tested, and obtain the genotype of each MNP marker. By comparing the genotypes of the influenza A, B and C viruses with the published reference sequence and the existing DNA fingerprint database, identify whether the influenza A, B and C viruses in the sample are existing types or new Type, to accurately type the detected influenza virus.
- the present invention has the following advantages:
- the invention provides a combination of MNP markers of influenza A, B and C viruses, a combination of primer pairs, a kit and applications thereof.
- the provided 265 MNP markers of influenza A, B and C and their primer combinations can be used for multiplex PCR amplification, and the next-generation sequencing platform can be used to sequence the amplified products to meet the one-time detection of influenza A, B and C.
- the requirements for high-throughput, high-efficiency, high-accuracy, and high-sensitivity detection and differentiation of 3 influenza viruses meet the needs of accurate detection of genetic variation of influenza A, B and C; meet the requirements for the construction of influenza A, B and C A standard, shareable fingerprint database for the needs of epidemic strain monitoring.
- the invention is the first in the field of influenza A, B, and C viruses, and there are no related literature reports; MNP markers are mainly developed based on reference sequences, based on the resequencing of nearly one million influenza A, B, and C viruses that have been reported
- the data can be used to mine large-scale MNP markers that are distinguished from other species, polymorphic within influenza A, B, and C subtypes, and flanked by sequence conservation; design MNP marker detection primers suitable for multiplex PCR amplification; and then pass positive
- a set of MNP markers, multiple PCR primer combinations and detection kits with the largest polymorphism, the highest specificity and the best primer compatibility were screened out, and used for the detection of influenza A, B and C viruses,
- DNA fingerprints genetic variation detection and other related applications, it provides technical support for the detection, scientific research and prevention of influenza A, B and C viruses.
- Figure 1 is a schematic diagram of MNP marker polymorphism
- Fig. 2 is the screening and primer design flowchart of A, B and C influenza virus MNP markers
- Fig. 3 is the detection flowchart of MNP mark
- Example 1 The screening of A, B and C influenza virus MNP marker combinations and the design of multiplex PCR amplification primers
- Influenza A, B, and C viruses contain eight single-stranded negative-strand RNAs such as H and N, and the H and N strands are variable regions, so the existing subtypes are mainly classified based on H and N, such as H7N9, H5N8 subtype.
- the MNP marker designed by the present invention not only includes the subtype-specific markers distributed in the H and N chains, but also includes the markers distributed in the conserved regions between the subtypes of other chains. All known influenza A, B and C subtypes were identified. Based on the genomes or partial fragment sequences of 764,386 influenza A viruses, 116,653 influenza B viruses, and 2,260 influenza C viruses of different subtypes published online, 265 MNP markers were obtained through sequence alignment.
- 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.
- high-throughput sequencing can be whole genome or simplified genome sequencing.
- the genome sequences of at least 10 genetically representative isolates are generally used as references.
- the screened 265 MNP markers are shown in Table 1:
- the step S1 specifically includes:
- step lengths can also be used when screening on the reference genome with a window of 100-300 bp.
- the step size is 1 bp, which is conducive to comprehensive screening.
- the multiple PCR amplification primers labeled with MNP 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 multiple PCR amplification, that is, all designed primers can be used in one amplification reaction. normal expansion.
- the primers used to identify the MNP marker are shown in Table 1.
- the detection method of the MNP marker combination is to amplify all the MNP markers at one time through multiplex PCR, sequence the amplified products through second-generation high-throughput sequencing, analyze the sequencing data, and evaluate the MNP markers according to the detected markers. Primer combination compatibility.
- influenza A virus H7N9, H1N1, H5N6, H9N2, H3N2, H6N1, H6N2 and H10N8
- nucleotide positive reference materials of influenza B and C viruses provided by Hubei Provincial Center for Disease Control and Prevention
- the single sample and the mixed sample mixed according to the concentration ratio were added to 1ml of throat swab washing liquid matrix without influenza virus to prepare simulated single samples and mixed samples of influenza A, B and C viruses, and the sub-sample in the mixed sample Type H7 has the lowest concentration, estimated at 10 copies/ML.
- the primer combination the simulated single sample and mixed samples were detected by the MNP marker detection method, and three repeated sequencing libraries were constructed for each sample.
- the designed primer combinations were screened, and finally 265 MNP-labeled primer pair combinations provided by the present invention were obtained through final screening, as shown in Table 1.
- the above results show that the primers do not conflict with each other, and can efficiently amplify by multiplex PCR, detect three influenza viruses in the sample at one time, and distinguish the subtypes of influenza A. Due to the high variability of Influenza A, the number of detected markers is related to the tested samples, so the markers in this embodiment are not all detected in the tested samples, but they are sufficient to detect and distinguish each subtype.
- nucleotides of influenza A virus H1N1 subtype, type B Victoria strain, and type C influenza virus with known copy numbers were added to human genomic DNA to prepare 1 copy/reaction and 10 copies/reaction and 100 copies/reaction of 3 influenza virus mock samples.
- 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 the detection continued for 4 days, that is, 12 sets of sequencing data were obtained for each sample, as shown in Table 3.
- the reproducibility of the assay was assessed based on the number of sequenced reads and the number of markers for MNP markers of influenza A virus subtype H1N1, influenza B and C detected in placebo and mock samples in 12 replicate experiments , accuracy, sensitivity, and formulate thresholds for contamination of the quality control system and detection of target pathogens.
- the kit can stably detect 16 influenza A viruses, 8 influenza B viruses, and 7 influenza C virus or more MNP sites in a sample of 10 copies/reaction respectively, While at most 1 MNP site can be detected in a small number of samples with 0 copies/reaction, the kit can clearly distinguish samples with 10 copies/reaction and 0 copies/reaction, with technical stability and the Detection sensitivity.
- the reproducibility and accuracy of the MNP marker detection method for detecting influenza A, B and C viruses were evaluated. Specifically, 12 sets of data of 100 copies of samples were compared in pairs, and the results are shown in Table 5.
- the quality control plan is as follows:
- the amount of sequencing data is greater than 80 megabases. The calculation is based on the fact that the maximum number of MNP markers detected by each sample is 265, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 80 megabases, most samples can be guaranteed to cover each marker in one experiment. The number of sequencing fragments reaches 1000 times, ensuring accurate analysis of the base sequence of each MNP marker.
- 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 influenza A, B and C viruses 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 influenza A, B and C viruses in the test sample.
- the average value of the noise index of the influenza A virus H1N1 subtype in the blank control is 0.05%, while the average value of the signal index in the sample with 1 copy is 0.27%, the sample with 1 copy and the blank
- the average value of the signal-to-noise ratio of the control is 5.82. 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 value of the signal-to-noise ratio of influenza A virus H1N1 in 10 copies of the sample and the blank control is 62.9, and in the 12 sets of data of 10 copies/reaction, at least 16 can be stably detected MNP markers, including H1N1 subtype-specific markers and influenza A conserved markers, accounted for 43.2% of the total markers. Therefore, in the case of ensuring accuracy and taking into account sensitivity, the criteria for judging the positiveness of influenza A virus in this patent is: when the signal-to-noise ratio of influenza A virus in the sample is greater than 31.5, and the marker detection rate is greater than or equal to 21.6%. , it is determined that the nucleotide of influenza A virus has been detected in the sample.
- the criteria for judging the positiveness of influenza B virus in this patent is: when the signal-to-noise ratio of influenza B virus in the sample is greater than 32.2, and the marker is detected When the rate is greater than or equal to 26.7%, it is determined that the nucleotide of influenza B virus has been detected in the sample.
- the standard for judging the positiveness of influenza C virus in this patent is: when the signal-to-noise ratio of influenza C virus in the sample is greater than 30.3, and the marker detection rate is greater than or equal to 23.4%, it is determined that the nucleus of influenza C virus has been detected in the sample. glycosides. Therefore, the kit provided by the present invention can accurately and sensitively detect the three types of influenza viruses A, B and C as low as 10 copies/reaction.
- influenza A virus shown in Table 2 The 8 subtypes (H7N9, H1N1, H5N6, H9N2, H3N2, H6N1, H6N2 and H10N8) of influenza A virus shown in Table 2 are artificially divided into influenza B and C and human parainfluenza virus and metapneumovirus , human rhinovirus, mumps virus, measles virus, respiratory syncytial virus, coronavirus, influenza A virus, influenza B virus, influenza C virus, avian influenza virus, Coxsackie virus, echovirus and Zika virus RNA was mixed together to prepare a mixed template, and a blank template was used as a control to detect pathogens in the mixed template using the kit provided by the present invention, and three repeated experiments were performed.
- influenza A, B and C viruses In three repeated experiments, the specific MNP marker sites of influenza A, B and C viruses could be detected, and the average number of MNP marker sites detected by influenza A, B and C viruses were 32 and 15 respectively. and 12. After analyzing according to the described quality control scheme and decision threshold, all can judge influenza A, B and C to be positive in 3 repeated experiments, show that MNP mark and described kit detect the high efficiency of target microorganism in complex template specificity.
- the kit was used to detect 6 backup strains of influenza B virus Victoria strains provided by the Hubei Center for Disease Control and Prevention.
- the samples were named S1-S6 in sequence, and the average sequencing coverage of each sample reached 1521 Times, each strain can detect 15 MNP markers (Table 7).
- the fingerprints of the 6 strains were compared in pairs, and the results are shown in Table 7. There was 1 copy (S-2) and 5 copies of influenza A, B and C detected together in the same batch, all of which had partial markers The main genotype difference (Table 7), there is variation among strains.
- kits to identify genetic variation between strains by detecting MNP markers can be used to ensure the genetic consistency of the same named influenza A, B and C strains in different laboratories, thereby ensuring the comparability of research results, This has important implications for the scientific study of influenza A, B and C viruses.
- the diagnostic scheme can be considered according to whether differential markers affect drug resistance.
- the authenticity evaluation of the secondary allelic type in this embodiment is carried out as follows: first, the allelic type with strand preference (the ratio of the number of sequencing sequences covered on the DNA double strand) is excluded according to the following rules: the strand preference is greater than 10 times, or more than 5 times different from the strand preference of the main allele type.
- 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 markers.
- Table 8 The critical value for determining the suballelic genotype under partial sequencing depth
- the nucleotides 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, artificial heterozygous samples were prepared, each sample was detected 3 times, and a total of 24 sequencing data were obtained.
- markers of heterozygous genotypes were detected in 24 artificial heterozygous samples, indicating that the developed influenza A, B and C Applicability of MNP marker assays for viruses to detect genetic variation within strain populations.
- the DNA of all strains or samples used to construct the DNA fingerprint database of influenza A, B and C viruses was extracted by conventional CTAB method, commercial kits, etc., and the quality of DNA was detected by agarose gel and UV spectrophotometer . If the ratio of the absorbance value of the extracted DNA at 260nm to 230nm is greater than 2.0, the ratio of the absorbance value at 260nm to 280nm is between 1.6 and 1.8, the main band of DNA electrophoresis is obvious, and there is no obvious degradation and RNA residue, it means that the genomic DNA has reached Relevant quality requirements, follow-up experiments can be carried out.
- the genotypes of each subtype at MNP markers were obtained, and these genotypes constituted the reference genotype library of MNP markers.
- the 8 subtypes of influenza A virus, type B and type C shown in Table 2 were detected by MNP markers, and the obtained sequencing data were compared with the reference genotype library to obtain each
- the main genotype of each marker of each strain forms the MNP fingerprint of each strain, and enters the database file to form the MNP fingerprint database of influenza A, B and C viruses.
- the MNP fingerprints of strains with different main genotypes can be entered into the constructed MNP fingerprint database.
- the constructed MNP fingerprint database can be continuously updated and enriched. Because the constructed database is based on the gene sequences of detected strains, it is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed, shared, and updateable at any time.
- the nucleotides of A, B and C influenza viruses are mixed together, and the blank sample is used as a control, and the kit provided by the present invention is used to treat the A, B and C influenza viruses in the mixed template. to test.
- the results of 3 repeated experiments showed that, according to the analysis of the quality control scheme and determination scheme, the kit can detect subtype-specific MNP markers, and can successfully identify A There are 8 subtypes of influenza virus.
- subtype-specific MNP markers could only be detected specifically in the subtype, indicating that the kit has high specificity in detecting target microorganisms in mixed templates, and can be used for A and B It can be used for the fine typing of each subtype of influenza A, B and C influenza virus.
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Abstract
本发明公开了一种甲、乙和丙型流感病毒的MNP标记组合、检测该MNP标记组合的引物对组合、试剂盒及其应用，所述MNP标记组合包括265个标记，具体的核苷酸序列如SEQ ID NO.1-SEQ ID NO.265所示；所述引物对组合包括265对引物，具体的引物核苷酸序列如SEQ ID NO.266-SEQ ID NO.795所示。所述MNP标记组合能特异的鉴定3种流感病毒并区分每种流感病毒不同的亚型；所述引物互不干扰。
Description
本发明实施例涉及生物技术领域，特别涉及一种甲、乙和丙型流感病毒的MNP标记组合、引物对组合、试剂盒及其应用。
流行性感冒病毒简称流感病毒，分为甲(A)、乙(B)、丙(C)三型，可引起人、禽、猪、马、蝙蝠等多种动物感染和发病，是人流感、禽流感、猪流感、马流感等人与动物疫病的病原。流感病毒主要通过空气中的飞沫、易感者与感染者之间的接触或与被污染物品的接触而传播。其中甲型流感病毒易发生变异，其遗传物质包含了H和N等八条单股负链RNA，H和N链是易变区，所以已有的亚型主要基于H和N进行亚型分类，根据H和N抗原不同，H可分为18个亚型(H1～H18)，N有11个亚型(N1～N11)。其中仅H1N1、H2N2、H3N2主要感染人类，其它许多亚型的自然宿主是多种禽类和动物，对禽类危害最大的为H5、H7和H9亚型毒株。在感染人类的三种流感病毒中，甲型流感病毒有着极强的变异性，乙型次之，而丙型流感病毒的抗原性非常稳定。甲型流感病毒的高变异性增大了人们应对流行性感冒的难度，人们无法准确预测即将流行的病毒亚型，便不能有针对性地进行预防性疫苗接种，严重威胁人类建康。
现有的流感病毒检测技术包括病毒分离鉴定、血清型鉴定、间接或直接免疫荧光法和检测遗传物质的分子检测技术，包括PCR、核苷酸杂交和测序技术。这些技术各有优势，但在时长、操作复杂度、检测通量、检测变异的准确性和灵敏度、成本等方面也存在一个或多个局限。比如病毒分离鉴定操作复杂、耗时长；血清型鉴定、间接或直接免疫荧光法容易出现交叉反应，导致检测不准确，且不能监测变异；PCR检测技术主要是针对一种病毒的单个亚型的1到2个标记进行检测，效率低下，且容易由于病毒的变异导致检测失败。宏基因组测序技术是另一种检测流感病毒的技术，但其往往包括大量的宿主测序数据，对低病毒载量的样本进行检测时，尤其需要超深度的测序，导致高的成本。因 此，开发快速、准确的、一次性高通量的检测分型甲、乙和丙型3种的流感病毒检测分析方法对于流感病毒的检测、科学研究、指纹数据库构建和流行株检测都具有重要意义。
本发明开发了新型分子标记MNP标记，并融合超多重PCR扩增和高通量测序技术分析检测MNP标记，一次性对成百上千份样本的数万个MNP标记进行分型，实现对甲、乙和丙型流感病毒的高通量、灵敏检测和精准分型，具有样本需要量少、同时覆盖3种病毒、诊断结果精确，节约数据量、检测亚型的优势。
MNP标记是指在基因组上一段区域内由多个核苷酸引起的多态性标记。与传统的SSR标记和SNP标记相比，MNP标记具有以下优势：(1)等位基因丰富，单个MNP标记上有2
n种等位基因，高于SSR和SNP；(2)物种区分能力强，只需要少量的MNP标记就能实现物种鉴定，减少了检测错误率。基于超多重PCR结合二代高通量测序技术检测MNP标记的MNP标记法具有以下优势：(1)输出的是碱基序列，无需平行实验，可构建标准化的数据库进行共享共用；(2)高效率，利用样品DNA条形码，突破测序样品数量的局限，可一次性对成百上千份样本的数万个MNP标记分型；(3)高灵敏度，利用多重PCR一次检测多个靶标，避免单个靶标扩增失败导致高的假阴性和低的灵敏度；(4)高准确性，利用二代高通量测序仪对扩增产物测序数百次。
鉴于以上优点和特性，MNP标记及其检测技术MNP标记法在同时鉴定分型多种病毒、指纹数据库构建、遗传变异检测等方面都具有应用潜力。目前在甲、乙和丙型流感病毒的检测中，尚未有关于MNP标记的报道，也缺乏相应的技术。本发明所开发的标记和引物组合也将用于制定病原体检测的国家标准(计划编号20201830-T-469)，该国家标准将于2021年底发布。
发明内容
本发明实施例目的是提供一种甲、乙和丙型流感病毒的MNP标记组合、引物对组合、试剂盒及其应用，可以一次性对甲、乙和丙型3种流感病毒进行鉴定和变异检测，具有多靶标、高通量、高灵敏和精细分型的效果。
在本发明的第一方面，提供了一种甲、乙和丙型流感病毒的MNP标记组合， 所述MNP标记是指在甲、乙和丙型流感病毒基因组上分别筛选的保守的、区分于其他物种且在物种内部具有多个核苷酸多态性的基因组区域，所述MNP标记组合包括甲、乙和丙型流感病毒参考序列上MNP-1～MNP-265的265个标记。
上述技术方案中，MNP-1～MNP-265的265个标记具体的核苷酸序列如SEQ ID NO.1-SEQ ID NO.265所示，为下引物说明书表1对其进一步地说明，表1中标注的所述MNP标记的起始和终止位置是基于表1中MNP同一行对应的参考序列确定的。
在本发明的第二方面，提供了一种用于检测所述MNP标记组合的多重PCR引物对组合，所述多重PCR引物对组合包括265对引物，具体的引物序列如SEQ ID NO.266-SEQ ID NO.795所示，其中ID NO.266-SEQ ID NO.530为上引物，ID NO.531-SEQ ID NO.795。
上述技术方案中，每个MNP标记的引物包括上引物和下引物，具体如说明书表1所示。
在本发明的第三方面，提供了一种用于检测所述甲、乙和丙型流感病毒MNP标记组合的检测试剂盒，所述试剂盒包括所述的引物对组合。
进一步地，所述试剂盒还包括多重PCR预混液。
以及所述的甲、乙和丙型流感病毒的MNP标记组合或所述的引物对组合或所述的检测试剂盒在非诊断目的甲、乙和丙型流感病毒检测中的应用，在制备甲、乙和丙型流感病毒检测产品中的应用。
在本发明的第四方面，提供了所述的甲、乙和丙型流感病毒的MNP标记组合或者所述的多重PCR引物对组合或者所述的检测试剂盒在甲、乙和丙型流感病毒的鉴定、遗传变异检测、DNA指纹数据库的构建和精细分型中的应用。
以上所述的应用中，首先是获取待测样本的病毒总RNA；利用商业化试剂盒对所述总RNA进行cDNA合成；利用本发明的试剂盒对所述cDNA和空白对照进行第一轮多重PCR扩增，循环数不高于25个；对扩增产物进行纯化后，进行基于第二轮PCR扩增的样本标签和二代测序接头添加；对第二轮扩增产物纯化后定量；检测多个毒株时通过将第二轮扩增产物等量混合后进行高通量测序；测序结果比对到所述的甲、乙和丙型流感病毒的参考序列上，获取在所述cDNA中的检测序列数目和基因型数据。根据在所述cDNA和所述空白对照获得的甲、 乙和丙型流感病毒测序序列数量和检出MNP标记的数目，对所述cDNA的测序数据进行数据质量控制和数据分析，获得在所述样本中检出的甲、乙和丙型流感病毒MNP标记数目、覆盖每个所述MNP标记的测序序列数目和所述MNP标记基因型数据。
当用于甲、乙和丙型流感病毒鉴定时，根据在待测样品和空白对照中检出的甲、乙和丙型流感病毒的测序序列数量和检出MNP位点的数目，进行质控后判定待测样品中是否含有甲、乙和丙型流感病毒的核酸。其中，所述的质控方案和判定方法是以拷贝数已知的甲、乙和丙型流感病毒核苷酸标准品为检测样本，评估所述试剂盒检测甲、乙和丙型流感病毒的灵敏度、准确性和特异性，制定所述试剂盒检出甲、乙和丙型流感病毒的质控方案和判定方法。并依次为依据，对实测样本中的流感病毒进行检测。
当用于甲、乙和丙型流感病毒遗传变异检测时，包括毒株间和毒株内部的遗传变异检测。毒株间的遗传变异检测包括利用所述的试剂盒和方法，获得待比较毒株各自在265所述部分或全部MNP标记的基因型数据。通过基因型比对，分析待比较毒株在265共同检出MNP标记上的主基因型是否存在差异。若待比较毒株在至少一个MNP标记的主基因型存在变异，则判定两者存在遗传变异。作为一种备选方案，也可以根据研究目的，通过单重PCR对待比较毒株的部分或全部265个MNP标记分别进行扩增，然后对扩增出的产物进行Sanger测序，获得序列后，对待比较毒株共同检出MNP标记的基因型进行比对。如果存在主基因型不一致的MNP标记，则待比较毒株之间存在变异。当检测毒株内部的遗传变异时，则通过统计模型判定在待测毒株所述的MNP标记是否检出主基因型以外的次基因型。若待测毒株在至少一个MNP标记存在次基因型，则判定待测毒株内部存在遗传变异。
当用于构建甲、乙和丙型流感病毒DNA指纹数据库时，将从样本中鉴定的甲、乙和丙型流感病毒的所述MNP标记的基因型数据，录入数据库文件，分别构成甲、乙和丙型流感病毒的DNA指纹数据库；每次鉴定不同的样本时，通过和所述甲、乙和丙型流感病毒的DNA指纹数据库比对，鉴定样本中的甲、乙和丙型流感病毒是否和数据库中的毒株在所述MNP标记存在主基因型(在一个MNP标记具有超过50％测序片段支持的基因型)的差异，在至少1个MNP标 记存在主基因型差异的甲、乙和丙型流感病毒即为新的变异类型，收录进DNA指纹数据库。
当用于甲、乙和丙型流感病毒分型时，是对待测样本中的甲、乙和丙型流感病毒进行鉴定，获得每个所述MNP标记的基因型。通过和所述甲、乙和丙型流感病毒的基因型同公开的参考序列和已有的DNA指纹数据库比对，鉴定样本中的甲、乙和丙型流感病毒是已有的类型还是新的类型，对检测到的流感病毒进行精准分型。与现有技术相比，本发明具有以下优点：
本发明提供了一种甲、乙和丙型流感病毒的MNP标记组合、引物对组合、试剂盒及其应用。所提供的甲、乙和丙型流感病毒的265个MNP标记和其引物组合，可进行多重PCR扩增，融合二代测序平台进行扩增产物的测序，满足一次性对甲、乙和丙型3种流感病毒进行高通量、高效率、高准确性和高灵敏度检测和区分的需求；满足准确检测甲、乙和丙型流感病毒遗传变异的需求；满足构建甲、乙和丙型流感病毒标准的、可共享的指纹数据库，用于流行株监测的需求。
本发明在甲、乙和丙型流感病毒领域属于首创，并未见相关文献报道；MNP标记主要基于参考序列开发，根据已报道的近百万条的甲、乙和丙型流感病毒的重测序数据可以挖掘大规模的区分于其他物种、在甲、乙和丙型流感病毒亚型内部多态、两侧序列保守的MNP标记；设计适用于多重PCR扩增的MNP标记检测引物；再通过阳性样本的测试，筛选出一套多态性最大、特异性最高、引物兼容性最好的一套MNP标记、多重PCR引物组合以及检测试剂盒，并用于甲、乙和丙型流感病毒的检测、DNA指纹图谱构建，遗传变异检测以及其他相关应用中，为甲、乙和丙型流感病毒的检测、科学研究和防治提供技术支撑。
图1为MNP标记多态性原理图；
图2为甲、乙和丙型流感病毒MNP标记的筛选和引物设计流程图；
图3为MNP标记的检测流程图；
为了便于理解本发明，下面结合附图和具体实施例，对本发明进行更详细的说明。附图中给出了本发明的较佳的实施例。但是，本发明可以以许多不同的形式来实现，并不限于本说明书所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
需要说明的是，除非另有定义，本说明书所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是用于限制本发明。
除非另有特别说明，本发明实施例中用到的各种原材料、试剂、仪器和设备等，均可通过市场购买得到或者可通过现有方法制备得到。
实施例1甲、乙和丙型流感病毒MNP标记组合的筛选和多重PCR扩增引物的设计
S1、甲、乙和丙型流感病毒MNP标记组合的筛选
甲、乙和丙型流感病毒包含了H和N等八条单股负链RNA，而H和N链是易变区，所以已有的亚型主要基于H和N进行亚型分类，比如H7N9、H5N8亚型。本发明设计的MNP标记即包含了分布在H和N链的亚型特异的标记，也包含了分布在其他链的亚型间保守区域的标记，所以，本发明提供的MNP标记一次性对已知的所有甲、乙和丙型流感病毒亚型进行鉴定。基于网上公开的764386个甲型流感病毒、116653乙型流感病毒和2260个丙型流感病毒不同亚型的基因组或是部分片段的序列，通过序列比对，获得265个MNP标记。
对于网上不存在基因组数据的物种，也可以通过高通量测序获得待检测微生物物种代表小种的基因组序列信息，其中高通量测序可以是全基因组或简化基因组测序。为了保证所筛选标记的多态性，一般使用至少10个遗传上具有代表性的分离株的基因组序列作为参考。
筛选的265个MNP标记如表1所示：
表1所述MNP标记以及检测引物在参考序列上的起始位置
所述步骤S1具体包括：
选择所述甲、乙和丙型流感病毒的一个代表株的基因组序列作为参考基因组，将所述基因组序列和所述参考基因组进行序列比对，获得所述甲、乙和丙型流感病毒的单核苷酸多态性标记；根据序列相似性将所述基因组序列分为若干个组别，然后在每个组别上筛选所述的MNP标记。
在所述参考基因组上，以100-300bp为窗口，以1bp为步长进行窗口平移，筛选获得多个候选MNP标记区域，其中，所述候选MNP标记区域含有≥2个所述单核苷酸变异标记，且两端各30bp的序列上均不存在所述单核苷酸多态性标记；
在所述候选多核苷酸多态性标记区域中筛选区分度DP≥0.2的区域作为MNP标记；其中，DP＝d/t，t是在所述候选多核苷酸多态性标记区域中所有小种两两比较时的比较对数，d是在所述候选多核苷酸多态性标记区域中至少两个单核苷酸多态性差异的样品对数。
作为一种可选的实施方式，在所述参考基因组上，以100-300bp为窗口进行筛选时，也可选用其他步长，本实施方式采用步长为1bp，有利于全面的筛选。
S2、多重PCR扩增引物的设计
通过引物设计软件设计所述MNP标记的多重PCR扩增引物，引物设计遵循引物间互不干扰，所有引物可以组合成引物池进行多重PCR扩增，即所有设计的引物可以在一个扩增反应中均正常扩增。
该实施方式中，用于鉴定所述MNP标记的引物，如表1所示。
S3、引物组合的检测效率评估
所述MNP标记组合的检测方法是通过多重PCR对所有MNP标记一次性进行扩增，通过二代高通量测序对扩增产物进行测序，对测序数据进行分析，根据检出的标记评价所述引物组合的兼容性。
将甲型流感病毒不同亚型(H7N9，H1N1，H5N6，H9N2，H3N2，H6N1，H6N2和H10N8)、乙和丙型流感病毒的核苷酸阳性参考品(由湖北省疾控预防控制中心提供)单样和按浓度比例混合的混合样，分别加入到1ml的不含流感病毒的咽拭子洗液基质中，制备甲、乙和丙型流感病毒模拟单样本和混合样本，混合样本中的亚型H7浓度最低，预估在10拷贝/ML。使用所述的引物组合，通过所述的MNP标记检测方法对模拟单样本和混合样本进行检测，每个样本构建3个重复的测序文库。根据检测结果对所设计的引物组合进行筛选，最终筛选获得本发明提供的265个MNP标记的引物对组合，具体如表1所示。使用本发明提供的引物对组合测试的结果如表2和表3所示，在测试的8个甲型流感病毒、1个乙型流感病毒和1个丙型流感病毒单样本中都检出了预期的亚型特异标记和保守标记，检出数目至少14个；在3个混合样本中都检出至少138个MNP标记，所有混入的甲型流感病毒亚型和乙型、丙型流感病毒全部检出。上述结果表明所述引物互相间不冲突，可以通过多重PCR进行高效的扩增，一次性的检出样本中的3种流感病毒，并对甲型流感的亚型进行区分。由于甲型流感的高变异，检出标记数和所测试的样本相关，所以本实施例中的标记在所测试的样本未全部检出，但足以检出和区分各亚型。
表1甲、乙和丙型流感病毒亚型MNP标记检测分析
表2表2中甲、乙和丙型流感病毒亚型混合物的MNP标记检测分析
实施例2所述MNP标记组合和引物组合鉴定甲、乙和丙型流感病毒的性能评估和阈值设置
本实施例中，将拷贝数已知的甲型流感病毒H1N1亚型、乙型Victoria系和丙型流感病毒的核苷酸分别加入到人基因组DNA中，制备1拷贝/反应、10拷贝/反应和100拷贝/反应的3种流感病毒模拟样本。同时设置的等体积的无菌水作为空白对照。共计4个样本，每个样本每天构建3个重复文库，连续检测4天，即每个样本获得12组测序数据，具体如表3所示。根据在12次重复实验中，在空白对照和模拟样品中检出的甲型流感病毒H1N1亚型、乙和丙型流感病毒的MNP标记的测序片段数和标记数，评估检测方法的重现性、准确性、灵敏度，制定质控体系污染和目标病原体检出的阈值。
MNP标记的检测流程如图3所示。
1、MNP标记组合检测试剂盒检测甲、乙和丙型流感病毒的稳定性和灵敏度评估
如表4所示，所述试剂盒能在10拷贝/反应的样本中稳定的分别检出16个甲型 流感病毒、8各乙型流感病毒和7个丙型流感病毒以上的MNP位点，而在0拷贝/反应的少数样本中最多检出1个MNP位点，所述试剂盒能够明显区分10拷贝/反应和0拷贝/反应的样品，具有技术稳定性和低至10拷贝/反应的检测灵敏度。
表4甲、乙和丙型流感病毒的MNP标记法的检测灵敏度、稳定性分析
1、MNP标记组合检测试剂盒检测甲、乙和丙型流感病毒的重现性和准确性评估
基于两次重复中，共同检出标记的基因型是否可重现，评估MNP标记检测方法检测甲、乙和丙型流感病毒的重现性和准确性。具体地，对100拷贝样品的12组数据分别进行两两比较，结果如表5所示，主基因型存在差异的MNP标记数目都为0；依据2次重复实验间可重现的基因型认为是准确的原则，准确率a＝1-(1-r)/2＝0.5+0.5r，r代表重现率，即主基因型可重现的标记数目占共有标记数目的比率。本项目重现性试验中每个样品不同文库间、不同建库批次间MNP标记主基因型的差异对数为0，重现率r＝100％，准确率a＝100％。
表5甲、乙和丙型流感病毒MNP标记检出方法的重现性和准确率评估
3、MNP标记组合检测试剂盒检出甲、乙和丙型流感病毒的阈值判定
如表4所示，在1个拷贝/反应的样本中多数能检出比对到甲、乙和丙型流感病毒的序列。而在部分空白对照中也检出了甲、乙和丙型流感病毒的序列。由于MNP标记检测方法的极度灵敏，因此检测过中的数据污染容易导致假阳性的产生。因此本实例中根据对连续4天不同拷贝数的阳性标准品的测试结果，制定如下质控方案。
质控方案具体如下：
1)测序数据量大于80兆碱基。测算依据是每个样品检测MNP标记的数目最多是265个，一条测序片段的长度是300个碱基，所以当数据量大于80兆碱基时，大部分样品一次实验可以保证覆盖每个标记的测序片段数量达到1000倍，保证对每个MNP标记碱基序列的精准分析。
2)根据测试样品中的甲、乙和丙型流感病毒的信号指数S和空白对照中甲、乙和丙型流感病毒的噪音指数P判定污染是否可接受，其中：
空白对照噪音指数P＝nc/Nc，其中nc和Nc分别代表空白对照中，甲、乙和丙型流感病毒的测序片段的数量和总测序片段数量。
测试样品的信号指数S＝nt/Nt，其中nt和Nt分别代表测试样品中，甲、乙和丙型流感病毒的测序片段的数量和总测序片段数量。
3)计算测试样品中MNP标记的检出率，指的是检出标记数和总设计标记数的比值。
如表6所示，甲型流感病毒H1N1亚型在空白对照中的噪音指数平均值是0.05％，而在1个拷贝的样品中的信号指数平均值是0.27％，1个拷贝的样品和空白对照的信噪比的平均值是5.82，因此，本发明规定当信噪比大于10倍时，可判定检测体系中的污染是可接受的。
如表6所示，在10个拷贝的样品和空白对照中甲型流感病毒H1N1的信噪比的平均值是62.9，在10拷贝/反应的12组数据中，能稳定的检出至少16个MNP标记，包括了H1N1亚型特异标记和甲型流感病毒保守标记，占总标记的43.2％。因此，在保证准确性并兼顾灵敏度的情况下，本专利对甲型流感病毒阳性的判定标准是：当样品中甲型流感病毒的信噪比大于31.5，且标记检出率大于等于21.6％时，判定样本中检出了甲型流感病毒的核苷酸。
同理，在10个拷贝的样品中，乙型和丙型流感病毒的信噪比平均值分别为64.4和60.5，在10拷贝/反应的12组数据中，能稳定的检出各自总标记的53.3％和46.7％，因此，在保证准确性并兼顾灵敏度的情况下，本专利对乙型流感病毒阳性的判定标准是：当样品中乙型流感病毒的信噪比大于32.2，且标记检出率大于等于26.7％时，判定样本中检出了乙型流感病毒的核苷酸。本专利对丙型流感病毒阳性的判定标准是：当样品中丙型流感病毒的信噪比大于30.3，且标记检出率大于等于23.4％时，判定样本中检出了丙型流感病毒的核苷酸。因此本发明所提供的试剂盒能准确、灵敏的检测到低至10copy/反应的甲、乙和丙型3种流感病毒。
表6待测样品中甲型流感病毒H1N1亚型的信噪比
4、MNP标记组合检测方法检测甲、乙和丙型流感病毒的特异性评估
人为的将表2所示的甲型流感病毒的8个亚型(H7N9，H1N1，H5N6，H9N2，H3N2，H6N1，H6N2和H10N8)乙型和丙型流感病毒和人副流感病毒、偏肺病毒、人鼻病毒、流行性腮腺炎病毒、麻疹病毒、呼吸道合胞病毒、冠状病毒、甲型流感病毒、乙型流感病毒、丙型流感病毒、禽流感病毒、柯萨奇病毒、埃可病毒和寨卡病毒的RNA混在一起，制备混合模板，以空白模板作为对照，采用本发明所提供的试剂盒对混合模板中的病原体进行检测，进行3个重复实验。在3个重复实验中都能检出甲、乙和丙型流感病毒的特异的MNP标记位点，甲、乙和丙型流感病毒检出的MNP标记位点数目分别平均为32个，15个和12个。 按照所述的质控方案和判定阈值进行分析后，在3个重复实验中都能判断甲、乙和丙型流感病毒阳性，表明MNP标记和所述试剂盒在复杂模板中检测目标微生物的高特异性。
实施例3、甲、乙和丙型流感病毒毒株间的遗传变异检测
利用所述的试剂盒对湖北疾控预防控制中心提供的乙型流感病毒Victoria系毒株的6份备份毒株进行检测，样本依次命名为S1-S6，每个样品的测序平均覆盖倍数达1521倍，每个毒株均可以检出15个MNP标记(表7)。将6个毒株的指纹图谱进行两两比对，结果如表7所示，有1份(S-2)和同批次一起检测的5份甲、乙和丙型流感病毒均存在部分标记的主基因型差异(表7)，存在毒株间变异。
所述的试剂盒通过检测MNP标记鉴定毒株间遗传变异的应用可以用于保证不同实验室相同命名甲、乙和丙型流感病毒毒株的遗传一致性，从而保证研究结果的可比较性，这对于甲、乙和丙型流感病毒的科学研究具有重要意义。而在临床上，可针对差异标记是否影响抗药性斟酌诊断方案。
表5 6个甲、乙和丙型流感病毒的检测分析
实施例4、甲、乙和丙型流感病毒毒株内部的遗传变异检测
作为群体生物，甲、乙和丙型流感病毒群体内部部分个体发生变异，使群 体不再纯合，形成异质的杂合群体，影响尤其是试验用微生物表型的稳定性和一致性。这种变异体在对群体进行分子标记检测时，表现为标记的主基因型外的等位基因型。当变异个体还未累积时，只占群体的极少部分，表现为低频率的等位基因型。低频率的等位基因型往往和技术错误混在一起，导致现有技术难以区分。本发明检测的是高多态性的MNP标记。基于多个错误同时发生的几率低于一个错误发生的几率，MNP标记的技术错误率显著低于SNP标记。
本实施例次等位基因型的真实性评估按如下进行：首先按照以下规则排除具有链偏好性(在DNA双链上覆盖的测序序列数的比值)的等位基因型：链偏好性大于10倍，或者与主等位基因型的链偏好性之差大于5倍。
不存在链偏好性的基因型基于表8测序序列数目和比例判定其真实性。表8列出了基于BINOM.INV函数计算在α＝99.9999％的概率保障下，e
max(n＝1)和e
max(n≥2)分别为1.03％和0.0994％时，在各个标记中次等位基因型测序序列数目的临界值，只有次等位基因型的测序序列数目超过临界值时判定为真实的次等位基因型。当存在多个候选次等位基因时，对各候选等位基因型的P值进行多重校正，FDR<0.5％的候选等位基因判定是真实的次等位基因型。
表8涉及到的参数e
max(n＝1)和e
max(n≥2)指的是携带n个SNP的错误等位基因的测序序列数占该标记总测序序列数的最高比例。e
max(n＝1)和e
max(n≥2)分别为1.03％和0.0994％是根据在930个纯合MNP标记检测到的所有次等位基因型的频率获得。
表8部分测序深度下进行判定次等位基因型的临界值
按照上述参数，将基因型存在差异的两个毒株的核苷酸按照以下8个比例1/1000，3/1000，5/1000，7/1000，1/100，3/100，5/100，7/100混合，制备人工杂合样本，每个样本检测3次重复，获得共计24个测序数据。通过和所述两个毒株的MNP标记的基因型进行精准比对，在24个人工杂合样本中均检测到了存在杂合基因型的标记，说明了所开发的甲、乙和丙型流感病毒的MNP标记检测方法在检测毒株群体内部遗传变异的适用性。
实施例5甲、乙和丙型流感病毒DNA指纹数据库的构建
利用常规CTAB法、商业化试剂盒等方法提取用于构建甲、乙和丙型流感病毒DNA指纹数据库的所有毒株或是样本的DNA，采用琼脂糖凝胶和紫外分光光度计检测DNA的质量。若所提取的DNA在260nm与230nm处的吸光度值的比值大于2.0，260nm与280nm吸光度值比值介于1.6与1.8之间，DNA电泳主带明显，无明显降解和RNA残留，则说明基因组DNA达到相关的质量要求，可进行后续实验。
在进行MNP标记筛选时，获得了每个亚型在MNP标记的基因型，这些基因型组成了MNP标记的参考基因型库。采用本发明提供的试剂盒，对表2所示的甲型流感病毒8个亚型、乙型和丙型流感病毒进行MNP标记检测，将获得测序数据同参考基因型库进行比对后获得每个毒株每个标记的主基因型，形成每个毒株的MNP指纹图谱，并录入数据库文件，形成甲、乙和丙型流感病毒的MNP指纹数据库。每次检测的样本或毒株的MNP指纹图谱同构建的MNP指纹数据库进行比对后，主基因型存在差异的毒株的MNP指纹图谱均可录入所构建的MNP指纹数据库。因此，理论上，所构建的MNP指纹数据库可以不断的更新和充实。因为所构建的数据库是基于检测的毒株的基因序列，因此和所有的高通量测序数据兼容，具有完全可共建共享、随时可更新的特征。
实施例6、甲、乙和丙型流感病毒的精细分型
如表3所示，将甲、乙和丙型流感病毒的核苷酸混合在一起，以空白样品作为对照，采用本发明所提供的试剂盒对混合模板中的甲、乙和丙型流感病毒进行检测。3个重复实验的结果表明，按照所述的质控方案和判定方案进行分析，所述试剂盒可以检出亚型特异的MNP标记，根据检出的亚型特异的MNP标记可以成功的鉴定甲型流感病毒的8个亚型。在3个重复实验中，亚型特异的MNP标记都仅能特异的在所述亚型中检出，表明所述试剂盒在混合模板中检测目标微生物的高特异性，可以用于甲、乙和丙型流感病毒各亚型的精细分型，从而可用于甲、乙和丙型流感病毒流行株的监测。
最后，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明实施例的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明实施例范围的所有变更和修改。
显然，本领域的技术人员可以对本发明实施例进行各种改动和变型而不脱离本发明实施例的精神和范围。这样，倘若本发明实施例的这些修改和变型属于本发明实施例权利要求及其等同技术的范围之内，则本发明实施例也意图包含这些改动和变型在内。
Claims (9)
- 一种甲、乙和丙型流感病毒的MNP标记组合，其特征在于，所述MNP标记组合，包括265个标记，具体的核苷酸序列如SEQ ID NO.1-SEQ ID NO.265所示。
- 一种用于检测权利要求1所述甲、乙和丙型流感病毒MNP标记组合的多重PCR引物对组合，其特征在于，所述多重PCR引物对组合包括265对引物，具体的引物核苷酸序列如SEQ ID NO.266-SEQ ID NO.795所示。
- 一种用于检测权利要求1所述甲、乙和丙型流感病毒MNP标记组合的检测试剂盒，其特征在于，所述试剂盒包括权利要求2所述的引物对组合。
- 根据权利要求3所述的检测试剂盒，其特征在于，所述试剂盒还包括多重PCR预混液。
- 权利要求1所述的甲、乙和丙型流感病毒的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在非诊断目的甲、乙和丙型流感病毒检测中的应用。
- 权利要求1所述的甲、乙和丙型流感病毒的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在制备甲、乙和丙型流感病毒检测产品中的应用。
- 权利要求1所述的甲、乙和丙型流感病毒的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在检测甲、乙和丙型流感病毒毒株内部和毒株间遗传变异中的应用。
- 权利要求1所述的甲、乙和丙型流感病毒的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在构建甲、乙和丙型流感病毒数据库中的应用。
- 权利要求1所述的甲、乙和丙型流感病毒的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在甲、乙和丙型流感病毒精细分型检测中的应用。
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