Primer design method and primer group

Abstract

The invention discloses a primer design method and a primer group. According to the application, through deep analysis of influenza monitoring network data nationwide, an important contribution is made to improvement of the identification accuracy and sensitivity of influenza viruses. The revision of the primer not only achieves a remarkable effect in an experiment, but also provides beneficial enlightenment for future flu monitoring and research. In-depth understanding of hereditary characteristics and epidemic rules of influenza viruses is of great significance in formulating scientific and effective prevention and control strategies. In disease prevention and public health management, continuous improvement of monitoring methods and strategies will bring greater benefits to timely coping with infectious diseases such as influenza.

Description

Technical Field

[0001] The invention belongs to the biological field, and particularly relates to a primer design method and a primer set. Background Art

[0002] At present, when gene capture is performed based on the polymerase chain reaction (PCR) technology, the primers used are designed based on the deoxyribonucleic acid (DNA) sequence on the reference genome. Among them, the sequence of DNA in the reference genome is detected by the human genome project, including the genetic group genes of most living organisms. However, based on the reference genome, mutations occur to varying degrees, resulting in reduced sensitivity of primers. Therefore, designing universal primers that can detect is of great importance in this field. Summary of the invention

[0003] To overcome the deficiencies of the prior art, the present invention provides a primer design method and a primer set.

[0004] To achieve the above object, the present invention adopts the following technical solution:

[0005] A first aspect of the present invention provides a primer design method, the method comprising:

[0006] Acquire a target gene database, wherein the target gene database includes a target gene sequence;

[0007] Screening target gene mutation sites;

[0008] Primers are designed according to target gene mutation sites, wherein the target gene mutation sites include one or more gene mutation sites and / or degenerate bases.

[0009] Furthermore, the primer is an influenza virus primer.

[0010] Furthermore, the influenza virus primer is an H3 influenza virus primer.

[0011] Furthermore, the target gene mutation sites include one or more of c.308T>C, c.357T>A, c.317A>G, c.364A>G, and c.389A>G.

[0012] Furthermore, the degenerate base includes one or more of R1 at position 317, R2 or Y at position 364.

[0013] Here, the degenerate base R represents A or G, and the degenerate base Y represents C or T.

[0014] A second aspect of the present invention provides a primer set for detecting influenza virus, the primer set comprising a primer pair shown in SEQ ID NOs: 4 and 6, a primer pair shown in SEQ ID NOs: 7 and 9, a primer pair shown in SEQ ID NOs: 10 and 12, a primer pair shown in SEQ ID NOs: 13 and 15, a primer pair shown in SEQ ID NOs: 16 and 18, a primer pair shown in SEQ ID NOs: 19 and 21, a primer pair shown in SEQ ID NOs: 22 and 24, a primer pair shown in SEQ ID NOs: 25 and 27, a primer pair shown in SEQ ID NOs: 28 and 30, a primer pair shown in SEQ ID NOs: 31 and 33, a primer pair shown in SEQ ID NOs: 34 and 36, a primer pair shown in SEQ ID NOs: 37 and 39, a primer pair shown in SEQ ID NOs: 40 and 42, a primer pair shown in SEQ ID NOs: 43 and 45, and a primer pair shown in SEQ ID NOs: 46 and 48. The primer pair has at least 90% identity.

[0015] Further, the primer set includes any one of the primer pairs shown in SEQ ID NOs: 4 and 6, the primer pair shown in SEQ ID NOs: 7 and 9, the primer pair shown in SEQ ID NOs: 10 and 12, the primer pair shown in SEQ ID NOs: 13 and 15, the primer pair shown in SEQ ID NOs: 16 and 18, the primer pair shown in SEQ ID NOs: 19 and 21, the primer pair shown in SEQ ID NOs: 22 and 24, the primer pair shown in SEQ ID NOs: 25 and 27, the primer pair shown in SEQ ID NOs: 28 and 30, the primer pair shown in SEQ ID NOs: 31 and 33, the primer pair shown in SEQ ID NOs: 34 and 36, the primer pair shown in SEQ ID NOs: 37 and 39, the primer pair shown in SEQ ID NOs: 40 and 42, the primer pair shown in SEQ ID NOs: 43 and 45, and the primer pair shown in SEQ ID NOs: 46 and 48.

[0016] Further, the primer set is selected from the primer pair shown in SEQ ID NOs: 16 and 18 and / or the primer pair shown in SEQ ID NOs: 37 and 39.

[0017] Further, the primer set is selected from the primer pair shown in SEQ ID NO:37 and 39.

[0018] Furthermore, the primer set also includes a probe.

[0019] Further, the probe comprises a sequence having at least 90% identity with any one of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, and SEQ ID NO:47.

[0020] Further, the probe includes any one group of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, and SEQ ID NO:47.

[0021] Further, the probe is selected from SEQ ID NO:17 and / or SEQ ID NO:38.

[0022] Further, the probe is selected from SEQ ID NO:38.

[0023] Furthermore, the influenza virus primer is an H3 influenza virus primer.

[0024] Furthermore, the primer pair includes a forward primer and a reverse primer.

[0025] Furthermore, at least one of the forward primer and the reverse primer is labeled with a labeling substance.

[0026] Furthermore, the labeling substance includes a fluorescent substance, a radioactive isotope or an enzyme.

[0027] Furthermore, the fluorescent substance includes TAMRAT TM , Alexa555, Alexa647, Cy3, Cy5 of the cyanine dye series, and fluorescein.

[0028] Furthermore, the radioisotope comprises 32 P. 33 P. 35 S.

[0029] Furthermore, the enzymes include alkaline phosphatase and horseradish peroxidase.

[0030] The third aspect of the present invention provides a kit for detecting influenza virus in a sample, the kit comprising the primer set according to the second aspect of the present invention.

[0031] Furthermore, the kit also includes enzymes for PCR, dNTPs, buffer solution, Mg 2+ at least one reactant.

[0032] Furthermore, the enzyme used for PCR includes DNA polymerase and / or RNA polymerase.

[0033] Furthermore, the buffer comprises a polymerase buffer and a Tris hydrochloric acid buffer.

[0034] Furthermore, the kit also includes instructions.

[0035] Furthermore, the influenza virus primer is an H3 influenza virus primer.

[0036] A fourth aspect of the present invention provides a method for detecting influenza virus in a sample, the method comprising amplifying using the primer set described in the second aspect of the present invention.

[0037] Furthermore, the influenza virus primer is an H3 influenza virus primer.

[0038] Furthermore, the method is a method for non-diagnostic purposes.

[0039] The fifth aspect of the present invention provides use of the primer set described in the second aspect of the present invention in preparing a kit for detecting influenza virus.

[0040] Furthermore, the influenza virus primer is an H3 influenza virus primer.

[0041] A sixth aspect of the present invention provides a primer design system, the system comprising:

[0042] An acquisition unit is used to acquire a target gene database, wherein the target gene database includes a target gene sequence;

[0043] Mutation site screening unit: used to screen the mutation sites of target genes;

[0044] Primer design unit: used to design primers according to target gene mutation sites, wherein the target gene mutation sites include one or more gene mutation sites and / or degenerate bases.

[0045] Furthermore, the primer is an influenza virus primer.

[0046] Furthermore, the influenza virus primer is an H3 influenza virus primer.

[0047] Furthermore, the target gene mutation sites include one or more of c.308T>C, c.357T>A, c.317A>G, c.364A>G, and c.389A>G.

[0048] Furthermore, the degenerate base includes one or more of R1 at position 317, R2 or Y at position 364.

[0049] Here, the degenerate base R represents A or G, and the degenerate base Y represents C or T.

[0050] The seventh aspect of the present invention provides an electronic device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the steps of the method described in the first aspect of the present invention when executing the computer program.

[0051] Advantages and beneficial effects of the present invention:

[0052] This application has made an important contribution to improving the accuracy and sensitivity of influenza virus identification through in-depth analysis of influenza surveillance network data across the country. The revision of primers not only achieved remarkable results in the experiment, but also provided useful inspiration for future influenza monitoring and research. A deep understanding of the genetic characteristics and epidemic patterns of influenza viruses is of great significance for the formulation of scientific and effective prevention and control strategies. In disease prevention and public health management, continuous improvement of monitoring methods and strategies will bring greater benefits to timely response to infectious diseases such as influenza. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is a primer design flow chart;

[0054] Figure 2 It is the distribution map of 92 samples in 30 provinces, cities and districts among the 34 provinces, cities and districts in my country;

[0055] Figure 3 It is the map of variant sites 317, 364 and 389;

[0056] Figure 4 This is the geographical location map of variant sites 317, 364, and 389;

[0057] Figure 5 This is the geographical distribution map after changing the three sites 317, 364 and 389;

[0058] Figure 6 This is the regional distribution statistics after changing the three sites 317, 364 and 389;

[0059] Figure 7 It is a design roadmap for 16 pairs of primers;

[0060] Figure 8 It is a statistical graph of the maximum and minimum values ​​contained in the 16 primer pairs;

[0061] Fig. 9 It is the arithmetic mean and variance statistics of the comparison results of 15 pairs of primers;

[0062] Fig.10 It is the clustering distribution diagram between primers. DETAILED DESCRIPTION

[0063] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] In some of the processes described in the specification and claims of this application and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish between different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this application are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to different types.

[0065] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0066] Figure 1 This is the primer design flow chart. The specific process is as follows:

[0067] 101: Obtain target gene database;

[0068] 102: Screening target gene mutation sites;

[0069] 103: Design primers based on the target gene mutation site.

[0070] In one embodiment, the target gene database is a collection of target genes, and the target gene can be any sequence. In a preferred embodiment, the target gene is a strain sequence, and the strain sequence can include the evolution process of the strain within a certain period and a certain range, that is, any strain sequence that has mutated. In a more preferred embodiment, the strain sequence is an influenza virus sequence. In a specific embodiment, the influenza virus sequence is an H3 subtype influenza virus sequence.

[0071] In one embodiment, the target gene mutation site can be any mutation site of the selected target gene. In a specific embodiment, the H3 subtype influenza virus mutation site includes one or more of c.308T>C, c.357T>A, c.317A>G, c.364A>G, and c.389A>G.

[0072] For example, the sequence can be subjected to c.308T>C and c.357T>A mutations, and primers can be designed, which are recorded as primer 1 (primer 1); on the basis of c.308T>C and c.357T>A, c.317A>G mutations can be performed, and primers can be designed, which are recorded as primer 2 (primer 2); on the basis of c.308T>C and c.357T>A, c.364A>G mutations can be performed, and primers can be designed, which are recorded as primer 3 (primer 3); on the basis of c.308T>C and c.357T>A, c.389A>G mutations can be performed, and primers can be designed, which are recorded as primer 4 (primer 4); on the basis of c.308T>C and c.357T>A, c.317A>G and c.364A>G mutations can be performed, and primers can be designed, which are recorded as primer 5 (primer 5); based on c.308T>C and c.357T>A, c.364A>G and c.389A>G mutations can be performed, and primers are designed, which are recorded as primer 6 (primer 6); based on c.308T>C and c.357T>A, c.317A>G and c.389A>G mutations can be designed, and primers are recorded as primer 7 (primer 7); based on c.308T>C and c.357T>A, c.317A>G, c.364A>G and c.389A>G mutations can be designed, and primers are recorded as primer 8 (primer 8); based on c.308T>C and c.357T>A, a degenerate base R1 (A / G) can be added to position 317, and primers can be designed, and primers are recorded as primer 9 (primer 9); based on c.308T>C and c.357T>A, a degenerate base R2 (A / G) can be added to position 364 to design a primer, which is recorded as primer 10 (primer 10); based on c.308T>C and c.357T>A, a degenerate base Y (C / T) can be added to position 308 to design a primer, which is recorded as primer 11 (primer 11); based on c.308T>C and c.357T>A, a degenerate base R1 (A / G) can be added to position 317 and a degenerate base R2 (A / G) can be added to position 364 to design a primer, which is recorded as primer 12 (primer 12); based on c.308T>C and c.357T>A, a degenerate base R1 (A / G) can be added to position 317 and a degenerate base Y (C / T) can be added to position 308 to design a primer, which is recorded as primer 13 (primer 13); based on c.308T>C and c.357T>A, a degenerate base R2 (A / G) can be added to position 364 and a degenerate base Y (C / T) can be added to position 308 to design a primer, which is recorded as primer 14 (primer 14); based on c.308T>C and c.357T>A, a degenerate base R2 (A / G) can be added to position 364 and a degenerate base Y (C / T) can be added to position 308 to design a primer, which is recorded as primer 14 (primer 14);On the basis of 357T>A, degenerate base R1 (A / G) was added to position 317, degenerate base R2 (A / G) was added to position 364, and degenerate base Y (C / T) was added to position 308, and primers were designed and recorded as primer 15.

[0073] The present invention provides a primer set for detecting influenza virus, wherein the primer set has at least 90% identity with the sequence shown in any one of the primer pairs shown above.

[0074] In one embodiment, primer refers to a short nucleic acid molecule. Can be annealed with complementary target nucleic acid molecules by nucleic acid hybridization, forming a hybrid between primer and target nucleic acid chain. Primer can be extended along target nucleic acid molecules by polymerase. Therefore, primer can be used for amplification of target nucleic acid molecules, wherein the sequence of primer is specific for target nucleic acid molecules, so for example primer will hybridize with target nucleic acid molecules under very high stringency hybridization conditions.

[0075] In one embodiment, primers and primer pairs can be used interchangeably, and the primer pair comprises at least one forward primer and at least one reverse primer, wherein the primer is specific for the amplification of influenza virus nucleic acid molecules. The forward or upstream primer is a primer in the 5' direction relative to a reference point on the nucleic acid sequence; the reverse or downstream primer is a primer in the 3' direction relative to a reference point on the nucleic acid sequence. In general, at least one forward and one reverse primer are included in the amplification reaction.

[0076] At least one of the forward primer and the reverse primer is labeled with a labeling substance.

[0077] The labeling substances include, but are not limited to, fluorescent substances, radioactive isotopes or enzymes.

[0078] Among them, the fluorescent material includes but is not limited to TAMRAT TM , Alexa555, Alexa647, Cy3, Cy5 of the cyanine dye series, and fluorescein.

[0079] Radioisotopes include but are not limited to 32 P. 33 P. 35 S.

[0080] Enzymes include, but are not limited to, alkaline phosphatase, horseradish peroxidase.

[0081] In the primers involved in the present application that are labeled with a labeling substance, the labeling substance may be directly bound to the primer or may be bound via a linker. As a linker, any linker commonly used in the field may be used, specifically, for example, preferably a nucleic acid of 1 to 3 bases, more preferably a DNA of 1 to 3 bases, further preferably a DNA of 2 bases, and particularly preferably the 2 bases of adenine (A)-adenine (A).

[0082] The method of labeling the primers involved in the present application with a fluorescent substance may be carried out according to a method generally known in the art. Specifically, for example, a method of incorporating a nucleotide labeled with fluorescent substance into a primer according to a method known in the art may be mentioned.

[0083] As a method for labeling the primer involved in the present application with a radioisotope, it is only necessary to carry out according to a method known per se that is usually carried out in the field. Specifically, for example, a method of labeling by incorporating a nucleotide labeled with a radioisotope, etc. can be cited. Specifically, a random primer method, a gap translation method, a 5' end labeling method based on T4 polynucleotide kinase, a 3' end labeling method based on terminal deoxynucleotidyl transferase, etc. can be cited. The primer set includes at least one modified nucleotide.

[0084] As a method for labeling the primer involved in the present application with an enzyme, it is sufficient to perform it according to a known method generally performed in the field. Specifically, for example, a direct labeling method in which an enzyme molecule such as alkaline phosphatase or horseradish peroxidase is directly covalently bonded to the primer to be labeled can be cited.

[0085] In one embodiment, identity / similarity refers to the identity / similarity between two or more nucleic acid sequences, or two or more amino acid sequences, expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequence. When aligned using standard methods, homologs or orthologs of nucleic acid or amino acid sequences have a relatively high level of sequence identity / similarity. The primers disclosed herein are not limited to the exact sequence shown, and as will be appreciated by those skilled in the art, the sequences may be altered, if desired, without significantly affecting the ability of the primers to function.

[0086] The present invention provides a kit for detecting influenza virus in a sample, and the kit comprises the above primer set.

[0087] In one embodiment, a suitable amount of one or more primers is provided in one or more containers, or fixed on a substrate, and the primer can be provided to be suspended in an aqueous solution, or, for example, as a freeze-dried or lyophilized powder. The container in which the nucleic acid is provided can be any conventional container capable of accommodating the provided form, such as a microcentrifuge tube, an ampoule or a bottle. The kit can include labeled or unlabeled probes for detecting influenza virus nucleotide sequences.

[0088] In some applications, one or more primers (as described above) can be provided in pre-measured single-use amounts in separate, typically disposable tubes or equivalent containers. Using such an arrangement, a sample to be tested for the presence of influenza virus can be added to a separate tube and amplified directly.

[0089] The amount of nucleic acid primers provided in the test kit can be any suitable amount and can depend on the target market for the product. For example, if the test kit is suitable for research or clinical applications, the amount of each nucleic acid primer provided can be an amount sufficient to trigger several PCR amplification reactions. General guidance for determining suitable amounts can be found in the literature of Innis et al., Sambrook et al., and Ausubel et al. The test kit can contain more than two primers to facilitate PCR amplification of larger amounts of influenza virus nucleotide sequences.

[0090] In certain embodiments, the kit may contain the necessary reagents for performing a PCR amplification reaction, including DNA sample preparation reagents, enzymes for PCR, buffers, Mg 2+ and deoxyribonucleotides (dNTPs).

[0091] The enzymes of PCR include DNA polymerase and / or RNA polymerase.

[0092] The DNA polymerase includes but is not limited to Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, and Klenow fragment.

[0093] The buffer is used to adjust the pH of the sample to be subjected to PCR analysis to pH 7.0 to 10.0 (more preferably pH 8.0 to 9.0).

[0094] The buffer includes, but is not limited to, polymerase buffer and Tris-HCl buffer.

[0095] dNTP is a nucleoside source for PCR-based DNA amplification, and dATP, dGTP, dCTP, and dTTP are necessary. In addition, dNTP can be chemically modified for use in the hot start method, for example, CleanAmp manufactured by TriLink BioTechnologies, Inc. can be used. TM dNTPs.

[0096] In PCR-based DNA amplification, Mg 2+ It is necessary as Mg 2+ Sources include, but are not limited to, MgCl2, MgSO4, etc. MgCl2 is preferred.

[0097] The kit also includes a fluorescent pigment.

[0098] In one embodiment, a variety of known fluorescent pigments can be used. For example, a method using an intercalator with a labeling function, a method using a probe combining a fluorescent substance on a nucleotide specifically hybridized to a relatively amplified DNA sequence, etc. can be cited. As an intercalator, ethidium bromide, SYBRGreenI as an unsaturated fluorescent pigment, Resolight, and EvaGreen as saturated fluorescent pigments can be cited. Preferred intercalators are SYBR Green I as an unsaturated fluorescent pigment, EvaGreen as a saturated fluorescent pigment, and EvaGreen and Resolight as saturated fluorescent pigments, more preferably EvaGreen and Resolight as saturated fluorescent pigments. For usage, the recommendation of the manufacturer of the fluorescent pigment used is followed.

[0099] The kit also includes instructions, which may include guidance on obtaining the sample and processing the sample.

[0100] Furthermore, the kit may contain bacterial genomic DNA used as a positive control for PCR and sterile water used as a negative control.

[0101] When implementing the present application, other necessary instruments include pipettes, pipette tips, 1.5 ml microtubes, and other instruments widely used in molecular biology experiments. Devices include PCR, clean benches, tube centrifuges, and other instruments widely used in molecular biology experiments.

[0102] The present invention provides a primer design system, the system comprising:

[0103] An acquisition unit is used to acquire a target gene database, wherein the target gene database includes a target gene sequence;

[0104] Mutation site screening unit: used to screen the mutation sites of target genes;

[0105] Primer design unit: used to design primers according to target gene mutation sites, wherein the target gene mutation sites include one or more gene mutation sites and / or degenerate bases.

[0106] In one embodiment, the system includes a processor, which may be a single-core or multi-core processor or more than one processor for parallel processing. The system also includes a memory (e.g., random access memory, read-only memory, flash memory), an electronic storage unit (e.g., a hard disk), a communication interface (e.g., a network adapter) and peripheral devices for communicating with one or more other systems, such as a cache memory, other memories, data storage and / or an electronic display adapter. The memory, electronic storage unit, communication interface and peripheral devices communicate with the processor via a communication bus (solid line), such as a motherboard. The storage unit may be a data storage unit (or data repository) for storing data. The system may be operably coupled to a computer network by means of a communication interface. The network may be the Internet, an intranet and / or an extranet, or an intranet and / or an extranet communicating with the Internet. In some cases, the network is a communication and / or data network. The network may include one or more computer servers, which may support distributed computing, such as cloud computing. In some cases, with the aid of the system, the network may implement a peer-to-peer network, which may enable the device coupled to the system to operate as a client or server.

[0107] In one embodiment, the processor may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as a memory. The instructions may be directed to the processor, which may then program or otherwise configure the processor to implement the method of the present application. Examples of operations performed by the processor may include reading, decoding, executing, and writing back.

[0108] In one embodiment, the processor may be part of a circuit such as an integrated circuit, and one or more other components of the system may be included in the circuit, which in some cases is an application specific integrated circuit.

[0109] In one embodiment, the electronic storage unit can store files, such as drivers, libraries, and saved programs. The electronic storage unit can store user data, such as user preferences and user programs. In some cases, the system can include one or more additional data storage units that are external to the computer system, such as located on a remote server that communicates with the system via an intranet or the Internet.

[0110] In one embodiment, the system can communicate with one or more remote computer systems via a network. For example, the system can communicate with a remote computer system of a user (e.g., a physician). Examples of remote computer systems include personal computing, tablet or tablet PCs, phones, smart phones, or personal digital assistants. Users can access the system via a network.

[0111] The present invention provides an electronic device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the steps of the above method when executing the computer program.

[0112] In one embodiment, the electronic device is a computer device, which may be a terminal, including a processor and a memory connected via a system bus; and also including a network interface, a display screen and an input device. Among them, the processor of the computer device is used to provide computing and control capabilities. The memory of the computer device includes a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system and a computer program. The internal memory provides an environment for the operation of the operating system and the computer program in the non-volatile storage medium. The network interface of the computer device is used to communicate with an external terminal through a network connection. The display screen of the computer device may be a liquid crystal display screen or an electronic ink display screen, and the input device of the computer device may be a touch layer covered on the display screen, or a key, trackball or touchpad provided on the housing of the computer device, or an external keyboard, touchpad or mouse, etc.

[0113] The above-mentioned devices are only partial structures related to the scheme of the present application and do not constitute a limitation on the computer devices to which the scheme of the present application is applied. The specific computer devices may include more or fewer components than those shown in the figure, or combine certain components, or have different component arrangements.

[0114] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0115] Example

[0116] The process of primer screening is as follows Figure 1 shown.

[0117] 1 Specimen source

[0118] The focus of the study is on 92 reported samples from 2022 to June 2023, spanning 30 provinces. To ensure the representativeness of the samples, clever sample extraction was carried out according to the data proportions of each province. In this carefully designed sample set, there are 4 samples from Anhui Province, Fujian Province, Jiangsu Province, Qinghai Province and Chongqing City, 3 samples from Beijing, Gansu Province, Guangdong Province, Guangxi Zhuang Autonomous Region, Guizhou Province, Hebei Province, Henan Province, Heilongjiang Province, Hubei Province, Hunan Province, Jilin Province, Jiangxi Province, Liaoning Province, Inner Mongolia Autonomous Region, Shandong Province, Shanxi Province, Shaanxi Province, Shanghai, Sichuan Province, Tianjin City, Yunnan Province, and Zhejiang Province. At the same time, Hainan Province, Ningxia Hui Autonomous Region and Xinjiang Uygur Autonomous Region each have 2 samples. Such meticulous sample distribution ensures full coverage of influenza virus samples from all over the country, providing reliable and comprehensive basic data for the research, such as Figure 2 shown.

[0119] 2. Nucleic Acid Extraction

[0120] For the respiratory specimens of the first confirmed case received, total viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the instructions and stored at -80°C until use.

[0121] 3 Whole genome sequencing

[0122] FluA universal amplification primers and a one-step amplification enzyme system were used to perform whole-genome specific amplification of the extracted viral total RNA. The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen, Germany). The DNA sequencing library was constructed according to the Nextera XT DNA Library Preparation Kit (Illumina, USA), and the whole-genome deep sequencing was performed using the Miniseq sequencer of the Illumina sequencing platform. The CLC Genomics Workbench (Version 20.0) software was used to sequence the raw data from the sequencing machine.

[0123] 4PCR primer design

[0124] First, after nucleic acid extraction, library construction, machine sequencing and data analysis of the strain samples reported by various provinces, municipalities and autonomous regions in China, a total of 755 consistent sequences were obtained; the sequences were aligned using the Nextclade website, and homology analysis was performed with the help of GeneiousPrime software, and the aligned sequences were compared with the original primer sequences. 98.55% (744 / 755) of the sequences mutated at the 308 (TC) site and 97.62% (737 / 755) of the sequences mutated at the 357 (TA) site. Based on this feature, the first pair of new primers was designed. On this basis, it was found that the three sites 317 (AG), 364 (AG) and 389 (AG) had mutated in more provinces ( Figure 3 ), three pairs of primers were designed for these three sites.

[0125] According to the geographical location of the three different variant sites ( Figure 4 ), after marking on the map, it was found that 317 variant sites were mainly concentrated in southern China, with Guizhou Province being the most prominent, followed by Guangdong Province; 364 variant sites were mainly concentrated in central and northern cities in China, with Sichuan Province being the most prominent, followed by Inner Mongolia Autonomous Region; 389 variant sites were mainly concentrated in coastal cities, with Fujian Province, Jiangsu Province, Shanghai and Shandong Province being more prominent.

[0126] Using 92 samples collected from the national reported samples, the old primers and 4 new primers were experimentally verified. It was found that after changing the three sites 317, 364 and 389, no obvious regional distribution appeared. The CT value did not show regular changes ( Figure 5 , Figure 6 ).

[0127] The sensitivity of the four new pairs of primers designed by changing a single base did not change significantly. Therefore, based on the first pair of new primers, four more pairs of primers were designed by permuting and combining the three mutated base sites (317, 364 and 389), including simultaneous changes of sites 317 and 364, simultaneous changes of sites 317 and 389, simultaneous changes of sites 364 and 389, and simultaneous changes of three sites 317, 364 and 389; and on this basis, degenerate bases were introduced for design.

[0128] According to the design method of single base site, 7 pairs of primers were successfully derived by permutation and combination. A total of 15 pairs of primers were carefully designed in this study, such as Figure 7 This series of primers is designed to improve the accuracy and sensitivity of identification, laying a solid foundation for subsequent experimental research.

[0129] The specific primer design method is:

[0130] (1) The subtype is selected as H3.

[0131] (2) Primers for identifying the H3 subtype were designed on the HA fragment, and the HA segment was used as the target gene for primer design.

[0132] (3) Use tools (such as MUFFT, muscle, etc.) to align the downloaded H3 subtype fragments and generate an alignment file.

[0133] (4) Use the software Geneious Primer to perform a match test on the existing primers using the alignment file generated in the previous step.

[0134] (5) After the program is finished, an overview diagram with mismatch information will appear. The result of the most ideal primer is mismatch = 0. Therefore, according to the number of mismatches and their distribution positions in the primers, primers for single-base changes, multiple-base changes and degenerate bases are designed respectively.

[0135] (6) Repeat step (3) using the modified primers to verify whether it is as expected (the number of mismatches is reduced).

[0136] (7) Experimental verification: QPCR experiments were conducted on H3 subtype influenza viruses nationwide. The results were summarized and analyzed and it was found that in the same sample, by reducing the mismatch sites, the CT value can be effectively reduced on the basis of the original primers. In this set of designed primers, the most suitable primers were selected. These primers can effectively detect strains from different geographical locations and reduce their CT values.

[0137] Table 116 pairs of primer sequences

[0138]

[0139]

[0140] 5. Detection Methods

[0141] (1) Sample preparation: 92 original samples of sufficient volume (volume > 1 ml, 200 μl was used for Tianlong nucleic acid extraction). (2) Experimental reagents: Tianlong nucleic acid extraction, FastVirus one-step method, Tianlong PCR instrument.

[0142] (3) Dilute the 16 primer pairs to a concentration of 10 μmol.

[0143] (4) Configuration system (Table 2).

[0144] Table 2 Configuration system

[0145]

[0146] (5) Mix the prepared system and add it to a 96-well plate, adding 20 μl to each well.

[0147] (6) Add samples as shown in Table 3. The total volume of each well is 25 μl. N represents different primers.

[0148] Table 3 Different samples and primer mixtures

[0149]

[0150] (7) Use a high-transmittance membrane to seal the 96-well plate.

[0151] (8) Mix well and centrifuge for 1 min.

[0152] (9) On-line: Place the 96-well plate into the fluorescence quantitative PCR instrument and run the program.

[0153] (10) Amplification procedure:

[0154] ① Create a new program and select the fluorescence channel (FAM) according to the fluorescent label of the probe.

[0155] ② Set the reaction conditions. The specific reaction conditions are shown in Table 4.

[0156] Table 4 Reaction conditions

[0157]

[0158] 6 Primer CT value rules

[0159] 92 samples from 30 provinces and cities across the country were tested using the QPCR method, generating 1472 CT value results. These 1472 experimental results were carefully classified, and the maximum value (expressed as the maximum CT value) and minimum value (expressed as the minimum CT value) under each reaction system were counted to obtain a series of data.

[0160] Through comprehensive analysis of the results of different primers ( Figure 8 ), it was found that under the amplification conditions of primers 5 and 12, the number of minimum values ​​was the largest, 22 and 24 respectively, accounting for 23.91% and 26.08% respectively. Through detailed statistics of the maximum and minimum values, among the 16 pairs of primers, primers 5 and 12 performed more significantly in reducing the CT value than other primers. This result provides a strong experimental basis for the selection and use of primers.

[0161] 7 Mean value between primers

[0162] Considering that the H3 identification primer provided by the current domestic influenza surveillance network is primer 0, taking primer 0 as the benchmark, the differences between 15 pairs of primers and primer 0 are calculated, and the arithmetic mean and variance of these differences are calculated, as follows: Fig. 9 As shown. From the perspective of arithmetic mean, the differences of 6 pairs of primers (primers 3, 5, 6, 10, 12 and 15) are all greater than 1, namely 1.17, 1.23, 1.12, 1.15, 1.37 and 1.18 respectively. Among them, primer 12 has the largest difference among these 6 pairs of primers, with an arithmetic mean of 1.37, followed by primer 5, with an arithmetic mean of 1.23. Therefore, primers 12 and primers 5 are the most significant in reducing CT values.

[0163] From the perspective of variance, the variance of primer 5 is 2.17, while the variance of primer 12 is 0.82. This result shows that primer 12 performs better than primer 5 in terms of variance. In summary, through the comprehensive analysis of arithmetic mean and variance, it is found that primer 12 is better than primer 5 in significantly reducing the CT value.

[0164] Discrete distribution of 8 primers

[0165] The variance of the differences between primer 1, primer 5, primer 12 and primer 0 was analyzed by discrete variance analysis ( Fig.10 ), clearly showing the wide distribution of data points within the entire coordinate axis range. Primer 1 has the highest numerical polymerization degree, all near zero, indicating that the difference of primer 1 does not change much and remains at a relatively stable level; on the contrary, primer 5 shows a large dispersion, with positive and negative deviations. The trend of data fluctuating around zero shows that primer 5 exhibits unstable characteristics in some cases. Most of the values ​​of primer 12 are higher than zero, showing that the overall trend of the data is developing towards positive values. This shows that primer 12 shows a significant effect in reducing the CT value, showing the good performance of the primer.

[0166] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A primer design method, characterized in that: The method comprises: Acquire a target gene database, wherein the target gene database includes a target gene sequence; Screening target gene mutation sites; Primers are designed according to target gene mutation sites, wherein the target gene mutation sites include one or more gene mutation sites and / or degenerate bases.

2. The method according to claim 1, characterized in that: The primer is an influenza virus primer; Preferably, the influenza virus primer is an H3 influenza virus primer; Preferably, the target gene mutation site includes one or more of c.308T>C, c.357T>A, c.317A>G, c.364A>G, c.389A>G; Preferably, the degenerate bases include one or more of R1 at position 317, R2 at position 364, or Y; Here, the degenerate base R represents A or G, and the degenerate base Y represents C or T.

3. A primer set for detecting influenza virus, characterized in that: The primer set comprises a primer pair shown in SEQ ID NOs: 4 and 6, a primer pair shown in SEQ ID NOs: 7 and 9, a primer pair shown in SEQ ID NOs: 10 and 12, a primer pair shown in SEQ ID NOs: 13 and 15, a primer pair shown in SEQ ID NOs: 16 and 18, a primer pair shown in SEQ ID NOs: 19 and 21, a primer pair shown in SEQ ID NOs: 22 and 24, a primer pair shown in SEQ ID NOs: 25 and 27, a primer pair shown in SEQ ID NOs: 28 and 30, a primer pair shown in SEQ ID NOs: 31 and 33, a primer pair shown in SEQ ID NOs: 34 and 36, a primer pair shown in SEQ ID NOs: 37 and 39, a primer pair shown in SEQ ID NOs: 40 and 42, a primer pair shown in SEQ ID NOs: 43 and 45, and a primer pair shown in SEQ ID NOs: 46 and 48, which have at least 90% identity; Preferably, the primer set includes any one of the primer pair shown in SEQ ID NOs: 4 and 6, the primer pair shown in SEQ ID NOs: 7 and 9, the primer pair shown in SEQ ID NOs: 10 and 12, the primer pair shown in SEQ ID NOs: 13 and 15, the primer pair shown in SEQ ID NOs: 16 and 18, the primer pair shown in SEQ ID NOs: 19 and 21, the primer pair shown in SEQ ID NOs: 22 and 24, the primer pair shown in SEQ ID NOs: 25 and 27, the primer pair shown in SEQ ID NOs: 28 and 30, the primer pair shown in SEQ ID NOs: 31 and 33, the primer pair shown in SEQ ID NOs: 34 and 36, the primer pair shown in SEQ ID NOs: 37 and 39, the primer pair shown in SEQ ID NOs: 40 and 42, the primer pair shown in SEQ ID NOs: 43 and 45, and the primer pair shown in SEQ ID NOs: 46 and 48; Preferably, the primer set is selected from the primer pair shown in SEQ ID NOs: 16 and 18 and / or the primer pair shown in SEQ ID NOs: 37 and 39; Preferably, the primer set is selected from the primer pair shown in SEQ ID NO:37 and 39.

4. The primer set according to claim 3, characterized in that: The primer set also includes a probe; Preferably, the probe comprises a sequence having at least 90% identity to any one of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, and SEQ ID NO:47; Preferably, the probe comprises any one of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, and SEQ ID NO:47; Preferably, the probe is selected from SEQ ID NO: 17 and / or SEQ ID NO: 38; Preferably, the probe is selected from SEQ ID NO: 38; Preferably, the influenza virus primer is an H3 influenza virus primer.

5. The primer set according to claim 3, characterized in that: The primer pair includes a forward primer and a reverse primer; Preferably, at least one of the forward primer and the reverse primer is labeled with a labeling substance; Preferably, the labeling substance includes a fluorescent substance, a radioactive isotope or an enzyme; Preferably, the fluorescent substance comprises TAMRAT TM , Alexa555, Alexa647, Cy3, Cy5 of the cyanine dye series, and fluorescein; Preferably, the radioisotope comprises 32 P. 33 P. 35 S; Preferably, the enzyme comprises alkaline phosphatase, horseradish peroxidase.

6. A kit for detecting influenza virus in a sample, characterized in that: The kit comprises the primer set according to any one of claims 3 to 5; Preferably, the kit also includes enzymes for PCR, dNTPs, buffer solution, Mg 2+ at least one reactant; Preferably, the enzyme used for PCR comprises DNA polymerase and / or RNA polymerase; Preferably, the buffer comprises polymerase buffer and Tris hydrochloric acid buffer; Preferably, the kit further comprises instructions; Preferably, the influenza virus primer is an H3 influenza virus primer.

7. A method for detecting influenza virus in a sample, characterized in that: The method comprises amplifying using the primer set according to any one of claims 3 to 5; Preferably, the influenza virus primer is an H3 influenza virus primer.

8. Use of the primer set according to any one of claims 3 to 5 in preparing a kit for detecting influenza virus; Preferably, the influenza virus primer is an H3 influenza virus primer.

9. A primer design system, characterized in that: The system comprises: An acquisition unit is used to acquire a target gene database, wherein the target gene database includes a target gene sequence; Mutation site screening unit: used to screen the mutation sites of target genes; Primer design unit: used to design primers according to the target gene mutation site, wherein the target gene mutation site includes one or more gene mutation sites and / or degenerate bases; Preferably, the primer is an influenza virus primer; Preferably, the influenza virus primer is an H3 influenza virus primer; Preferably, the target gene mutation site includes one or more of c.308T>C, c.357T>A, c.317A>G, c.364A>G, c.389A>G; Preferably, the degenerate bases include one or more of R1 at position 317, R2 at position 364, or Y; Here, the degenerate base R represents A or G, and the degenerate base Y represents C or T.

10. An electronic device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the computer program, the steps of the method according to claim 1 or 2 are implemented.

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