Pneumonic genome detection system and method based on throat swab sample

Abstract

The invention relates to the technical field of molecular biology, in particular to a pneumonia genome detection system and method based on a throat swab sample. The method comprises the following steps: obtaining a pharynx throat swab sample solution, and adding lysozyme for sample digestion to obtain a sterile throat swab sample; the method comprises the following steps: putting a sterile throat swab sample into a nucleic acid extraction kit, and carrying out nucleic acid quality screening treatment on the sterile throat swab sample to obtain a sterile throat swab nucleic acid high-quality sample; the method comprises the following steps: obtaining a pneumonia related pathogen historical genome sequence, and carrying out related pathogen primer design and PCR amplification library construction to obtain a throat swab nucleic acid PCR amplification sequence library; and performing high-throughput sequencing treatment and genomic sequence pathogen comparison detection on each group of throat swab nucleic acid pneumonia gene sequences in the throat swab nucleic acid PCR amplification sequence library to obtain a throat swab sample pneumonia pathogen genome detection result. According to the invention, accurate identification and quantitative analysis of pneumonia pathogens can be realized.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a pneumonia genome detection system and method based on throat swab samples. Background Art

[0002] In recent years, the development of genomic detection technology has provided new possibilities for the rapid identification of pathogens, especially genomic detection methods based on polymerase chain reaction (PCR) and high-throughput sequencing (NGS), which can simultaneously detect multiple pathogens in a short time. These technologies not only improve the sensitivity and specificity of detection, but also can identify pathogens that cannot be detected by conventional culture methods. By non-invasive throat swab sampling, upper respiratory tract samples can be obtained, so that sample processing can be carried out in time to avoid sample degradation, and the genomic DNA of pathogens can be quickly extracted from throat swab samples by using optimized extraction kits. By improving the extraction efficiency and purity, the accuracy of subsequent PCR and sequencing can be ensured. At the same time, by using multiple PCR technology to amplify specific gene fragments of multiple pathogens in the same reaction, this technology can identify common pathogens that cause pneumonia in a short time, including bacteria, viruses and fungi, greatly improving the detection efficiency, and high-throughput sequencing of PCR amplification products to obtain comprehensive genomic information, so that pathogens can be quickly identified through bioinformatics analysis and corresponding drug sensitivity information can be provided. However, traditional pneumonia genomic detection methods mainly rely on culture, serology and imaging, and have problems such as insufficient accuracy and limited detection capabilities for different pathogens. Summary of the invention

[0003] Based on this, it is necessary for the present invention to provide a pneumonia genome detection system and method based on throat swab samples to solve at least one of the above technical problems.

[0004] To achieve the above purpose, a method for detecting pneumonia genome based on throat swab samples comprises the following steps:

[0005] Step S1: obtaining a pharyngeal swab sample solution, and adding lysozyme to digest the bacteria in the pharyngeal swab sample solution to obtain a sterile pharyngeal swab sample; placing the sterile pharyngeal swab sample in a nucleic acid extraction kit containing an RNA / DNA protective agent, and performing nucleic acid quality screening on the nucleic acid sample corresponding to the sterile pharyngeal swab sample by agarose gel electrophoresis to obtain a sterile pharyngeal swab nucleic acid high-quality sample;

[0006] Step S2: Obtain historical genome sequences of pneumonia-related pathogens, and design primers for related pathogens based on the historical genome sequences of pneumonia-related pathogens to obtain specific primers for pneumonia-related pathogens; construct a PCR amplification library for high-quality nucleic acid samples of sterile throat swabs using specific primers for pneumonia-related pathogens and ligase to obtain a PCR amplification sequence library for nucleic acid of throat swabs;

[0007] Step S3: performing high-throughput sequencing on each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library to obtain a high-throughput sequencing sequence corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0008] Step S4: Based on the historical genome sequences of pneumonia-related pathogens, the high-throughput sequencing sequences corresponding to each group of throat swab nucleic acid pneumonia genes are compared and tested for genome sequence pathogens to obtain the genome test results of pneumonia pathogens in throat swab samples.

[0009] Further, step S1 includes the following steps:

[0010] Step S11: obtaining an initial sample of a pharyngeal swab, placing the initial sample of the pharyngeal swab in a sterile test tube, adding an appropriate amount of physiological saline, and shaking the tube thoroughly to obtain a pharyngeal swab sample solution;

[0011] Step S12: measuring the optical density of the pharyngeal swab sample solution to obtain the bacterial concentration of the pharyngeal swab sample solution;

[0012] Step S13: performing an optimization analysis on the bacterial digestion conditions of the corresponding pharyngeal swab sample solution based on the bacterial concentration of the pharyngeal swab sample solution to obtain the optimal digestion conditions of the pharyngeal swab sample bacteria;

[0013] Step S14: performing a sample digestion operation on the corresponding bacteria in the pharyngeal swab sample solution based on the optimal digestion conditions of the pharyngeal swab sample bacteria and adding lysozyme to obtain a pharyngeal swab sample solution after bacterial digestion; performing sterile filtration and testing on the pharyngeal swab sample solution after bacterial digestion to obtain a sterile pharyngeal swab sample;

[0014] Step S15: The sterile throat swab sample is placed in a nucleic acid extraction kit containing an RNA / DNA protective agent, and the nucleic acid sample corresponding to the sterile throat swab sample is screened for nucleic acid quality using agarose gel electrophoresis to obtain a sterile throat swab nucleic acid high-quality sample.

[0015] Further, step S12 includes the following steps:

[0016] Step S121: using a spectrophotometer to perform a preliminary optical property evaluation on the pharyngeal swab sample solution to obtain a pharyngeal swab sample optical property data set at different wavelengths, wherein the pharyngeal swab sample optical property data set includes the pharyngeal swab sample optical transmittance and the pharyngeal swab sample optical absorbance value;

[0017] Step S122: performing wavelength selection optimization analysis on the corresponding pharyngeal swab sample solution based on the pharyngeal swab sample optical characteristic data set at different wavelengths to obtain the optimal measurement wavelength parameter of the pharyngeal swab sample solution;

[0018] Step S123: using a spectrophotometer to measure the optical density of the corresponding pharyngeal swab sample solution based on the optimal measurement wavelength parameter of the pharyngeal swab sample solution, to obtain the optical density measurement result of the pharyngeal swab sample solution;

[0019] Step S124: obtaining a standard bacterial solution of known concentration, and performing linear regression analysis and standard curve construction on the standard bacterial solution of known concentration based on the optimal measurement wavelength parameter of the throat swab sample solution, so as to obtain a mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration;

[0020] Step S125: performing reverse calculation of the bacterial concentration on the optical density measurement result of the throat swab sample solution according to the mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration, so as to obtain the bacterial concentration of the throat swab sample solution.

[0021] Further, step S15 includes the following steps:

[0022] Step S151: performing cold chain storage activity recovery treatment on the sterile throat swab sample to obtain a sterile throat swab activity recovery sample;

[0023] Step S152: placing the sterile throat swab activity recovery sample into a nucleic acid extraction kit containing an RNA / DNA protective agent for sample dilution treatment to obtain a sterile throat swab nucleic acid extraction solution system;

[0024] Step S153: centrifuging the sterile throat swab nucleic acid extraction solution system to obtain a sterile throat swab preliminary nucleic acid extraction solution;

[0025] Step S154: using agarose gel electrophoresis to perform nucleic acid mass electrophoresis analysis on the corresponding nucleic acid samples in the preliminary nucleic acid extract of the sterile throat swab, so as to obtain a nucleic acid fragment mass spectrum corresponding to each sterile throat swab nucleic acid sample;

[0026] Step S155: Based on the nucleic acid fragment mass spectrum corresponding to each sterile throat swab nucleic acid sample, the corresponding nucleic acid sample in the sterile throat swab preliminary nucleic acid extract is subjected to nucleic acid quality screening processing to obtain a sterile throat swab nucleic acid high-quality sample.

[0027] Further, step S155 includes the following steps:

[0028] The nucleic acid fragment mass spectrum corresponding to each sterile throat swab nucleic acid sample is subjected to nucleic acid fragment size and band clarity extraction processing to obtain the nucleic acid fragment size and nucleic acid fragment band clarity corresponding to each sterile throat swab nucleic acid sample;

[0029] Based on the size of nucleic acid fragments and the clarity of nucleic acid fragment bands corresponding to each sterile throat swab nucleic acid sample, the molecular weight cutoff calculation formula of nucleic acid fragments is used to perform a quantitative calculation of the molecular weight cutoff of the corresponding nucleic acid fragment mass spectrum to obtain the molecular weight cutoff value of the sterile throat swab nucleic acid fragment;

[0030] Based on the molecular weight cutoff value of the sterile throat swab nucleic acid fragment, membrane filtration technology is used to perform nucleic acid quality screening on the corresponding nucleic acid samples in the sterile throat swab preliminary nucleic acid extract to obtain a sterile throat swab nucleic acid high-quality sample.

[0031] Furthermore, the molecular weight cutoff calculation formula of the nucleic acid fragment is specifically as follows:

[0032]

[0033] Where M r is the molecular weight cutoff value of nucleic acid fragments from sterile throat swabs, V t is the total volume of the sterile throat swab nucleic acid sample, L is the size of the nucleic acid fragment corresponding to the sterile throat swab nucleic acid sample, C(x) is the clarity of the nucleic acid fragment band at position x of the sterile throat swab nucleic acid sample, M p (x) is the molecular weight of the nucleic acid fragment at position x in the sterile throat swab nucleic acid sample, M m is the reference molecular weight of the sterile throat swab nucleic acid sample, α is the influence index of the nucleic acid fragment molecular weight, β is the attenuation coefficient of the nucleic acid fragment molecular weight, and L c is the standard threshold value of the position size of the nucleic acid fragment, and η is the correction coefficient of the molecular weight cutoff value of the sterile throat swab nucleic acid fragment.

[0034] Further, step S2 includes the following steps:

[0035] Step S21: Obtain historical genome sequences of pneumonia-related pathogens;

[0036] Step S22: Systematically collect and organize historical genome sequences of pneumonia-related pathogens to obtain a historical genome database of pneumonia-related pathogens;

[0037] Step S23: performing sequence alignment and conservative region identification analysis on different historical genome sequences of pneumonia-related pathogens based on the historical genome database of pneumonia-related pathogens to obtain conservative segments of the historical genome of pneumonia-related pathogens;

[0038] Step S24: designing primers for the corresponding historical genome sequences of pneumonia-related pathogens based on the conserved segments of the historical genomes of pneumonia-related pathogens to obtain primers specific to pneumonia-related pathogens;

[0039] Step S25: setting primer annealing PCR reaction conditions for pneumonia-related pathogen-specific primers to obtain pneumonia-related pathogen primer PCR reaction system conditions; performing nucleic acid PCR amplification reaction on sterile throat swab nucleic acid high-quality samples based on the pneumonia-related pathogen primer PCR reaction system conditions to obtain throat swab sample nucleic acid PCR amplification sequence fragments; using ligase to perform library connection construction on throat swab sample nucleic acid PCR amplification sequence fragments to obtain throat swab nucleic acid PCR amplification sequence library.

[0040] Further, step S3 includes the following steps:

[0041] Step S31: performing gene band separation and excision processing on each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library to obtain the gene sequence target band corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0042] Step S32: performing gene sequence characteristic analysis on the gene sequence target bands corresponding to each group of pharyngeal swab nucleic acid pneumonia genes to obtain gene sequence characteristic data corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0043] Step S33: setting the sequencing reaction conditions for the corresponding gene sequence target bands based on the gene sequence characteristic data corresponding to each group of pharyngeal swab nucleic acid pneumonia genes, so as to generate a gene sequencing reaction condition system corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0044] Step S34: Perform high-throughput sequencing on each group of throat swab nucleic acid pneumonia gene sequences in the throat swab nucleic acid PCR amplification sequence library according to the gene sequencing reaction condition system corresponding to each group of throat swab nucleic acid pneumonia genes, so as to obtain a high-throughput sequencing sequence corresponding to each group of throat swab nucleic acid pneumonia genes.

[0045] Further, step S4 includes the following steps:

[0046] Step S41: performing sequence quality control preprocessing on the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes to obtain standard sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0047] Step S42: Perform gene sequence biometric analysis on the historical genome sequence of pneumonia-related pathogens and the standard sequencing sequence corresponding to each group of throat swab nucleic acid pneumonia genes to obtain the historical gene sequence biometrics of pneumonia-related pathogens and each group of throat swab nucleic acid pneumonia gene sequence biometrics;

[0048] Step S43: using a sequence similarity comparison calculation formula to perform sequence similarity comparison calculation on the historical gene sequence biological characteristics of pneumonia-related pathogens and each group of throat swab nucleic acid pneumonia gene sequence biological characteristics, so as to obtain a sequence similarity comparison value between each group of throat swab pneumonia genes and the historical genes of related pathogens;

[0049] Among them, the sequence similarity comparison calculation formula is specifically as follows:

[0050]

[0051] In the formula, S(A,B) is the sequence similarity comparison value between the pharyngeal swab pneumonia gene and the historical gene of the relevant pathogen, A is the historical gene sequence of pneumonia-related pathogens, B is the pharyngeal swab nucleic acid pneumonia gene sequence, n is the total number of biological sub-features in the historical gene sequence of pneumonia-related pathogens, i is the item-order measurement parameter of the biological sub-features of the historical gene sequence, m is the total number of biological sub-features in the pharyngeal swab nucleic acid pneumonia gene sequence, j is the item-order measurement parameter of the biological sub-features of the pharyngeal swab nucleic acid pneumonia gene sequence, a i is the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens, b j is the jth biological sub-feature in the pharyngeal swab nucleic acid pneumonia gene sequence, f(a i ,b j ) is the feature similarity distance value between the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens and the j-th biological sub-feature in the gene sequence of throat swab nucleic acid pneumonia, δ1 is the feature similarity distance weight parameter, g(a i ,b j ) is the additional physicochemical feature similarity contribution value between the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens and the j-th biological sub-feature in the gene sequence of throat swab nucleic acid pneumonia, δ2 is the additional feature similarity contribution weight parameter, P(a i ) is the physicochemical property parameter of the i-th biological sub-characteristic in the historical gene sequence of pneumonia-related pathogens, P(b j ) is the physicochemical property parameter of the jth biological sub-feature in the pharyngeal swab nucleic acid pneumonia gene sequence, gap(A,B) is the number of gaps in the sequence alignment between the historical gene sequence A of pneumonia-related pathogens and the pharyngeal swab nucleic acid pneumonia gene sequence B, and λ is the gap penalty coefficient;

[0052] Step S44: Based on the sequence similarity comparison value between each group of pharyngeal swab pneumonia genes and the relevant pathogen historical genes, the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes are subjected to pathogen comparison detection to obtain the pharyngeal swab sample pneumonia pathogen genome detection results.

[0053] Furthermore, the present invention also provides a pneumonia genome detection system based on a pharyngeal swab sample, which is used to perform the pneumonia genome detection method based on a pharyngeal swab sample as described above, and the pneumonia genome detection system based on a pharyngeal swab sample comprises:

[0054] The sterile throat swab nucleic acid sample screening module is used to obtain a throat swab sample solution, and add lysozyme to digest the bacteria in the throat swab sample solution to obtain a sterile throat swab sample; by placing the sterile throat swab sample in a nucleic acid extraction kit containing an RNA / DNA protective agent, and using agarose gel electrophoresis to perform nucleic acid quality screening on the nucleic acid sample corresponding to the sterile throat swab sample, thereby obtaining a sterile throat swab nucleic acid high-quality sample;

[0055] The throat swab nucleic acid sequence library amplification module is used to obtain the historical genome sequence of pneumonia-related pathogens, and to design primers for related pathogens based on the historical genome sequence of pneumonia-related pathogens to obtain specific primers for pneumonia-related pathogens; the PCR amplification library of high-quality nucleic acid samples of sterile throat swabs is constructed using specific primers for pneumonia-related pathogens and ligase, thereby obtaining a throat swab nucleic acid PCR amplification sequence library;

[0056] The throat swab nucleic acid pneumonia genome sequencing module is used to perform high-throughput sequencing on each group of throat swab nucleic acid pneumonia gene sequences in the throat swab nucleic acid PCR amplification sequence library to obtain high-throughput sequencing sequences corresponding to each group of throat swab nucleic acid pneumonia genes;

[0057] The pharyngeal swab pneumonia genome comparison and detection module is used to perform genome sequence pathogen comparison detection on the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes based on the historical genome sequences of pneumonia-related pathogens, thereby obtaining the pharyngeal swab sample pneumonia pathogen genome detection results.

[0058] Beneficial effects of the present invention:

[0059] 1. The pneumonia genome detection method based on pharyngeal swab samples proposed in the present invention has the beneficial effect of obtaining a pharyngeal swab sample solution and adding lysozyme to digest the bacteria in the pharyngeal swab sample solution, which can effectively lyse the cell walls of the original bacteria in the pharyngeal swab sample solution, thereby eliminating the bacteria originally present in the pharyngeal swab sample solution. The pharyngeal swab sample solution after bacterial digestion is sterile filtered and tested. Sterile filtration is an important step to further ensure the purity of the sample. By removing undigested bacteria and cell debris, a clearer sample can be obtained, which not only reduces interference in subsequent analysis, but also can effectively improve the purity of nucleic acid extraction, thereby ensuring the reliability of subsequent experimental results. At the same time, by placing the sterile throat swab sample in a nucleic acid extraction kit containing RNA / DNA protective agent, the protective agent can effectively prevent the degradation of nucleic acid during the extraction process, ensuring the high quality of the extraction results, and by using agarose gel electrophoresis to screen the nucleic acid quality of the nucleic acid sample corresponding to the sterile throat swab sample, the integrity and purity of the nucleic acid can be visually observed. This step not only helps to confirm whether the extracted nucleic acid meets the requirements of the subsequent application process, but also provides researchers with a visual data support for comparison and analysis. Secondly, by obtaining the historical genome sequence of pneumonia-related pathogens, the historical genome sequence corresponding to the historical pneumonia-related pathogens can be collected through public databases (such as NCBI, EBI), which can deeply analyze the genome characteristics of different pathogens. This systematic collection not only helps to construct the genome information map of pathogens, but also provides basic data for subsequent research. By designing primers for related pathogens based on the historical genome sequence of pneumonia-related pathogens, this step can ensure the specificity of primers between different pathogens, thereby improving the accuracy and sensitivity of PCR amplification, which not only optimizes the detection process, but also enhances the reliability of the subsequent processing process. The success or failure of primer design directly affects the quality of subsequent experimental results. Accurate primers can significantly reduce the risk of nonspecific amplification. By using pneumonia-related pathogen-specific primers and ligases to construct a PCR amplification library for high-quality nucleic acid samples from sterile throat swabs, specific nucleic acid fragments can be integrated into the library for subsequent sequencing and analysis. This step is an indispensable part of genomics and transcriptomics research. The successfully constructed library can provide rich genetic information and assist in subsequent high-throughput sequencing. The construction of the library not only improves the availability of samples, but also makes data analysis of multiple samples and multiple time points possible, thereby improving the monitoring and analysis capabilities of pathogens. This link can provide important tools and data support for a comprehensive understanding of pneumonia-related pathogens.Then, each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library is subjected to high-throughput sequencing. This step, by applying advanced sequencing technology, can quickly and accurately sequence thousands of DNA fragments at the same time. The key to this process is that it greatly improves the throughput and efficiency of sequencing, making large-scale sample genome research feasible, and can also provide rich genome data, which helps to fully understand the genome characteristics of pathogens related to diseases such as pneumonia, thereby accelerating the progress of pneumonia genome research and the transformation of results. Finally, based on the historical genome sequences of pneumonia-related pathogens, the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes are compared with the genome sequence pathogens. This process can compare the standard sequences obtained by high-throughput sequencing with the known pathogen genomes in the database to identify potential infectious pathogens. By calculating the comparison value, quantitative information about the pathogens in the sample can be obtained to help determine their relative abundance and epidemic characteristics, which can significantly improve the detection ability and recognition accuracy of pneumonia pathogens and promote the in-depth development of related research.

[0060] 2. The pneumonia genome detection system based on pharyngeal swab samples proposed in the present invention is composed of a sterile pharyngeal swab nucleic acid sample screening module, a pharyngeal swab nucleic acid sequence library amplification module, a pharyngeal swab nucleic acid pneumonia genome sequencing module and a pharyngeal swab pneumonia genome comparison and detection module. It can realize the pneumonia genome detection method based on pharyngeal swab samples described in the present invention, and is used to combine the operations between the computer programs running on each module to realize the pneumonia genome detection method based on pharyngeal swab samples. The internal structures of the system cooperate with each other, which can greatly reduce duplication of work and manpower investment, and can quickly and effectively provide a more accurate and efficient pneumonia genome detection process based on pharyngeal swab samples, thereby simplifying the operation process of the pneumonia genome detection system based on pharyngeal swab samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments thereof made with reference to the following drawings:

[0062] Figure 1 This is a schematic diagram of the steps of the pneumonia genome detection method based on throat swab samples of the present invention;

[0063] Figure 2 for Figure 1 Detailed step flow diagram of step S1;

[0064] Figure 3 for Figure 2 Detailed step flow diagram of step S12;

[0065] Figure 4This is an example diagram of an experimental record table of optical property data of throat swab samples at different wavelengths of the present invention;

[0066] Figure 5 This is an example diagram of the experimental record table for the bacterial digestion condition optimization analysis process of the present invention. DETAILED DESCRIPTION

[0067] The technical method of the present invention is described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0068] In addition, the accompanying drawings are only schematic illustrations of the present invention and are not necessarily drawn to scale. The same reference numerals in the figures represent the same or similar parts, and their repeated description will be omitted. Some of the block diagrams shown in the accompanying drawings are functional entities and do not necessarily correspond to physically or logically independent entities. The functional entities can be implemented in software form, or implemented in one or more hardware modules or integrated circuits, or implemented in different networks and / or processor methods and / or microcontroller methods.

[0069] It should be understood that, although the terms "first", "second", etc. may be used herein to describe various units, these units should not be limited by these terms. These terms are used only to distinguish one unit from another unit. For example, without departing from the scope of the exemplary embodiments, the first unit may be referred to as the second unit, and similarly the second unit may be referred to as the first unit. The term "and / or" used herein includes any and all combinations of one or more of the listed associated items.

[0070] To achieve this, please refer to Figures 1 to 3 The present invention provides a method for detecting the pneumonia genome based on a throat swab sample, the method comprising the following steps:

[0071] Step S1: obtaining a pharyngeal swab sample solution, and adding lysozyme to digest the bacteria in the pharyngeal swab sample solution to obtain a sterile pharyngeal swab sample; placing the sterile pharyngeal swab sample in a nucleic acid extraction kit containing an RNA / DNA protective agent, and performing nucleic acid quality screening on the nucleic acid sample corresponding to the sterile pharyngeal swab sample by agarose gel electrophoresis to obtain a sterile pharyngeal swab nucleic acid high-quality sample;

[0072] Step S2: Obtain historical genome sequences of pneumonia-related pathogens, and design primers for related pathogens based on the historical genome sequences of pneumonia-related pathogens to obtain specific primers for pneumonia-related pathogens; construct a PCR amplification library for high-quality nucleic acid samples of sterile throat swabs using specific primers for pneumonia-related pathogens and ligase to obtain a PCR amplification sequence library for nucleic acid of throat swabs;

[0073] Step S3: performing high-throughput sequencing on each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library to obtain a high-throughput sequencing sequence corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0074] Step S4: Based on the historical genome sequences of pneumonia-related pathogens, the high-throughput sequencing sequences corresponding to each group of throat swab nucleic acid pneumonia genes are compared and tested for genome sequence pathogens to obtain the genome test results of pneumonia pathogens in throat swab samples.

[0075] In the embodiment of the present invention, please refer to Figure 1 As shown, it is a schematic diagram of the steps of the pneumonia genome detection method based on throat swab samples of the present invention. In this example, the pneumonia genome detection method based on throat swab samples includes the following steps:

[0076] Step S1: obtaining a pharyngeal swab sample solution, and adding lysozyme to digest the bacteria in the pharyngeal swab sample solution to obtain a sterile pharyngeal swab sample; placing the sterile pharyngeal swab sample in a nucleic acid extraction kit containing an RNA / DNA protective agent, and performing nucleic acid quality screening on the nucleic acid sample corresponding to the sterile pharyngeal swab sample by agarose gel electrophoresis to obtain a sterile pharyngeal swab nucleic acid high-quality sample;

[0077] In an embodiment of the present invention, the corresponding initial pharyngeal swab sample is obtained by gently wiping the patient's pharynx, and the collected initial pharyngeal swab sample is immediately placed in a sterile test tube, followed by adding an appropriate amount of normal saline (generally 1-2 ml) to ensure that no additional bacteria are introduced, and after sealing the test tube with a test tube lid, the test tube is gently shaken up and down to ensure that the pharyngeal swab sample is fully mixed with the normal saline, thereby forming a uniform pharyngeal swab sample solution. The fully mixed pharyngeal swab sample solution is taken out and the optical density is measured by using a spectrophotometer, and different wavelengths of the spectrophotometer are set, such as 280nm, 260nm, 240nm and other different wavelengths. After the instrument is preheated, the instrument is calibrated with a blank sample (normal saline), and the pharyngeal swab sample solution is loaded into the cuvette of the photometer to read its optical density value. According to the standard curve of the optical density value and the known bacterial concentration, the bacterial concentration in the pharyngeal swab sample solution can be calculated, and the digestion conditions of the corresponding pharyngeal swab sample solution are optimized and analyzed by combining the bacterial concentration obtained by the previous measurement, so as to set up multiple experimental groups by selecting a variety of different digestion reagent concentrations (such as protease, lysozyme, etc.) and different digestion times and temperatures. For each group, an equal amount of pharyngeal swab sample solution is taken for treatment, and the reaction conditions under different conditions are recorded. The bacterial digestion effect under different digestion conditions is evaluated by regularly sampling and measuring the optical density value of each group (such as Figure 5 As shown), the optimal digestion conditions are selected. At the same time, the corresponding bacteria in the pharyngeal swab sample solution are digested by using the previously determined optimal digestion conditions and preparing a lysozyme solution of corresponding concentration, and the solution is added to the pharyngeal swab sample solution according to the temperature and time of the optimal digestion conditions. The constant temperature is maintained in a constant temperature water bath, and the solution is gently stirred at regular intervals to promote the reaction between the enzyme and the bacteria. After the digestion is completed, the pharyngeal swab sample solution after the bacteria digestion is sterile filtered using a sterile filtration device (such as a 0.22 micron filter membrane) to remove undigested bacteria and large particle impurities, thereby obtaining a sterile pharyngeal swab sample. Then, the sterile throat swab sample obtained after sterile filtration is placed in a nucleic acid extraction kit containing a corresponding RNA / DNA protective agent, and the operation is performed according to the instructions of the kit. During the extraction process, different reagents need to be added according to the steps to ensure that the nucleic acid in the sample is effectively released. Subsequently, the corresponding extracted nucleic acid sample in the sterile throat swab sample is analyzed by agarose gel electrophoresis to prepare 1% agarose gel, use an electrophoresis device to perform electrophoresis, observe and record the migration of nucleic acids, and evaluate the integrity and concentration of nucleic acids based on the electrophoresis results, thereby screening out high-quality sterile throat swab nucleic acid samples, and finally obtaining high-quality sterile throat swab nucleic acid samples.

[0078] Step S2: Obtain historical genome sequences of pneumonia-related pathogens, and design primers for related pathogens based on the historical genome sequences of pneumonia-related pathogens to obtain specific primers for pneumonia-related pathogens; construct a PCR amplification library for high-quality nucleic acid samples of sterile throat swabs using specific primers for pneumonia-related pathogens and ligase to obtain a PCR amplification sequence library for nucleic acid of throat swabs;

[0079] In an embodiment of the present invention, historical genome sequences of pneumonia-related pathogens are retrieved through online databases (such as NCBI GenBank and EMBL-EBI) to ensure that the acquired sequences cover a variety of pneumonia-related pathogens, including bacteria (such as Streptococcus pneumoniae, Legionella pneumophila) and viruses (such as new coronavirus, influenza virus), and by using Bioinformatics tools (such as Entrez Programming Utilities) to write scripts to automatically download genome sequences under specific conditions, thereby obtaining historical genome sequences of pneumonia-related pathogens. By systematically arranging the historical genome sequences of pneumonia-related pathogens previously obtained, the sequences are classified according to pathogen type, genome length and genome characteristics, and a historical genome database is created, and by combining the previously sorted historical genome databases using Clustal Sequence alignment tools such as Omega or MUSCLE perform multiple alignment analyses on historical genome sequences of different pneumonia-related pathogens, and set appropriate alignment parameters (such as Gap opening and closing penalties) to ensure the reliability of the alignment results. After the alignment is completed, software such as PhyloSuite is used to identify and analyze the conservative regions, extract highly conservative regions, and generate a list of conservative segments. At the same time, primer design software such as Primer3 or OligoAnalyzer is used to design pathogen primers for the corresponding pneumonia-related pathogen historical genome sequences by combining the conservative segments of the historical genome obtained by the previous identification analysis to design corresponding specific primers, and set primer design parameters, including primer length (generally 18-24bp), GC content (40%-60%), Tm value (≥55°C), etc., to ensure the specificity and effectiveness of the primers. The BLAST tool is used for alignment to ensure that the designed primers have no significant similarity with non-target pathogen sequences, thereby obtaining pneumonia-related pathogen-specific primers.Then, the primer annealing PCR reaction conditions of the pneumonia-related pathogen-specific primers designed previously were optimized to prepare the PCR reaction system, including DNA template, primer, dNTP, Taq DNA polymerase, buffer and MgCl2, and the gradient PCR method was used to adjust the reaction temperature and primer concentration, and different cycle conditions (such as denaturation temperature, annealing temperature, extension time) were set by the PCR instrument to determine the optimal reaction conditions, and the corresponding sterile throat swab nucleic acid high-quality samples obtained after quality screening were subjected to PCR amplification reaction treatment by combining the previously determined PCR reaction system conditions, so as to ensure that the quality of the extracted nucleic acid meets the PCR requirements by using kits such as QIAamp Viral RNAMini Kit, add the optimized PCR reaction system to the extracted nucleic acid sample, use a thermal cycler for PCR amplification, set the cycle parameters (usually 95°C denaturation, 55-60°C annealing, 72°C extension), and after completion, analyze the PCR product by gel electrophoresis to confirm the amplified nucleic acid fragment, and use ligase to construct the library of the amplified sequence fragment of the throat swab sample previously amplified by PCR, and use T4 DNA ligase is used for ligation reaction. A ligation reaction system is prepared, including the amplified PCR fragment, a linearized vector (such as pUC19) and an appropriate amount of ligase. The reaction conditions (such as temperature and time) are set according to the enzyme instructions. Usually, the reaction is incubated at 16°C overnight to ensure the high efficiency of the ligation. After the reaction is completed, the constructed library is transferred into Escherichia coli for screening by a transformation method to obtain the corresponding PCR amplification sequence library, and finally the throat swab nucleic acid PCR amplification sequence library is obtained.

[0080] Step S3: performing high-throughput sequencing on each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library to obtain a high-throughput sequencing sequence corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0081] In an embodiment of the present invention, high-throughput sequencing is performed on each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library previously obtained by polymerase chain reaction (PCR) amplification, so as to prepare a sequencing reaction system, mix each group of nucleic acid samples with specific primers, sequencing enzymes and reaction buffers thereof to form a reaction system, and add the reaction system to the reaction tank of the sequencing platform, start the high-throughput sequencing program, and after the sequencing is completed, use data analysis software to perform preliminary processing of the sequence data, including data cleaning, quality control and other steps, so as to obtain the corresponding high-throughput sequencing sequence, and finally obtain the high-throughput sequencing sequence corresponding to each group of pharyngeal swab nucleic acid pneumonia genes.

[0082] Step S4: Based on the historical genome sequences of pneumonia-related pathogens, the high-throughput sequencing sequences corresponding to each group of throat swab nucleic acid pneumonia genes are compared and tested for genome sequence pathogens to obtain the genome test results of pneumonia pathogens in throat swab samples.

[0083] In an embodiment of the present invention, high-throughput sequencing sequence data of each group of pharyngeal swab nucleic acid pneumonia genes are obtained, and the sequencing results are evaluated for quality by using professional sequencing quality control software, such as FastQC. The evaluation content includes sequence quality score, sequence repetition rate and joint contamination, and low-quality sequences are trimmed by using tools such as Trimmomatic to remove low-quality bases and joint sequences at the ends of the sequences, thereby generating a standard sequencing sequence that has undergone quality control and trimming, and the historical genome sequences of pneumonia-related pathogens and the standard sequencing sequences of each group of pharyngeal swab samples are compared and analyzed by using bioinformatics tools. Software such as BLAST or BWA can be used to compare gene sequences and extract biological features of each gene sequence, such as gene length, GC content, conserved regions, etc. At the same time, a suitable sequence similarity ratio is formed by combining the biological sub-features corresponding to the historical gene sequences of pneumonia-related pathogens and the biological sub-features corresponding to the standard sequencing sequences of each group of pharyngeal swab samples and related parameters. The calculation formula is used to perform sequence similarity comparison calculations on the biological characteristics of the historical gene sequences of pneumonia-related pathogens and the biological characteristics of each group of throat swab nucleic acid pneumonia gene sequences to evaluate the similarity between the gene sequences of each group of throat swab samples and the historical gene sequences of pneumonia-related pathogens. Then, by combining the sequence similarity comparison values ​​between each group of throat swab pneumonia genes and the historical genes of related pathogens obtained by previous quantitative calculations, a detailed pathogen comparison test is performed on the high-throughput sequencing sequences corresponding to each group of throat swab nucleic acid pneumonia genes, so as to use bioinformatics analysis platforms such as Geneious or Galaxy to perform comprehensive comparison analysis. This analysis will combine historical genome data to calculate the similarity and difference between each sample and the genome of known pneumonia pathogens, so as to ultimately determine the pathogens and their genomic characteristics present in the sample, and determine the pathogen gene with the highest corresponding similarity comparison value as the pathogen present in the throat swab pneumonia gene to be tested, thereby providing accurate pathogen detection results, and finally obtaining the throat swab sample pneumonia pathogen genome detection results.

[0084] Further, step S1 includes the following steps:

[0085] Step S11: obtaining an initial sample of a pharyngeal swab, placing the initial sample of the pharyngeal swab in a sterile test tube, adding an appropriate amount of physiological saline, and shaking the tube thoroughly to obtain a pharyngeal swab sample solution;

[0086] Step S12: measuring the optical density of the pharyngeal swab sample solution to obtain the bacterial concentration of the pharyngeal swab sample solution;

[0087] Step S13: performing an optimization analysis on the bacterial digestion conditions of the corresponding pharyngeal swab sample solution based on the bacterial concentration of the pharyngeal swab sample solution to obtain the optimal digestion conditions of the pharyngeal swab sample bacteria;

[0088] Step S14: performing a sample digestion operation on the corresponding bacteria in the pharyngeal swab sample solution based on the optimal digestion conditions of the pharyngeal swab sample bacteria and adding lysozyme to obtain a pharyngeal swab sample solution after bacterial digestion; performing sterile filtration and testing on the pharyngeal swab sample solution after bacterial digestion to obtain a sterile pharyngeal swab sample;

[0089] Step S15: The sterile throat swab sample is placed in a nucleic acid extraction kit containing an RNA / DNA protective agent, and the nucleic acid sample corresponding to the sterile throat swab sample is screened for nucleic acid quality using agarose gel electrophoresis to obtain a sterile throat swab nucleic acid high-quality sample.

[0090] As an embodiment of the present invention, refer to Figure 2 As shown, Figure 1 Detailed step flow diagram of step S1 in FIG. 1 , in this embodiment, step S1 includes the following steps:

[0091] Step S11: obtaining an initial sample of a pharyngeal swab, placing the initial sample of the pharyngeal swab in a sterile test tube, adding an appropriate amount of physiological saline, and shaking the tube thoroughly to obtain a pharyngeal swab sample solution;

[0092] In an embodiment of the present invention, the corresponding pharyngeal swab initial sample is obtained by gently wiping the patient's pharynx, and the collected pharyngeal swab initial sample is immediately placed in a sterile test tube, and then an appropriate amount of physiological saline (generally 1-2 ml) is added to ensure that no additional bacteria are introduced. After the test tube is sealed with a test tube lid, the test tube is gently shaken up and down to ensure that the pharyngeal swab sample and the physiological saline are fully mixed to form a uniform pharyngeal swab sample solution. This process ensures that the original bacteria and viruses in the pharyngeal swab sample are fully dissolved, and finally a pharyngeal swab sample solution is obtained.

[0093] Step S12: measuring the optical density of the pharyngeal swab sample solution to obtain the bacterial concentration of the pharyngeal swab sample solution;

[0094] In an embodiment of the present invention, a fully mixed pharyngeal swab sample solution is taken out and the optical density is measured by using a spectrophotometer, by setting different wavelengths of the spectrophotometer, such as 280nm, 260nm, 240nm and other different wavelengths, and after the instrument is preheated, a blank sample (normal saline) is used to calibrate the instrument, and the pharyngeal swab sample solution is loaded into the cuvette of the photometer, and its optical density value is read. According to the standard curve of the optical density value and the known bacterial concentration, the bacterial concentration of the pharyngeal swab sample solution can be calculated, and finally the bacterial concentration of the pharyngeal swab sample solution is obtained.

[0095] Step S13: performing an optimization analysis on the bacterial digestion conditions of the corresponding pharyngeal swab sample solution based on the bacterial concentration of the pharyngeal swab sample solution to obtain the optimal digestion conditions of the pharyngeal swab sample bacteria;

[0096] In the embodiment of the present invention, the digestion conditions of the corresponding pharyngeal swab sample solution are optimized and analyzed by combining the previously determined bacterial concentration of the pharyngeal swab sample solution, so as to set up multiple experimental groups by selecting a variety of different digestion reagent concentrations (such as protease, lysozyme, etc.) and different digestion times and temperatures. For each group, an equal amount of pharyngeal swab sample solution is taken for treatment, and the reaction conditions under different conditions are recorded. The bacterial digestion effect under different digestion conditions is evaluated by regularly sampling and measuring the optical density value of each group (such as Figure 5 As shown), the optimal digestion conditions are selected, under which the bacterial digestion effect is the best, and finally the optimal digestion conditions for the bacteria in the throat swab sample are obtained.

[0097] Step S14: performing a sample digestion operation on the corresponding bacteria in the pharyngeal swab sample solution based on the optimal digestion conditions of the pharyngeal swab sample bacteria and adding lysozyme to obtain a pharyngeal swab sample solution after bacterial digestion; performing sterile filtration and testing on the pharyngeal swab sample solution after bacterial digestion to obtain a sterile pharyngeal swab sample;

[0098] In an embodiment of the present invention, by using the previously determined optimal digestion conditions for the throat swab sample bacteria and preparing a lysozyme solution of corresponding concentration to digest the corresponding bacteria in the throat swab sample solution, add it to the throat swab sample solution according to the temperature and time of the optimal digestion conditions, and use a constant temperature water bath to maintain a constant temperature, and stir gently at regular intervals to promote the reaction between the enzyme and the bacteria. After digestion is completed, a throat swab sample solution after bacterial digestion is obtained. At the same time, the throat swab sample solution after bacterial digestion is sterile filtered by using a sterile filtration device (such as a 0.22 micron filter membrane) to remove undigested bacteria and large particle impurities, and finally obtain a sterile throat swab sample.

[0099] Step S15: The sterile throat swab sample is placed in a nucleic acid extraction kit containing an RNA / DNA protective agent, and the nucleic acid sample corresponding to the sterile throat swab sample is screened for nucleic acid quality using agarose gel electrophoresis to obtain a sterile throat swab nucleic acid high-quality sample.

[0100] In an embodiment of the present invention, a sterile throat swab sample previously obtained after sterile filtration is placed in a nucleic acid extraction kit containing a corresponding RNA / DNA protective agent, and the operation is performed according to the instructions of the kit. During the extraction process, different reagents need to be added according to the steps to ensure that the nucleic acid in the sample is effectively released. Subsequently, the corresponding extracted nucleic acid sample in the sterile throat swab sample is analyzed by agarose gel electrophoresis to prepare 1% agarose gel, use an electrophoresis device to perform electrophoresis, observe and record the migration of the nucleic acid, and evaluate the integrity and concentration of the nucleic acid based on the electrophoresis results, thereby screening out high-quality sterile throat swab nucleic acid samples, and finally obtaining sterile throat swab nucleic acid high-quality samples.

[0101] Further, step S12 includes the following steps:

[0102] Step S121: using a spectrophotometer to perform a preliminary optical property evaluation on the pharyngeal swab sample solution to obtain a pharyngeal swab sample optical property data set at different wavelengths, wherein the pharyngeal swab sample optical property data set includes the pharyngeal swab sample optical transmittance and the pharyngeal swab sample optical absorbance value;

[0103] Step S122: performing wavelength selection optimization analysis on the corresponding pharyngeal swab sample solution based on the pharyngeal swab sample optical characteristic data set at different wavelengths to obtain the optimal measurement wavelength parameter of the pharyngeal swab sample solution;

[0104] Step S123: using a spectrophotometer to measure the optical density of the corresponding pharyngeal swab sample solution based on the optimal measurement wavelength parameter of the pharyngeal swab sample solution, to obtain the optical density measurement result of the pharyngeal swab sample solution;

[0105] Step S124: obtaining a standard bacterial solution of known concentration, and performing linear regression analysis and standard curve construction on the standard bacterial solution of known concentration based on the optimal measurement wavelength parameter of the throat swab sample solution, so as to obtain a mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration;

[0106] Step S125: performing reverse calculation of the bacterial concentration on the optical density measurement result of the throat swab sample solution according to the mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration, so as to obtain the bacterial concentration of the throat swab sample solution.

[0107] As an embodiment of the present invention, refer to Figure 3 As shown, Figure 2 Detailed step flow diagram of step S12 in the embodiment, step S12 includes the following steps:

[0108] Step S121: using a spectrophotometer to perform a preliminary optical property evaluation on the pharyngeal swab sample solution to obtain a pharyngeal swab sample optical property data set at different wavelengths, wherein the pharyngeal swab sample optical property data set includes the pharyngeal swab sample optical transmittance and the pharyngeal swab sample optical absorbance value;

[0109] In an embodiment of the present invention, a preliminary optical property evaluation is performed on a previously obtained pharyngeal swab sample solution by using a spectrophotometer. First, a pharyngeal swab sample solution is prepared, and the pharyngeal swab sample is placed in physiological saline and subjected to appropriate centrifugation to remove large particles and obtain a clear supernatant. Subsequently, the sample is measured at different wavelengths such as 280 nm, 260 nm, and 240 nm using a spectrophotometer, and at each wavelength, the light transmittance and absorbance values ​​are recorded to ensure that the zero point and light source stability of the spectrophotometer are calibrated during the operation to obtain an accurate optical property data set, and finally a pharyngeal swab sample optical property data set at different wavelengths is obtained. This data set will include the optical transmittance and absorbance values ​​of the pharyngeal swab sample at different wavelengths (such as Figure 4 shown).

[0110] Step S122: performing wavelength selection optimization analysis on the corresponding pharyngeal swab sample solution based on the pharyngeal swab sample optical characteristic data set at different wavelengths to obtain the optimal measurement wavelength parameter of the pharyngeal swab sample solution;

[0111] In an embodiment of the present invention, the wavelength selection of the corresponding pharyngeal swab sample solution is optimized by combining the optical property data set of pharyngeal swab samples at different wavelengths obtained by previous evaluation and analysis, so as to perform statistical analysis on the optical transmittance and absorbance values ​​at different wavelengths by using data analysis software (such as Excel or Matlab), draw a curve of optical transmittance and absorbance changing with wavelength, and identify the wavelength corresponding to the highest absorbance by comparing the absorbance values ​​at different wavelengths. This wavelength will be determined as the optimal measurement wavelength parameter. During the analysis process, attention should be paid to the repeatability experiment of absorbance to ensure that the finally selected wavelength has a higher signal-to-noise ratio and lower background interference, so as to finally obtain the optimal measurement wavelength parameter of the pharyngeal swab sample solution.

[0112] Step S123: using a spectrophotometer to measure the optical density of the corresponding pharyngeal swab sample solution based on the optimal measurement wavelength parameter of the pharyngeal swab sample solution, to obtain the optical density measurement result of the pharyngeal swab sample solution;

[0113] In an embodiment of the present invention, the optical density of the pharyngeal swab sample solution is measured again using a spectrophotometer in combination with the optimal measurement wavelength parameters of the pharyngeal swab sample solution previously determined experimentally. During this process, the spectrophotometer is adjusted to the optimal wavelength previously determined, and a sample container is prepared to ensure that the light path is clear and free of impurities. During the measurement process, multiple readings should be taken continuously and their average values ​​calculated to improve the accuracy of the measurement results, thereby obtaining the corresponding optical density value, and finally obtaining the optical density measurement result of the pharyngeal swab sample solution.

[0114] Step S124: obtaining a standard bacterial solution of known concentration, and performing linear regression analysis and standard curve construction on the standard bacterial solution of known concentration based on the optimal measurement wavelength parameter of the throat swab sample solution, so as to obtain a mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration;

[0115] In an embodiment of the present invention, after obtaining a standard bacterial solution of known concentration in advance, a linear regression analysis and a standard curve construction are performed on the standard bacterial solution of known concentration based on the previously determined optimal measurement wavelength parameters of the throat swab sample solution. A series of standard bacterial solutions of different concentrations are configured, and the optical density is measured using the same spectrophotometer. The optical density value corresponding to each concentration is recorded, and the optical density value and the bacterial concentration data are input into the statistical analysis software. The linear regression method is used to calculate the slope and intercept of the best fitting straight line, and a standard curve is constructed. The standard curve will form a mathematical relationship model between the bacterial concentration and the optical density, and finally a mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration is constructed.

[0116] Step S125: performing reverse calculation of the bacterial concentration on the optical density measurement result of the throat swab sample solution according to the mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration, so as to obtain the bacterial concentration of the throat swab sample solution.

[0117] In an embodiment of the present invention, based on the constructed mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration, the bacterial concentration is reversely calculated according to the optical density measurement result of the throat swab sample solution obtained previously, so as to substitute the previously obtained optical density value into the equation of the standard curve and use the equation to calculate the corresponding bacterial concentration. In this process, attention should be paid to the unit consistency of the data, and it should be ensured that the optical density value is within the range of the standard curve. Through this calculation process, the concentration of microorganisms in the throat swab sample solution is obtained, and finally the bacterial concentration of the throat swab sample solution is obtained.

[0118] Further, step S15 includes the following steps:

[0119] Step S151: performing cold chain storage activity recovery treatment on the sterile throat swab sample to obtain a sterile throat swab activity recovery sample;

[0120] In an embodiment of the present invention, the sterile throat swab sample obtained after aseptic digestion is placed in a suitable refrigeration device, the temperature is controlled at 2-8 degrees Celsius, and the processing is maintained within this temperature range for 24 hours. Before processing, the throat swab sample is placed in a biological safety cabinet to ensure a sterile environment during the operation, and the sample is transferred to a resuscitation fluid for incubation using a culture medium containing specific resuscitation components, so that the surviving microorganisms in the sample can be reactivated, ensuring that the activity of the sample is maintained, and finally a sterile throat swab activity-recovered sample is obtained.

[0121] Step S152: placing the sterile throat swab activity recovery sample into a nucleic acid extraction kit containing an RNA / DNA protective agent for sample dilution treatment to obtain a sterile throat swab nucleic acid extraction solution system;

[0122] In an embodiment of the present invention, a sterile throat swab activity recovery sample that has been previously restored is placed in a commercial nucleic acid extraction kit, which should contain an RNA / DNA protective agent to prevent nucleic acid degradation during the extraction process, and a certain amount of the sterile throat swab activity recovery sample is added to the diluent in the kit in accordance with the requirements of the kit instructions, usually in a ratio of 1:5 or 1:10, and the specific ratio depends on the experimental design. The mixture is gently inverted several times to ensure that the sample is evenly mixed and to avoid the generation of bubbles. This process needs to be carried out on ice to reduce the effect of temperature on the sample, and finally a sterile throat swab nucleic acid extraction solution system is obtained.

[0123] Step S153: centrifuging the sterile throat swab nucleic acid extraction solution system to obtain a sterile throat swab preliminary nucleic acid extraction solution;

[0124] In an embodiment of the present invention, the sterile throat swab nucleic acid extraction solution system obtained by previous dilution is centrifuged to transfer the mixed nucleic acid extraction solution to a pre-cooled centrifuge tube, and the centrifuge speed is set to 8000 rpm and the centrifugation time is 10 minutes. This step is intended to separate the cellular components in the sample from the nucleic acids in the solution through the action of centrifugal force to form a precipitate. After the centrifugation is completed, the centrifuge tube is carefully removed, the supernatant is removed, ensuring that the precipitate is not disturbed, and the precipitate is re-suspended with an appropriate amount of extraction buffer and gently mixed to form a preliminary nucleic acid extract, thereby finally obtaining a sterile throat swab preliminary nucleic acid extract.

[0125] Step S154: using agarose gel electrophoresis to perform nucleic acid mass electrophoresis analysis on the corresponding nucleic acid samples in the preliminary nucleic acid extract of the sterile throat swab, so as to obtain a nucleic acid fragment mass spectrum corresponding to each sterile throat swab nucleic acid sample;

[0126] In an embodiment of the present invention, agarose gel electrophoresis is used to perform nucleic acid mass electrophoresis analysis on the corresponding nucleic acid samples in the preliminary nucleic acid extract of the sterile throat swab obtained after centrifugation, so as to prepare agarose gel, the concentration of which is generally 1%-2%, and solidify it in an electrophoresis tank, and inject an electrophoresis buffer (such as TAE or TBE) into the electrophoresis tank to ensure that the electrodes are well connected. Subsequently, an appropriate amount of the preliminary nucleic acid extract is mixed with the loading buffer in a ratio of 1:1, and a micropipette is used to carefully add the mixture to the wells of the agarose gel, and the electrophoresis conditions are set, such as a voltage of 100V and an electrophoresis time of about 30-60 minutes, and the migration of the nucleic acid sample is observed. After the electrophoresis is completed, the gel is irradiated with ultraviolet light, and a quality spectrum is taken to evaluate the integrity and concentration of the nucleic acid in each sample, and finally a nucleic acid fragment quality spectrum corresponding to each sterile throat swab nucleic acid sample is obtained.

[0127] Step S155: Based on the nucleic acid fragment mass spectrum corresponding to each sterile throat swab nucleic acid sample, the corresponding nucleic acid sample in the sterile throat swab preliminary nucleic acid extract is subjected to nucleic acid quality screening processing to obtain a sterile throat swab nucleic acid high-quality sample.

[0128] In an embodiment of the present invention, the quality of the corresponding nucleic acid samples in the preliminary nucleic acid extract of the sterile throat swab is screened by combining the nucleic acid fragment mass map corresponding to each sterile throat swab nucleic acid sample previously obtained by agarose gel electrophoresis analysis, so as to identify high-quality nucleic acid samples by comparing the intensity and integrity of each nucleic acid band in the map. For samples with clear bands and moderate intensity, their characteristics are recorded and subsequently processed, while samples with blurred bands or no bands are marked as low-quality samples and excluded from further analysis. After screening, all high-quality samples are stored in a low-temperature refrigerator at -80°C to ensure their stability, and finally high-quality nucleic acid samples of sterile throat swabs are screened.

[0129] Further, step S155 includes the following steps:

[0130] The nucleic acid fragment mass spectrum corresponding to each sterile throat swab nucleic acid sample is subjected to nucleic acid fragment size and band clarity extraction processing to obtain the nucleic acid fragment size and nucleic acid fragment band clarity corresponding to each sterile throat swab nucleic acid sample;

[0131] In an embodiment of the present invention, the nucleic acid fragment mass spectrum is analyzed by gel electrophoresis technology for each sterile throat swab nucleic acid sample, the extracted nucleic acid sample is electrophoresed through agarose gel, and agarose gel of appropriate concentration (e.g., 1% to 2%) is selected to achieve the best separation effect. During the electrophoresis, a suitable voltage (e.g., 80-120V) is used to continuously perform electrophoresis for about 30-60 minutes. After the nucleic acid fragments migrate in the gel, the gel is stained with a nucleic acid dye (e.g., ethidium bromide or SYBR Green). After staining, the image is recorded by an ultraviolet imaging system, and the banding pattern and size of each nucleic acid fragment are analyzed by using image processing software, thereby forming comprehensive data on the size of the nucleic acid fragment and the clarity of the banding pattern, and finally obtaining the size of the nucleic acid fragment and the clarity of the nucleic acid fragment banding pattern corresponding to each sterile throat swab nucleic acid sample.

[0132] Preferably, based on the nucleic acid fragment size and nucleic acid fragment band clarity corresponding to each sterile throat swab nucleic acid sample, the molecular weight cutoff calculation formula of the nucleic acid fragment molecular weight cutoff is used to perform a quantitative calculation of the molecular weight cutoff of the corresponding nucleic acid fragment mass spectrum to obtain the sterile throat swab nucleic acid fragment molecular weight cutoff value;

[0133] In an embodiment of the present invention, a suitable nucleic acid fragment molecular weight cutoff calculation formula is formed by combining the total volume of the sterile pharyngeal swab nucleic acid sample, the size of the nucleic acid fragment corresponding to the sterile pharyngeal swab nucleic acid sample, the clarity of the nucleic acid fragment banding corresponding to the sterile pharyngeal swab nucleic acid sample, the molecular weight of the nucleic acid fragment corresponding to the sterile pharyngeal swab nucleic acid sample, the reference molecular weight of the sterile pharyngeal swab nucleic acid sample, the nucleic acid fragment molecular weight influence index, the nucleic acid fragment molecular weight attenuation coefficient, the nucleic acid fragment position size standard threshold and related parameters to perform a molecular weight cutoff quantitative calculation on the corresponding nucleic acid fragment mass spectrum, so as to quantitatively calculate the molecular weight cutoff value corresponding to each sterile pharyngeal swab nucleic acid sample, ensure that it can reflect the distribution of nucleic acid fragments in a specific mass spectrum, and finally obtain the sterile pharyngeal swab nucleic acid fragment molecular weight cutoff value.

[0134] Preferably, based on the molecular weight cutoff value of the sterile pharyngeal swab nucleic acid fragment, the corresponding nucleic acid sample in the sterile pharyngeal swab preliminary nucleic acid extract is subjected to nucleic acid quality screening processing using membrane filtration technology to obtain a sterile pharyngeal swab nucleic acid high-quality sample.

[0135] In an embodiment of the present invention, the quality of the corresponding nucleic acid sample in the preliminary nucleic acid extract of the sterile pharyngeal swab is screened by using membrane filtration technology in combination with the molecular weight cutoff value of the sterile pharyngeal swab nucleic acid fragment obtained by quantitative calculation previously, so as to select a suitable membrane filtration device, place the preliminary nucleic acid extract in the membrane filtration device, ensure that the membrane pore size used matches the molecular weight cutoff value calculated previously, and pass the extract through the filter membrane under negative pressure or air pressure, so that nucleic acid fragments smaller than the preset molecular weight are retained on the other side of the membrane, collect the liquid that passes through the membrane, and extract the high-quality nucleic acid sample, so as to finally obtain a high-quality nucleic acid sample of the sterile pharyngeal swab.

[0136] Furthermore, the molecular weight cutoff calculation formula of the nucleic acid fragment is specifically as follows:

[0137]

[0138] Where M r is the molecular weight cutoff value of nucleic acid fragments from sterile throat swabs, V t is the total volume of the sterile throat swab nucleic acid sample, L is the size of the nucleic acid fragment corresponding to the sterile throat swab nucleic acid sample, C(x) is the clarity of the nucleic acid fragment band at position x of the sterile throat swab nucleic acid sample, M p (x) is the molecular weight of the nucleic acid fragment at position x in the sterile throat swab nucleic acid sample, M m is the reference molecular weight of the sterile throat swab nucleic acid sample, α is the influence index of the nucleic acid fragment molecular weight, β is the attenuation coefficient of the nucleic acid fragment molecular weight, and L c is the standard threshold value of the position size of the nucleic acid fragment, and η is the correction coefficient of the molecular weight cutoff value of the sterile throat swab nucleic acid fragment.

[0139] The present invention obtains a nucleic acid fragment molecular weight cutoff calculation formula by using a specific mathematical model and after verification, which is used to perform molecular weight cutoff quantitative calculation on the corresponding nucleic acid fragment mass spectrum. The nucleic acid fragment molecular weight cutoff calculation formula is comprehensively evaluated by multiple parameters such as nucleic acid fragment size, band clarity and molecular weight, and can more accurately quantify the quality of nucleic acid fragments. By adjusting parameters such as α, β and η, it can be optimized for the characteristics of different experimental samples, improve the sensitivity and specificity of detection, and reduce the risk of false positives and false negatives. By calculating the molecular weight cutoff value, combined with membrane filtration technology, low-quality or degraded nucleic acid fragments are effectively removed to ensure that the obtained nucleic acid sample is of high purity. The calculation formula can be applied to different types of nucleic acid samples (such as DNA and RNA), so that it has a wide range of applicability in various experiments and researches, and the quantified molecular weight cutoff value can be used as the basis for experimental decision-making, helping researchers select the best nucleic acid sample for subsequent analysis and experiments. In summary, the formula fully considers the molecular weight cutoff value M of sterile throat swab nucleic acid fragments. r, the total volume of sterile throat swab nucleic acid sample V t , the size of the nucleic acid fragment corresponding to the sterile throat swab nucleic acid sample L, the clarity of the nucleic acid fragment banding C(x) at position x of the sterile throat swab nucleic acid sample, the molecular weight M of the nucleic acid fragment at position x of the sterile throat swab nucleic acid sample p (x), reference molecular weight of sterile throat swab nucleic acid sample M m , nucleic acid fragment molecular weight influence index α, nucleic acid fragment molecular weight attenuation coefficient β, nucleic acid fragment position size standard threshold L c , the correction coefficient η of the molecular weight cutoff value of the sterile throat swab nucleic acid fragment is based on the molecular weight cutoff value M of the sterile throat swab nucleic acid fragment. r The correlation between the above parameters constitutes a functional relationship The formula can realize the quantitative calculation process of the molecular weight cutoff of the corresponding nucleic acid fragment mass spectrum. At the same time, the introduction of the correction coefficient η of the molecular weight cutoff value of the sterile pharyngeal swab nucleic acid fragment can be adjusted according to the errors occurring in the calculation process, thereby improving the accuracy and applicability of the calculation formula for the molecular weight cutoff of nucleic acid fragments.

[0140] Further, step S2 includes the following steps:

[0141] Step S21: Obtain historical genome sequences of pneumonia-related pathogens;

[0142] In an embodiment of the present invention, historical genome sequences of pneumonia-related pathogens are retrieved through online databases (such as NCBI GenBank and EMBL-EBI) to ensure that the acquired sequences cover a variety of pneumonia-related pathogens, including bacteria (such as Streptococcus pneumoniae, Legionella pneumophila) and viruses (such as new coronavirus, influenza virus), and by using Bioinformatics tools (such as Entrez Programming Utilities) to write scripts to automatically download genome sequences under specific conditions, the integrity and accuracy of the sequences are ensured, and finally the historical genome sequences of pneumonia-related pathogens are obtained.

[0143] Step S22: Systematically collect and organize historical genome sequences of pneumonia-related pathogens to obtain a historical genome database of pneumonia-related pathogens;

[0144] In an embodiment of the present invention, the historical genome sequences of pneumonia-related pathogens previously obtained are systematically sorted out, and format conversion and preliminary analysis are performed through Bioinformatics software (such as Geneious or MEGA), and the sequences are classified according to pathogen type, genome length and genome characteristics by using data sorting methods to create a historical genome database. The database should include metadata of the sequences, such as biological species, acquisition date and sample source, and finally a historical genome database of pneumonia-related pathogens is obtained.

[0145] Step S23: performing sequence alignment and conservative region identification analysis on different historical genome sequences of pneumonia-related pathogens based on the historical genome database of pneumonia-related pathogens to obtain conservative segments of the historical genome of pneumonia-related pathogens;

[0146] In an embodiment of the present invention, by combining the previously sorted historical genome database of pneumonia-related pathogens, multiple alignment analysis is performed on different historical genome sequences of pneumonia-related pathogens using sequence alignment tools such as Clustal Omega or MUSCLE, so as to set appropriate alignment parameters (such as Gap opening and closing penalty) to ensure the reliability of the alignment results. After the alignment is completed, software such as PhyloSuite is used to identify and analyze the conserved regions, and by calculating the conservation scores, highly conserved regions are extracted, and a list of conserved segments is generated, and finally the conserved segments of the historical genome of pneumonia-related pathogens are obtained.

[0147] Step S24: designing primers for the corresponding historical genome sequences of pneumonia-related pathogens based on the conserved segments of the historical genomes of pneumonia-related pathogens to obtain primers specific to pneumonia-related pathogens;

[0148] In an embodiment of the present invention, the pathogen primers are designed for the corresponding pneumonia-related pathogen historical genome sequences using primer design software such as Primer3 or OligoAnalyzer in combination with the conserved segments of the pneumonia-related pathogen historical genome obtained by the previous identification analysis, so as to design corresponding specific primers, and set primer design parameters, including primer length (generally 18-24bp), GC content (40%-60%), Tm value (≥55°C), etc., to ensure the specificity and effectiveness of the primers, and compare them using the BLAST tool to ensure that the designed primers have no significant similarity with the non-target pathogen sequences, so as to further improve the specificity of the detection, and finally obtain pneumonia-related pathogen-specific primers.

[0149] Step S25: setting primer annealing PCR reaction conditions for pneumonia-related pathogen-specific primers to obtain pneumonia-related pathogen primer PCR reaction system conditions; performing nucleic acid PCR amplification reaction on sterile throat swab nucleic acid high-quality samples based on the pneumonia-related pathogen primer PCR reaction system conditions to obtain throat swab sample nucleic acid PCR amplification sequence fragments; using ligase to perform library connection construction on throat swab sample nucleic acid PCR amplification sequence fragments to obtain throat swab nucleic acid PCR amplification sequence library.

[0150] In an embodiment of the present invention, an optimization experiment of primer annealing PCR reaction conditions is performed on the pneumonia-related pathogen-specific primers previously designed and generated, so as to prepare a PCR reaction system, including a DNA template, primers, dNTPs, Taq DNA polymerase, a buffer and MgCl2, and adjust the reaction temperature and primer concentration by a gradient PCR method, set different cycle conditions (such as denaturation temperature, annealing temperature, extension time) by a PCR instrument, and detect the specificity and band clarity of the PCR product by gel electrophoresis to determine the optimal reaction conditions, thereby obtaining the pneumonia-related pathogen primer PCR reaction system conditions. At the same time, by combining the previously determined pneumonia-related pathogen primer PCR reaction system conditions, the corresponding high-quality nucleic acid samples of sterile throat swabs obtained after quality screening are subjected to PCR amplification reaction treatment, so as to ensure that the quality of the extracted nucleic acid meets the PCR requirements by using kits such as QIAampViral RNAMini Kit. The optimized PCR reaction system is added to the extracted nucleic acid sample, and PCR amplification is performed using a thermal cycler, and the cycle parameters are set (usually 95°C denaturation, 55-60°C annealing, and 72°C extension). After completion, the PCR products are analyzed by gel electrophoresis to confirm the amplified nucleic acid fragments, thereby obtaining the PCR amplification sequence fragments of the throat swab sample nucleic acid. Then, the library is constructed by using a ligase to construct the PCR amplified sequence fragments of the throat swab sample nucleic acid that has been previously amplified by PCR, and T4 DNA ligase is used for ligation reaction. A ligation reaction system is prepared, including the amplified PCR fragments, a linearized vector (such as pUC19) and an appropriate amount of ligase, and the reaction conditions (such as temperature and time) are set according to the enzyme instructions. Usually, incubate at 16°C overnight to ensure the efficiency of the ligation. After the reaction is completed, the constructed library is transferred into Escherichia coli for screening by a transformation method to obtain the corresponding PCR amplification sequence library, and finally a throat swab nucleic acid PCR amplification sequence library is obtained.

[0151] Further, step S3 includes the following steps:

[0152] Step S31: performing gene band separation and excision processing on each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library to obtain the gene sequence target band corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0153] In an embodiment of the present invention, gene bands of each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library previously obtained by polymerase chain reaction (PCR) amplification are separated, and the PCR amplification products are separated by using agarose gel electrophoresis, agarose gel of appropriate concentration is prepared, and the PCR product is mixed with the loading buffer and loaded into the gel well, and an electric field is applied by an electrophoresis instrument. After the electrophoresis is completed, the gene bands are observed using an ultraviolet lamp, and the bands that match the target size are selected and cut using a gel extraction tool to obtain the corresponding gene sequence target bands, thereby ensuring that the specific gene sequence is separated from the complex background, and finally obtaining the gene sequence target bands corresponding to each group of pharyngeal swab nucleic acid pneumonia genes.

[0154] Step S32: performing gene sequence characteristic analysis on the gene sequence target bands corresponding to each group of pharyngeal swab nucleic acid pneumonia genes to obtain gene sequence characteristic data corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0155] In an embodiment of the present invention, the characteristics of the target gene sequence bands corresponding to each group of throat swab nucleic acid pneumonia genes previously obtained are analyzed, and DNA is extracted from the cut bands using a gel extraction kit, and then the concentration and purity of the extracted DNA are determined using a spectrophotometer. The extracted DNA samples are sent to the gene analysis software, and sequence alignment and feature extraction are performed using bioinformatics tools. The similarity between the target gene sequence and the known gene sequence is analyzed by using database tools such as BLAST (Basic Local Alignment Search Tool), the homology and variation sites are calculated, and the corresponding gene sequence characteristic data are generated in combination with sequence characteristics (such as GC content, length, etc.), and finally the gene sequence characteristic data corresponding to each group of throat swab nucleic acid pneumonia genes are obtained.

[0156] Step S33: setting the sequencing reaction conditions for the corresponding gene sequence target bands based on the gene sequence characteristic data corresponding to each group of pharyngeal swab nucleic acid pneumonia genes, so as to generate a gene sequencing reaction condition system corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0157] In an embodiment of the present invention, the sequencing conditions of the corresponding gene sequence target bands are set by combining the gene sequence characteristic data corresponding to each group of throat swab nucleic acid pneumonia genes obtained by previous analysis to set the conditions of the gene sequencing reaction. The specific operation includes selecting a suitable sequencing platform (such as Illumina, PacBio, etc.), and adjusting the various parameters in the sequencing reaction system according to the sequence characteristic data of the target gene, such as enzyme concentration, primer design and reaction time. By using the principles of synthetic biology, specific primers are designed to ensure that they can effectively bind to the target sequence. According to the sequencing experience in the literature and the laboratory, the appropriate number of amplification cycles and reaction temperature are determined to ensure that the interaction between the components is optimal, so as to generate a sequencing reaction condition system suitable for each group of throat swab nucleic acid pneumonia genes, and finally set the gene sequencing reaction condition system corresponding to each group of throat swab nucleic acid pneumonia genes.

[0158] Step S34: Perform high-throughput sequencing on each group of throat swab nucleic acid pneumonia gene sequences in the throat swab nucleic acid PCR amplification sequence library according to the gene sequencing reaction condition system corresponding to each group of throat swab nucleic acid pneumonia genes, so as to obtain a high-throughput sequencing sequence corresponding to each group of throat swab nucleic acid pneumonia genes.

[0159] In an embodiment of the present invention, a high-throughput sequencing process is performed on each group of pharyngeal swab nucleic acid pneumonia gene sequences corresponding to the pharyngeal swab nucleic acid PCR amplification sequence library according to the gene sequencing reaction condition system corresponding to each group of pharyngeal swab nucleic acid pneumonia genes previously set, so as to prepare a sequencing reaction system, mix each group of nucleic acid samples with specific primers, sequencing enzymes and reaction buffers thereof to form a reaction system, and add the reaction system to the reaction tank of the sequencing platform, start the high-throughput sequencing program, and after the sequencing is completed, use data analysis software to perform preliminary processing of the sequence data, including data cleaning, quality control and other steps, so as to obtain the corresponding high-throughput sequencing sequence, and finally obtain the high-throughput sequencing sequence corresponding to each group of pharyngeal swab nucleic acid pneumonia genes.

[0160] Further, step S4 includes the following steps:

[0161] Step S41: performing sequence quality control preprocessing on the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes to obtain standard sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes;

[0162] In an embodiment of the present invention, high-throughput sequencing sequence data of each group of pharyngeal swab nucleic acid pneumonia genes are obtained, and the sequencing results are evaluated for quality by using professional sequencing quality control software, such as FastQC. The evaluation content includes sequence quality score, sequence repetition rate, and connector contamination, and low-quality sequences are trimmed using tools such as Trimmomatic to remove low-quality bases and connector sequences at the ends of the sequences, thereby generating a standard sequencing sequence that has been quality controlled and trimmed, and finally obtaining a standard sequencing sequence corresponding to each group of pharyngeal swab nucleic acid pneumonia genes.

[0163] Step S42: Perform gene sequence biometric analysis on the historical genome sequence of pneumonia-related pathogens and the standard sequencing sequence corresponding to each group of throat swab nucleic acid pneumonia genes to obtain the historical gene sequence biometrics of pneumonia-related pathogens and each group of throat swab nucleic acid pneumonia gene sequence biometrics;

[0164] In an embodiment of the present invention, by using bioinformatics tools to compare and analyze the historical genome sequences of pneumonia-related pathogens and the standard sequencing sequences of each group of pharyngeal swab samples, software such as BLAST or BWA can be used to compare gene sequences and extract the biological characteristics of each gene sequence, such as gene length, GC content, conserved regions, etc. For each sample, its similarity and characteristic differences with the genome sequences of known pneumonia pathogens are recorded respectively. This process aims to deeply understand the pathogens and their genetic characteristics present in the samples, and finally obtain the biological characteristics of the historical gene sequences of pneumonia-related pathogens and the biological characteristics of each group of pharyngeal swab nucleic acid pneumonia gene sequences.

[0165] Step S43: using a sequence similarity comparison calculation formula to perform sequence similarity comparison calculation on the historical gene sequence biological characteristics of pneumonia-related pathogens and each group of throat swab nucleic acid pneumonia gene sequence biological characteristics, so as to obtain a sequence similarity comparison value between each group of throat swab pneumonia genes and the historical genes of related pathogens;

[0166] In an embodiment of the present invention, a suitable sequence similarity comparison calculation formula is formed by combining the historical gene sequence of pneumonia-related pathogens, the pharyngeal swab nucleic acid pneumonia gene sequence, the number parameters of biological sub-features, the feature similarity distance value between the historical gene sequence of pneumonia-related pathogens and the pharyngeal swab nucleic acid pneumonia gene sequence, the feature similarity distance weight parameter, the additional physicochemical feature similarity contribution value between the historical gene sequence of pneumonia-related pathogens and the pharyngeal swab nucleic acid pneumonia gene sequence, the additional feature similarity contribution weight parameter, the physicochemical property parameter, the number of sequence alignment gaps, the gap penalty coefficient and related parameters to perform sequence similarity comparison calculations on the biological features of the historical gene sequence of pneumonia-related pathogens and each group of pharyngeal swab nucleic acid pneumonia gene sequence biological features to evaluate and calculate the similarity between the gene sequence of each group of pharyngeal swab samples and the historical gene sequence of pneumonia-related pathogens, and finally obtain the sequence similarity comparison value between each group of pharyngeal swab pneumonia genes and the historical genes of related pathogens.

[0167] Among them, the sequence similarity comparison calculation formula is specifically as follows:

[0168]

[0169] In the formula, S(A,B) is the sequence similarity comparison value between the pharyngeal swab pneumonia gene and the historical gene of the relevant pathogen, A is the historical gene sequence of pneumonia-related pathogens, B is the pharyngeal swab nucleic acid pneumonia gene sequence, n is the total number of biological sub-features in the historical gene sequence of pneumonia-related pathogens, i is the item-order measurement parameter of the biological sub-features of the historical gene sequence, m is the total number of biological sub-features in the pharyngeal swab nucleic acid pneumonia gene sequence, j is the item-order measurement parameter of the biological sub-features of the pharyngeal swab nucleic acid pneumonia gene sequence, a i is the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens, b j is the jth biological sub-feature in the pharyngeal swab nucleic acid pneumonia gene sequence, f(a i ,b j ) is the feature similarity distance value between the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens and the j-th biological sub-feature in the gene sequence of throat swab nucleic acid pneumonia, δ1 is the feature similarity distance weight parameter, g(a i ,b j ) is the additional physicochemical feature similarity contribution value between the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens and the j-th biological sub-feature in the gene sequence of throat swab nucleic acid pneumonia, δ2 is the additional feature similarity contribution weight parameter, P(a i ) is the physicochemical property parameter of the i-th biological sub-characteristic in the historical gene sequence of pneumonia-related pathogens, P(b j) is the physicochemical property parameter of the jth biological sub-feature in the pharyngeal swab nucleic acid pneumonia gene sequence, gap(A,B) is the number of gaps in the sequence alignment between the historical gene sequence A of pneumonia-related pathogens and the pharyngeal swab nucleic acid pneumonia gene sequence B, and λ is the gap penalty coefficient;

[0170] The present invention obtains a sequence similarity comparison calculation formula by using a specific mathematical model and verifying it, which is used to perform sequence similarity comparison calculations on the biological characteristics of the historical gene sequences of pneumonia-related pathogens and the biological characteristics of each group of throat swab nucleic acid pneumonia gene sequences. The sequence similarity comparison calculation formula increases the accuracy of detection and reduces the false positive rate by considering both the biological characteristics similarity and the physicochemical properties. By adjusting the feature similarity distance weight and the gap penalty coefficient, it is possible to optimize the characteristics of different samples, improve the comparison effect, and also to punish the gaps in the comparison, thereby avoiding the similarity miscalculation caused by incomplete data, making the analysis more rigorous. This formula can be widely used in different types of pathogen analysis, is not limited to a specific type of pneumonia pathogen, not only uses the comparison of gene sequences, but also combines the physicochemical parameters of biological characteristics, so that the results are more reliable and scientific. In summary, this formula fully considers the sequence similarity comparison value S(A, B) between the pharyngeal swab pneumonia gene and the historical gene of the related pathogen, the historical gene sequence A of the pneumonia-related pathogen, the pharyngeal swab nucleic acid pneumonia gene sequence B, the total number of biological sub-features n in the historical gene sequence of the pneumonia-related pathogen, the item-order measurement parameter i of the biological sub-features of the historical gene sequence, the total number m of biological sub-features in the pharyngeal swab nucleic acid pneumonia gene sequence, the item-order measurement parameter j of the biological sub-features of the pharyngeal swab nucleic acid pneumonia gene sequence, and the i-th biological sub-feature a in the historical gene sequence of the pneumonia-related pathogen i , the jth biological sub-feature b in the pharyngeal swab nucleic acid pneumonia gene sequence j , the feature similarity distance value f(a) between the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens and the j-th biological sub-feature in the gene sequence of throat swab nucleic acid pneumonia i ,b j ), feature similarity distance weight parameter δ1, additional physicochemical feature similarity contribution value g(a) between the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens and the j-th biological sub-feature in the gene sequence of throat swab nucleic acid pneumonia i ,b j ), additional feature similarity contribution weight parameter δ2, physicochemical property parameter P(a) of the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens i ), the physicochemical property parameter P(b j), the number of gaps in the sequence alignment between the historical gene sequence A of pneumonia-related pathogens and the gene sequence B of pharyngeal swab nucleic acid pneumonia, gap(A,B), the gap penalty coefficient λ, where the item-order metric parameter i of the biological sub-feature of the historical gene sequence, the item-order metric parameter j of the biological sub-feature of the pharyngeal swab nucleic acid pneumonia gene sequence, the i-th biological sub-feature a in the historical gene sequence of pneumonia-related pathogens i , the jth biological sub-feature b in the pharyngeal swab nucleic acid pneumonia gene sequence j , the physicochemical property parameter P(a) of the i-th biological sub-characteristic in the historical gene sequence of pneumonia-related pathogens i ) and the physicochemical property parameters P(b j ) constitutes an additional physicochemical feature similarity contribution value g(a) between the i-th biological sub-feature in the historical gene sequence of a pneumonia-related pathogen and the j-th biological sub-feature in the gene sequence of pharyngeal swab nucleic acid pneumonia i ,b j ) According to the sequence similarity comparison value S(A,B) between the pneumonia gene of throat swab and the historical gene of related pathogens and the correlation between the above parameters, a functional relationship is formed:

[0171]

[0172] This formula can realize the sequence similarity comparison calculation process of the historical gene sequence biological characteristics of pneumonia-related pathogens and the gene sequence biological characteristics of each group of pharyngeal swab nucleic acid pneumonia, thereby improving the accuracy and applicability of the sequence similarity comparison calculation formula.

[0173] Step S44: Based on the sequence similarity comparison value between each group of pharyngeal swab pneumonia genes and the relevant pathogen historical genes, the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes are subjected to pathogen comparison detection to obtain the pharyngeal swab sample pneumonia pathogen genome detection results.

[0174] In an embodiment of the present invention, a detailed pathogen comparison test is performed on the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes by combining the sequence similarity comparison values ​​between each group of pharyngeal swab pneumonia genes and the historical genes of related pathogens obtained by previous quantitative calculation, so as to perform a comprehensive comparison analysis using a bioinformatics analysis platform, such as Geneious or Galaxy. This analysis will combine historical genome data to calculate the similarity and difference between each sample and the genome of known pneumonia pathogens, so as to ultimately determine the pathogens present in the sample and their genomic characteristics, and determine the pathogen gene with the highest corresponding similarity comparison value as the pathogen present in the pharyngeal swab pneumonia gene to be tested, thereby providing accurate pathogen detection results, and finally obtaining the pharyngeal swab sample pneumonia pathogen genome detection results.

[0175] Furthermore, the present invention also provides a pneumonia genome detection system based on a pharyngeal swab sample, which is used to perform the pneumonia genome detection method based on a pharyngeal swab sample as described above, and the pneumonia genome detection system based on a pharyngeal swab sample comprises:

[0176] The sterile throat swab nucleic acid sample screening module is used to obtain a throat swab sample solution, and add lysozyme to digest the bacteria in the throat swab sample solution to obtain a sterile throat swab sample; by placing the sterile throat swab sample in a nucleic acid extraction kit containing an RNA / DNA protective agent, and using agarose gel electrophoresis to perform nucleic acid quality screening on the nucleic acid sample corresponding to the sterile throat swab sample, thereby obtaining a sterile throat swab nucleic acid high-quality sample;

[0177] The throat swab nucleic acid sequence library amplification module is used to obtain the historical genome sequence of pneumonia-related pathogens, and to design primers for related pathogens based on the historical genome sequence of pneumonia-related pathogens to obtain specific primers for pneumonia-related pathogens; the PCR amplification library of high-quality nucleic acid samples of sterile throat swabs is constructed using specific primers for pneumonia-related pathogens and ligase, thereby obtaining a throat swab nucleic acid PCR amplification sequence library;

[0178] The throat swab nucleic acid pneumonia genome sequencing module is used to perform high-throughput sequencing on each group of throat swab nucleic acid pneumonia gene sequences in the throat swab nucleic acid PCR amplification sequence library to obtain high-throughput sequencing sequences corresponding to each group of throat swab nucleic acid pneumonia genes;

[0179] The pharyngeal swab pneumonia genome comparison and detection module is used to perform genome sequence pathogen comparison detection on the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes based on the historical genome sequences of pneumonia-related pathogens, thereby obtaining the pharyngeal swab sample pneumonia pathogen genome detection results.

[0180] Therefore, the embodiments should be regarded as illustrative and non-restrictive from all points, and the scope of the present invention is limited by the appended claims rather than the above description, and it is therefore intended that all changes falling within the meaning and range of equivalent elements of the application documents are included in the present invention.

[0181] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but should conform to the widest scope consistent with the principles and novel features invented herein.

Claims

1. A method for detecting pneumonia genome based on throat swab samples, characterized in that: The following steps are involved: Step S1: obtaining a pharyngeal swab sample solution, and adding lysozyme to digest the bacteria in the pharyngeal swab sample solution to obtain a sterile pharyngeal swab sample; placing the sterile pharyngeal swab sample in a nucleic acid extraction kit containing an RNA / DNA protective agent, and performing nucleic acid quality screening on the nucleic acid sample corresponding to the sterile pharyngeal swab sample by agarose gel electrophoresis to obtain a sterile pharyngeal swab nucleic acid high-quality sample; Step S2: Obtain historical genome sequences of pneumonia-related pathogens, and design primers for related pathogens based on the historical genome sequences of pneumonia-related pathogens to obtain primers specific for pneumonia-related pathogens; The PCR amplification library of high-quality nucleic acid samples of sterile throat swabs was constructed using pneumonia-related pathogen-specific primers and ligase to obtain a PCR amplification sequence library of nucleic acid of throat swabs; Step S3: performing high-throughput sequencing on each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library to obtain a high-throughput sequencing sequence corresponding to each group of pharyngeal swab nucleic acid pneumonia genes; Step S4: Based on the historical genome sequences of pneumonia-related pathogens, the high-throughput sequencing sequences corresponding to each group of throat swab nucleic acid pneumonia genes are compared and tested for genome sequence pathogens to obtain the genome test results of pneumonia pathogens in throat swab samples.

2. The method for detecting pneumonia genome based on throat swab samples according to claim 1, characterized in that: Step S1 includes the following steps: Step S11: obtaining an initial sample of a pharyngeal swab, placing the initial sample of the pharyngeal swab in a sterile test tube, adding an appropriate amount of physiological saline, and shaking the tube thoroughly to obtain a pharyngeal swab sample solution; Step S12: measuring the optical density of the pharyngeal swab sample solution to obtain the bacterial concentration of the pharyngeal swab sample solution; Step S13: performing an optimization analysis on the bacterial digestion conditions of the corresponding pharyngeal swab sample solution based on the bacterial concentration of the pharyngeal swab sample solution to obtain the optimal digestion conditions of the pharyngeal swab sample bacteria; Step S14: performing a sample digestion operation on the corresponding bacteria in the pharyngeal swab sample solution based on the optimal digestion conditions of the pharyngeal swab sample bacteria and adding lysozyme to obtain a pharyngeal swab sample solution after bacterial digestion; performing sterile filtration and testing on the pharyngeal swab sample solution after bacterial digestion to obtain a sterile pharyngeal swab sample; Step S15: The sterile throat swab sample is placed in a nucleic acid extraction kit containing an RNA / DNA protective agent, and the nucleic acid sample corresponding to the sterile throat swab sample is screened for nucleic acid quality using agarose gel electrophoresis to obtain a sterile throat swab nucleic acid high-quality sample.

3. The method for detecting pneumonia genome based on throat swab samples according to claim 2, characterized in that: Step S12 includes the following steps: Step S121: using a spectrophotometer to perform a preliminary optical property evaluation on the pharyngeal swab sample solution to obtain a pharyngeal swab sample optical property data set at different wavelengths, wherein the pharyngeal swab sample optical property data set includes the pharyngeal swab sample optical transmittance and the pharyngeal swab sample optical absorbance value; Step S122: performing wavelength selection optimization analysis on the corresponding pharyngeal swab sample solution based on the pharyngeal swab sample optical characteristic data set at different wavelengths to obtain the optimal measurement wavelength parameter of the pharyngeal swab sample solution; Step S123: using a spectrophotometer to measure the optical density of the corresponding pharyngeal swab sample solution based on the optimal measurement wavelength parameter of the pharyngeal swab sample solution, to obtain the optical density measurement result of the pharyngeal swab sample solution; Step S124: obtaining a standard bacterial solution of known concentration, and performing linear regression analysis and standard curve construction on the standard bacterial solution of known concentration based on the optimal measurement wavelength parameter of the throat swab sample solution, so as to obtain a mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration; Step S125: performing reverse calculation of the bacterial concentration on the optical density measurement result of the throat swab sample solution according to the mathematical model of the standard curve relationship between the optical density of the throat swab sample and the bacterial concentration, so as to obtain the bacterial concentration of the throat swab sample solution.

4. The method for detecting pneumonia genome based on throat swab samples according to claim 2, characterized in that: Step S15 includes the following steps: Step S151: performing cold chain storage activity recovery treatment on the sterile throat swab sample to obtain a sterile throat swab activity recovery sample; Step S152: placing the sterile throat swab activity recovery sample into a nucleic acid extraction kit containing an RNA / DNA protective agent for sample dilution treatment to obtain a sterile throat swab nucleic acid extraction solution system; Step S153: centrifuging the sterile throat swab nucleic acid extraction solution system to obtain a sterile throat swab preliminary nucleic acid extraction solution; Step S154: using agarose gel electrophoresis to perform nucleic acid mass electrophoresis analysis on the corresponding nucleic acid samples in the preliminary nucleic acid extract of the sterile throat swab, so as to obtain a nucleic acid fragment mass spectrum corresponding to each sterile throat swab nucleic acid sample; Step S155: Based on the nucleic acid fragment mass spectrum corresponding to each sterile throat swab nucleic acid sample, the corresponding nucleic acid sample in the sterile throat swab preliminary nucleic acid extract is subjected to nucleic acid quality screening processing to obtain a sterile throat swab nucleic acid high-quality sample.

5. The method for detecting pneumonia genome based on throat swab samples according to claim 4, characterized in that: Step S155 includes the following steps: The nucleic acid fragment mass spectrum corresponding to each sterile throat swab nucleic acid sample is subjected to nucleic acid fragment size and band clarity extraction processing to obtain the nucleic acid fragment size and nucleic acid fragment band clarity corresponding to each sterile throat swab nucleic acid sample; Based on the size of nucleic acid fragments and the clarity of nucleic acid fragment bands corresponding to each sterile throat swab nucleic acid sample, the molecular weight cutoff calculation formula of nucleic acid fragments is used to perform a quantitative calculation of the molecular weight cutoff of the corresponding nucleic acid fragment mass spectrum to obtain the molecular weight cutoff value of the sterile throat swab nucleic acid fragment; Based on the molecular weight cutoff value of the sterile throat swab nucleic acid fragment, membrane filtration technology is used to perform nucleic acid quality screening on the corresponding nucleic acid samples in the sterile throat swab preliminary nucleic acid extract to obtain a sterile throat swab nucleic acid high-quality sample.

6. The method for detecting pneumonia genome based on throat swab samples according to claim 5, characterized in that: The nucleic acid fragment molecular weight cutoff calculation formula is specifically: Where M r is the molecular weight cutoff value of nucleic acid fragments from sterile throat swabs, V t is the total volume of the sterile throat swab nucleic acid sample, L is the size of the nucleic acid fragment corresponding to the sterile throat swab nucleic acid sample, C(x) is the clarity of the nucleic acid fragment band at position x of the sterile throat swab nucleic acid sample, M p (x) is the molecular weight of the nucleic acid fragment at position x in the sterile throat swab nucleic acid sample, M m is the reference molecular weight of the sterile throat swab nucleic acid sample, α is the influence index of the nucleic acid fragment molecular weight, β is the attenuation coefficient of the nucleic acid fragment molecular weight, and L c is the standard threshold value of the position size of the nucleic acid fragment, and η is the correction coefficient of the molecular weight cutoff value of the sterile throat swab nucleic acid fragment.

7. The method for detecting pneumonia genome based on throat swab samples according to claim 1, characterized in that: Step S2 includes the following steps: Step S21: Obtain historical genome sequences of pneumonia-related pathogens; Step S22: Systematically collect and organize historical genome sequences of pneumonia-related pathogens to obtain a historical genome database of pneumonia-related pathogens; Step S23: performing sequence alignment and conservative region identification analysis on different historical genome sequences of pneumonia-related pathogens based on the historical genome database of pneumonia-related pathogens to obtain conservative segments of the historical genome of pneumonia-related pathogens; Step S24: designing primers for the corresponding historical genome sequences of pneumonia-related pathogens based on the conserved segments of the historical genomes of pneumonia-related pathogens to obtain primers specific to pneumonia-related pathogens; Step S25: setting primer annealing PCR reaction conditions for pneumonia-related pathogen-specific primers to obtain pneumonia-related pathogen primer PCR reaction system conditions; performing nucleic acid PCR amplification reaction on sterile throat swab nucleic acid high-quality samples based on the pneumonia-related pathogen primer PCR reaction system conditions to obtain throat swab sample nucleic acid PCR amplification sequence fragments; using ligase to perform library connection construction on throat swab sample nucleic acid PCR amplification sequence fragments to obtain throat swab nucleic acid PCR amplification sequence library.

8. The method for detecting pneumonia genome based on throat swab samples according to claim 1, characterized in that: Step S3 includes the following steps: Step S31: performing gene band separation and excision processing on each group of pharyngeal swab nucleic acid pneumonia gene sequences in the pharyngeal swab nucleic acid PCR amplification sequence library to obtain the gene sequence target band corresponding to each group of pharyngeal swab nucleic acid pneumonia genes; Step S32: performing gene sequence characteristic analysis on the gene sequence target bands corresponding to each group of pharyngeal swab nucleic acid pneumonia genes to obtain gene sequence characteristic data corresponding to each group of pharyngeal swab nucleic acid pneumonia genes; Step S33: setting the sequencing reaction conditions for the corresponding gene sequence target bands based on the gene sequence characteristic data corresponding to each group of pharyngeal swab nucleic acid pneumonia genes, so as to generate a gene sequencing reaction condition system corresponding to each group of pharyngeal swab nucleic acid pneumonia genes; Step S34: Perform high-throughput sequencing on each group of throat swab nucleic acid pneumonia gene sequences in the throat swab nucleic acid PCR amplification sequence library according to the gene sequencing reaction condition system corresponding to each group of throat swab nucleic acid pneumonia genes, so as to obtain a high-throughput sequencing sequence corresponding to each group of throat swab nucleic acid pneumonia genes.

9. The method for detecting pneumonia genome based on throat swab samples according to claim 1, characterized in that: Step S4 includes the following steps: Step S41: performing sequence quality control preprocessing on the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes to obtain standard sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes; Step S42: Perform gene sequence biometric analysis on the historical genome sequence of pneumonia-related pathogens and the standard sequencing sequence corresponding to each group of throat swab nucleic acid pneumonia genes to obtain the historical gene sequence biometrics of pneumonia-related pathogens and each group of throat swab nucleic acid pneumonia gene sequence biometrics; Step S43: using a sequence similarity comparison calculation formula to perform sequence similarity comparison calculation on the historical gene sequence biological characteristics of pneumonia-related pathogens and each group of throat swab nucleic acid pneumonia gene sequence biological characteristics, so as to obtain a sequence similarity comparison value between each group of throat swab pneumonia genes and the historical genes of related pathogens; Among them, the sequence similarity comparison calculation formula is specifically as follows: In the formula, S(A, B) is the sequence similarity comparison value between the pneumonia gene of the throat swab and the historical gene of the related pathogen, A is the historical gene sequence of the pneumonia-related pathogen, B is the throat swab nucleic acid pneumonia gene sequence, n is the total number of biological sub-features in the historical gene sequence of the pneumonia-related pathogen, i is the item-order measurement parameter of the biological sub-feature of the historical gene sequence, m is the total number of biological sub-features in the throat swab nucleic acid pneumonia gene sequence, j is the item-order measurement parameter of the biological sub-feature of the throat swab nucleic acid pneumonia gene sequence, a i is the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens, b j is the jth biological sub-feature in the pharyngeal swab nucleic acid pneumonia gene sequence, f(a i , b j ) is the feature similarity distance value between the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens and the j-th biological sub-feature in the gene sequence of throat swab nucleic acid pneumonia, δ1 is the feature similarity distance weight parameter, g(a i , b j ) is the additional physicochemical feature similarity contribution value between the i-th biological sub-feature in the historical gene sequence of pneumonia-related pathogens and the j-th biological sub-feature in the gene sequence of throat swab nucleic acid pneumonia, δ2 is the additional feature similarity contribution weight parameter, P(a i ) is the physicochemical property parameter of the i-th biological sub-characteristic in the historical gene sequence of pneumonia-related pathogens, P(b j ) is the physicochemical property parameter of the jth biological sub-feature in the pharyngeal swab nucleic acid pneumonia gene sequence, gap(A, B) is the number of gaps in the sequence alignment between the historical gene sequence A of pneumonia-related pathogens and the pharyngeal swab nucleic acid pneumonia gene sequence B, and λ is the gap penalty coefficient; Step S44: Based on the sequence similarity comparison value between each group of pharyngeal swab pneumonia genes and the relevant pathogen historical genes, the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes are subjected to pathogen comparison detection to obtain the pharyngeal swab sample pneumonia pathogen genome detection results.

10. A pneumonia genome detection system based on throat swab samples, characterized in that: Used to perform the pneumonia genome detection method based on throat swab samples as claimed in claim 1, the pneumonia genome detection system based on throat swab samples comprises: The sterile throat swab nucleic acid sample screening module is used to obtain a throat swab sample solution, and add lysozyme to digest the bacteria in the throat swab sample solution to obtain a sterile throat swab sample; by placing the sterile throat swab sample in a nucleic acid extraction kit containing an RNA / DNA protective agent, and using agarose gel electrophoresis to perform nucleic acid quality screening on the nucleic acid sample corresponding to the sterile throat swab sample, thereby obtaining a sterile throat swab nucleic acid high-quality sample; The throat swab nucleic acid sequence library amplification module is used to obtain the historical genome sequence of pneumonia-related pathogens, and to design primers for related pathogens based on the historical genome sequence of pneumonia-related pathogens to obtain specific primers for pneumonia-related pathogens; the PCR amplification library of high-quality nucleic acid samples of sterile throat swabs is constructed using specific primers for pneumonia-related pathogens and ligase, thereby obtaining a throat swab nucleic acid PCR amplification sequence library; The throat swab nucleic acid pneumonia genome sequencing module is used to perform high-throughput sequencing on each group of throat swab nucleic acid pneumonia gene sequences in the throat swab nucleic acid PCR amplification sequence library to obtain high-throughput sequencing sequences corresponding to each group of throat swab nucleic acid pneumonia genes; The pharyngeal swab pneumonia genome comparison and detection module is used to perform genome sequence pathogen comparison detection on the high-throughput sequencing sequences corresponding to each group of pharyngeal swab nucleic acid pneumonia genes based on the historical genome sequences of pneumonia-related pathogens, thereby obtaining the pharyngeal swab sample pneumonia pathogen genome detection results.

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