A quality control for detecting respiratory pathogen nucleic acid and a preparation method thereof

Abstract

The application provides a quality control for detecting respiratory pathogen nucleic acid, which comprises any one or more of the following respiratory pathogens: coronavirus, influenza virus, adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, rhinovirus or / and Legionella pneumophila. The full-length genome sequence of the coronavirus is divided into 6 target fragments, each with a length of 4000-5500 bp. The quality control of the application covers all detection target sequences (or target points) of the coronavirus, has a wide coverage, comprehensively detects target points, and does not have the phenomenon of missed detection. The quality control of the application contains main respiratory pathogen infections, has a wide range, and uses real virus samples and lentivirus samples as raw materials, and has more accurate detection target points. The preparation of the coronavirus quality control of the application integrates the target gene sequence into the host genome through the lentivirus vector, knocks out the self-replication gene in the preparation process, and has a "self-inactivation" ability, so that the recombinant lentivirus cannot replicate in the target cells and infect other cells.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a quality control material for detecting nucleic acids of respiratory pathogens and its preparation method. Background Technology

[0002] Respiratory infections are the most common infectious diseases, caused by respiratory pathogens. These pathogens primarily infect the respiratory system, including the nasal cavity and trachea. Respiratory viruses are a large class of viruses that invade through the respiratory tract, proliferating on the respiratory mucosal epithelial cells and causing acute respiratory tissue infections or other organ lesions. Statistics show that over 90% of primary acute upper respiratory tract infections are caused by respiratory viruses, and these viruses cause lower respiratory tract infections in up to 62% of cases. Studies have shown that some respiratory pathogens, such as Middle East Respiratory Syndrome (MERS) virus and avian influenza virus, spread rapidly and widely due to their respiratory transmission, resulting in high morbidity rates and a high risk of epidemics and outbreaks, significantly impacting society. Because there are many types of pathogens that can cause respiratory infections, patients may carry more than one pathogen. The clinical signs and symptoms of different viruses and viruses and bacteria often overlap, making it difficult to diagnose the cause of the disease based solely on clinical manifestations. This can lead to ineffective conventional treatment, antibiotic overuse, and iatrogenic cross-infection. Rapid detection of respiratory pathogens can help with diagnosis and accurate medication. Therefore, it is necessary to develop corresponding quality control materials to control the quality of the rapid detection process and ensure the accuracy and reliability of the test results.

[0003] Timely and accurate diagnosis of the pathogen in the early stages of viral infection helps medical staff accurately grasp the patient's condition and is also an important guarantee for the smooth implementation of acute infectious disease prevention and control. The first method to detect new pathogens was next-generation sequencing (NGS) technology, which quickly determined the nucleic acid sequence of the novel coronavirus. On March 3, 2020, the National Health Commission (NHC) released the Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 7), requiring confirmed cases to simultaneously meet one of the following etiological or serological evidence: positive real-time fluorescent RT-PCR test for novel coronavirus nucleic acid; viral gene sequencing showing high homology with known novel coronaviruses; or positive serum detection of novel coronavirus-specific IgM and IgG antibodies. Viral nucleic acid testing has advantages such as a short window period, low time consumption, high sensitivity, and strong specificity, enabling rapid diagnosis in the early stages of the virus and dynamic observation of the effectiveness of antiviral treatment. However, nucleic acid testing also has a certain false negative rate and potential false positive results. A "false negative" at the laboratory level refers to a situation where the viral load in the collected sample is higher than the detection limit (also known as analytical sensitivity) of the test reagent, but the laboratory fails to detect it. To minimize the risk of false negatives, laboratories must meet the following requirements: (1) select reliable in vitro diagnostic reagents; and (2) conduct standardized clinical nucleic acid testing (reasonable zoning, capable testing personnel, and a strict quality management system).

[0004] Digital PCR (dPCR) is a groundbreaking quantitative analysis technique that has gained significant attention and developed rapidly in recent years. In 1992, Sykes et al., when detecting low-abundance IgH heavy chain mutant genes in complex backgrounds, utilized limiting dilution of samples to obtain only a single template molecule in each well. By calculating the amplification signal after PCR, they aimed to accurately determine the number of starting molecules. Although the concept of "digital PCR" was not explicitly proposed, the basic experimental procedure for digital PCR was established, and a crucial principle in digital PCR detection—using the presence or absence of an endpoint signal as the quantitative method—was determined. This was the prototype of digital PCR. In 1999, Vogelstein and Kinzler et al. encountered bottlenecks in detection sensitivity and resolution due to interference from somatic cell genes when detecting BRAF-specific mutant genes in fecal cancerous tissue detached from cells. They adopted a method of extremely diluting the sample amount in each reaction well in a 384-well plate and increasing the number of reaction wells for detection, thus proposing the concept of digital PCR. They also suggested that using more wells would result in higher detection sensitivity, thus pointing out the development direction of digital PCR systems.

[0005] Currently, the UK National Institute for Biological Standards and Control (NIBSC), as a WHO-designated international standard laboratory, is the world's leading producer and distributor of international standard products and reference materials, producing over 95% of international standard products. While my country has a large quantity of quality control materials, the variety is incomplete, the categories are lacking, and the structure is unreasonable. The materials are mainly concentrated in the steel, mining, and building materials sectors, while quality control materials in important development areas such as bioengineering, food components, clinical chemistry, new materials, and new energy are relatively scarce. The development of relevant quality control materials is relatively lagging, making it impossible to test many products and necessitating imports to meet laboratory testing requirements. Because the manufacturers of these imported quality control materials implement different management systems, the accuracy and traceability of their measurements also differ, making it difficult to ensure the validity and consistency of my country's measurement results. With the increasing national supervision and management of in vitro diagnostic reagents and the continuous improvement of testing technology requirements, the demand for and reliance on in vitro diagnostic reagent quality control materials is becoming increasingly strong. Therefore, strengthening the construction of in vitro diagnostic reagent quality control materials is not only conducive to the accuracy and consistency of diagnostic results, making test results from different hospitals comparable, but also a powerful guarantee for effective technical supervision of in vitro diagnostic reagents.

[0006] Because the activity of biological samples is complex and variable, nucleic acids are highly susceptible to degradation by buffered environments, especially RNA, which is easily degraded by ubiquitous nucleases. Furthermore, performance validation experiments for nucleic acid detection kits, particularly those evaluating detection limits (sensitivity) and linearity, often require different concentrations of manufacturer reference materials, positive controls, and quality control materials for effective kit performance evaluation. Therefore, developing an economical, stable, reliable, and biohazard-free quality control material, including its value determination and stability testing, is an essential process and a crucial step in quality control material development, as well as a key part of this invention. Summary of the Invention

[0007] The first technical problem to be solved by the present invention is to provide a quality control material for detecting nucleic acids of respiratory pathogens.

[0008] The second technical problem to be solved by the present invention is to provide a method for preparing a quality control material for detecting nucleic acids of respiratory pathogens.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A quality control material for detecting respiratory pathogen nucleic acids includes any one or more of the following respiratory pathogen nucleic acids: coronavirus, influenza virus, respiratory syncytial virus, adenovirus, rhinovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, and / or Legionella pneumophila. The quality control material of this invention covers a broad range of major respiratory pathogens and uses both real and spurious virus samples as raw materials, resulting in more accurate detection targets.

[0011] Preferably, the coronavirus nucleic acid consists of six 4000-5500 bp fragments. This invention divides the full-length genome sequence of the coronavirus into six target fragments, each 4000-5500 bp in length. Therefore, the quality control material of this invention covers all target sequences (or targets) for coronavirus detection, providing broad coverage and comprehensive detection, preventing missed detections. Furthermore, overlap is designed at the ends of each fragment to effectively prevent the probe sequence of the detection reagent from falling into the segmented position and affecting the final detection result.

[0012] Preferably, the coronaviruses include: human coronavirus HCoV-NL63, human coronavirus HCoV-229E, human coronavirus HCoV-HKU1, human coronavirus HCoV-OC43, SRSA-CoV, MERS-CoV, or / and novel coronavirus.

[0013] Preferably, the influenza virus includes: influenza A virus H1N1, influenza A virus H5N1, influenza A virus H7N9, influenza A virus H9N2, influenza B virus (IVB), respiratory syncytial virus (RSV) and / or parainfluenza virus (PIV).

[0014] Preferably, the diluent comprises the following components: dimethyl sulfoxide, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, sodium dodecyl sulfate, bovine serum albumin, guanidine isothiocyanate, Tris-HCl, ethylenediaminetetraacetic acid, and glycerol; the pH of the diluent is 7.2–7.6. The diluent of the present invention has the effect of protecting nucleic acids and improving their stability.

[0015] Preferably, the standard value of human coronavirus HCoV-NL63 in the quality control material of the respiratory pathogen nucleic acid is 1.2 × 10⁻⁶. 4 The standard value for human coronavirus HCoV-229E is 1.9 × 10 copies / μL. 4 The standard value for human coronavirus HCoV-HKU1 is 1.3 × 10 copies / μL. 4 The standard value for human coronavirus HCoV-OC43 is 2.0 × 10 copies / μL. 4 The standard value for severe acute respiratory syndrome coronavirus (SRSA-CoV) is 2.0 × 10 copies / μL. 4 The standard value for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is 1.8 × 10 copies / μL. 4 The standard value for the novel coronavirus is 1.6 × 10⁶ copies / μL. 4The standard value for influenza A virus H1N1 is 1.1 × 10 copies / μL. 4 The standard value for influenza A virus H5N1 is 1.9 × 10 copies / μL. 4 The standard value for influenza A virus H7N9 is 1.5 × 10 copies / μL. 4 The standard value for influenza A virus H9N2 is 1.3 × 10 copies / μL. 4 The standard value for influenza B virus is 2.3 × 10 copies / μL. 4 The standard value for respiratory syncytial virus (RSV) is 2.2 × 10⁻⁶ copies / μL. 4 The standard value for parainfluenza virus is 1.5 × 10 copies / μL. 4 The standard value for adenovirus is 1.3 × 10⁻⁶ copies / μL. 4 The standard value for Mycoplasma pneumoniae is 1.9 × 10⁻⁶ copies / μL. 4 The standard value for Streptococcus pneumoniae is 1.3 × 10⁻⁶ copies / μL. 4 The standard value for rhinovirus is 2.3 × 10⁻⁶ copies / μL. 4 The standard value for Legionella pneumophila is 2.1 × 10⁻⁶ copies / μL. 4 Copies / μL. The quality control material of this invention contains nucleic acids from various pathogens, which can interact with each other. Nucleic acids are extremely unstable biological macromolecules; higher concentrations are more prone to degradation. However, the product protected by this invention, as a quality control material, needs to have a certain concentration value to be of reference value. Therefore, its standard value (i.e., concentration) cannot be too low. Thus, through extensive experimental research, the inventors discovered that when the standard values ​​(i.e., concentrations) of each nucleic acid are as described above, the homogeneity and stability of the quality control material are optimal, allowing it to be stored at low temperatures (-20±5℃) for 12 months without degradation.

[0016] The present invention also provides a method for preparing the above-mentioned quality control material, comprising the following steps:

[0017] S1. Prepare respiratory pathogen cultures and extract pathogen nucleic acids; the respiratory pathogens include coronavirus, influenza virus, respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, rhinovirus and / or Legionella pneumophila.

[0018] S2 uses real-time PCR to detect the nucleic acid content obtained in step S1, and obtains the original concentration value of nucleic acid for each pathogen;

[0019] S3 uses the diluent to dilute the respiratory pathogen nucleic acid from step S2 to the required concentration value, thereby obtaining the quality control material of the respiratory pathogen containing the concentration standard value.

[0020] The coronavirus in this invention integrates the target gene into the host genome via a retroviral vector and can be persistently expressed. Its advantage lies in its ability to control the quality of the entire process in nucleic acid detection, including sample processing, nucleic acid extraction, and viral reverse transcription and amplification. Simultaneously, it reduces biosafety risks for laboratory personnel. This invention obtains the target gene sequence of the coronavirus from the NCBI database, obtains the target fragment, artificially constructs an expression vector, and then extracts nucleic acid containing the target fragment.

[0021] Preferably, step S1 is implemented using the following method:

[0022] (A) Extraction of the coronavirus nucleic acid: First, the full-length genome sequence of the coronavirus is divided into 6 fragments, which are then ligated to lentiviral expression vector plasmids using a seamless cloning method to obtain 6 expression plasmids, which are then transfected into expression host bacteria; then, the expression plasmids that have been identified as having qualified sequences are transfected into packaging cells along with lentiviral packaging plasmids to obtain coronavirus fluid. At this point, the extracted coronavirus nucleic acid consists of 6 nucleic acid fragments containing partial full-length genome sequences.

[0023] (B) Extraction of nucleic acids from the influenza virus, respiratory syncytial virus, adenovirus and rhinovirus: After mixing and culturing the influenza virus solution, respiratory syncytial virus solution, adenovirus solution and rhinovirus solution with infected host cells respectively, the viruses are harvested and their nucleic acids are extracted.

[0024] (C) Extraction of nucleic acids from Mycoplasma pneumoniae, Legionella pneumophila and Streptococcus pneumoniae: Mycoplasma pneumoniae, Legionella pneumophila and Streptococcus pneumoniae were inoculated into the culture medium, and their culture solutions were obtained and their nucleic acids were extracted.

[0025] Preferably, the coronaviruses include: human coronavirus HCoV-NL63, human coronavirus HCoV-229E, human coronavirus HCoV-HKU1, human coronavirus HCoV-OC43, SRSA-CoV, MERS-CoV, or / and novel coronavirus.

[0026] Preferably, the influenza virus includes: influenza A virus H1N1, influenza A virus H5N1, influenza A virus H7N9, influenza A virus H9N2, influenza B virus (IVB), respiratory syncytial virus (RSV) and / or parainfluenza virus (PIV).

[0027] Preferably, the nucleic acid is extracted using a magnetic bead-based nucleic acid extraction kit.

[0028] Preferably, the 100 mL nucleic acid diluent in step S3 comprises the following components: 5% dimethyl sulfoxide (DMSO), 0.5 M ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 0.1% sodium dodecyl sarcosinate (SDS), 1% bovine serum albumin (BSA), 1% guanidine isothiocyanate, 1.21% Tris-HCl, 5 mol / L ethylenediaminetetraacetic acid (EDTA), and 5% glycerol.

[0029] The beneficial effects of this invention are as follows: The quality control materials obtained by this invention cover 19 common respiratory pathogens. Using real viral samples and lentiviral samples as raw materials, the quality control materials are quantified using an absolute quantitative droplet digital PCR method. Inactivation is achieved using an organic solvent / detergent (S / D) treatment method, which effectively improves the stability of the quality control materials, especially protecting the nucleic acids in low-concentration samples. They can be stored at -20℃ for more than 12 months. This invention is applicable to common nucleic acid extraction methods such as the "boiling alkaline lysis method," column extraction, magnetic bead extraction, and one-step method (sample release agent). The added glycerol, BSA, and DMSO can act as PCR amplification enhancers, improving the specificity and yield of the "one-step method" and the "boiling alkaline lysis method" PCR amplification.

[0030] This invention can be used for internal / inter-laboratory quality control in laboratories, effectively monitoring instrument status, personnel operation, reagent kit validity, etc., to ensure the accuracy and reliability of test results. It can also be used for evaluation experiments on the specificity, detection limit (sensitivity), linearity, etc. of nucleic acid reagent kits.

[0031] The quality control material of the present invention has the following advantages:

[0032] (1) By simulating the natural structure of the virus or directly using real inactivated viruses, the entire process of sample processing, nucleic acid extraction and reverse transcription and amplification of the virus particles can be quality controlled.

[0033] (2) There are no biosafety issues or infectious risks. The inactivation treatment is carried out using organic solvent / detergent (S / D) treatment.

[0034] (3) Stable values: The sample diluent can protect the RNA / DNA within it from the action of nucleases and prevent nucleic acid degradation.

[0035] (4) It has traceability: it can be traced back to the natural unit "1" through digital PCR molecular counting method.

[0036] (5) It can be used for rapid detection by fluorescence quantitative PCR and quality control of high-throughput sequencing experiments.

[0037] (6) It can be used to verify the analytical specificity of nucleic acid detection kits for respiratory pathogens.

[0038] (7) It has a wide range of applications and can be used with most nucleic acid test kits on the market. It also helps to enhance the specificity and yield of "one-step" and "boiling alkaline lysis" PCR amplification. Attached Figure Description

[0039] Figure 1 Schematic diagram of vector plasmid spectrum

[0040] Figure 2 H1N1 standard amplification curve

[0041] Figure 3 H1N1 standard curve

[0042] Figure 4 Droplet formation / distribution diagram of H1N1 quality control material setter 1

[0043] Figure 5 Droplet formation / distribution diagram of H1N1 quality control material setter 2

[0044] Figure 6 Droplet formation / distribution diagram of H1N1 quality control material setter 3

[0045] Figure 7 Droplet formation / distribution diagram of H1N1 quality control material setter 4 Detailed Implementation

[0046] To more concisely and clearly demonstrate the technical solution, purpose, and advantages of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0047] Example 1: Obtaining coronavirus cultures

[0048] I. Obtaining the coronavirus genome sequence

[0049] 1. Download the following gene sequences from the National Center for Biotechnology Information (NCBI): HCoV-NL63, HCoV-229E, HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and Novel Coronavirus.

[0050] 2. The full-length genome sequence was divided into 6 target genes: Based on the publicly available coronavirus genome sequence, the full-length sequence was divided into 6 segments. Six target genes were synthesized using gene synthesis techniques, with overlaps designed at the beginning and end of each segment, each approximately 70 bp in length. Information on the 6 target genes is shown in Table 1 below:

[0051] Table 1 Information on 6 target genes

[0052]

[0053]

[0054] II. Construction of Coronavirus Lentivirals

[0055] 1. The target gene is ligated to the expression vector using seamless cloning ligation technology. The specific steps are as follows:

[0056] (a) The expression vector pCDH-CMV-MCS-EF1A-CopGFP-T2A-puro was linearized by enzyme digestion. The enzyme digestion products were identified by agarose gel electrophoresis, and the enzyme digestion products were recovered and purified according to the instructions of the gel recovery kit to obtain the linearized plasmid vector.

[0057] (b) Insert the six gene fragments synthesized in step one above into the aforementioned vector, and their plasmid patterns are shown below. Figure 1 As shown. Using multi-fragment homologous recombination, the vector and fragments were mixed with the recombinase in the ClonExpress Ultra One Step Cloning Kit at the calculated optimal amount according to the system ratio. After directional cloning at 50℃ for 15 min, six expression plasmids containing the target gene were obtained.

[0058] 2. Screening and Identification of Positive Clones: Six expression plasmids were transformed into Stb13 *E. coli* competent cells, plated on 2×YT solid medium containing AMP, and incubated upside down at 37°C. The next day, six single colonies were picked and expanded in 4 mL of 2×YT liquid medium containing AMP, and incubated at 37°C with shaking at 250 rpm for 14-16 hours to obtain amplified bacterial solutions. Plasmids were extracted from the bacterial solutions, and after electrophoresis, plasmids of appropriate height were selected for sequencing confirmation. Confirmed clones were amplified in large quantities, and lentiviral expression plasmids were extracted. The autonomous replication gene in the lentiviral vector viral genome has been knocked out, giving it the ability to "self-inactivate," thus preventing the lentivirus from replicating in target cells and infecting other cells.

[0059] 3. Lentiviral packaging

[0060] (a) Preparation of 293T cells: 18-24 hours before transfection, seed 4.5 × 10⁻⁶ cells. 6 293 T cells to 10cm 2 Add complete cell culture medium to the cell culture flask and incubate at 37°C and 5% CO2. Periodically remove the flask and observe the cells under a microscope. When the cells reach 80% confluence, transfection can be performed. Discard the existing culture medium and add 10 mL of virus packaging culture medium.

[0061] (b) Thoroughly mix serum-free Opti-MEM culture medium, lentiviral packaging helper plasmid (containing elements such as HIVgag, REV and VSV-G) and the above-mentioned pCDH-CMV-M-EF1A-copGFP-T2A-puro plasmid (containing the above-mentioned target gene fragment).

[0062] (c) Add serum-free Opti-MEM culture medium and Lipo2000 transfection reagent and mix well.

[0063] (d) Then, serum-free Opti-MEM culture medium containing plasmid DNA was added to serum-free Opti-MEM culture medium containing Lipo2000, gently inverted to mix, and incubated at room temperature for 10 minutes to obtain the transfection complex.

[0064] (e) Add the transfection complex to 293T cells and gently shake the culture dish back and forth to mix the complex. Incubate at 37°C in a 5% CO2 saturated humidity incubator; replace with fresh culture medium 4-6 hours after transduction.

[0065] (f) Collect the culture medium into sterile 50 mL centrifuge tubes at 48 and 72 hours after incubation (at this time the culture medium contains lentivirus particles), centrifuge at 3000 rpm for 5-10 minutes at 4°C to remove cell debris, filter through a 0.45 μm filter into 50 mL centrifuge tubes; add the corresponding amount of concentrate according to the filtrate volume, mix thoroughly, and let it settle overnight at 4°C.

[0066] (g) Take out the virus solution, centrifuge at 3000 rpm for 30 minutes at 4°C, remove the supernatant, and a white precipitate will be visible at the bottom of the tube. Resuspend the virus with an appropriate volume of HBSS, and aliquot 200 μL of virus solution into 0.5 mL cryovials (before aliquoting, attach a label with the virus name and batch number to the outside of the cryovials) and store at -80°C.

[0067] Example 2: Obtaining cultures of other respiratory pathogens

[0068] I. Preparation of cultures of influenza A virus H1N1 (ATCC VR-1520), influenza A virus H5N1, influenza A virus H7N9, influenza A virus H9N2, influenza B virus (ATCC VR-1931), respiratory syncytial virus (ATCC VR-1540), parainfluenza virus (ATCC VR-3), adenovirus (ATCC VR-846), and rhinovirus (ATCC VR-1177):

[0069] (a) Take the cell line out of the liquid nitrogen tank and immediately place the bottle into a 37°C water bath and shake it rapidly until it is completely dissolved. The rewarming process should be completed within 2 minutes. After it is completely dissolved, quickly remove it from the water bath and sterilize the bottle by wiping it with 70% alcohol.

[0070] (b) Transfer the cell line solution to a 15 mL centrifuge tube, balance the solution, centrifuge at 1000 rpm for 5 min to suspend the cells, and discard the supernatant; add 5-10 mL of culture medium, gently mix, and transfer the cell suspension to a T-25 cell culture flask; incubate at 37°C in a 5% carbon dioxide incubator; observe the cell growth under an inverted microscope, and also observe the color change of the culture medium.

[0071] (c) After obtaining a monolayer of cells with about 90% good growth, first pour out the culture medium in the culture flask, then add 1 mL of virus solution, shake to allow the virus solution to fully contact the cells, incubate at 37°C for 10 min, then take it out and shake it again; after incubating for 30 min, add 9 mL of 2% fetal bovine serum culture medium (cell maintenance medium).

[0072] (d) Incubate at 37°C in a 5% CO2 incubator. After a period of incubation, observe the cytopathic effects under an inverted microscope. Harvest the virus by repeating the freeze-thaw cycle three times.

[0073] II. Culture of Mycoplasma pneumoniae (ATCC VR-29085)

[0074] (a) Using a Pasteur pipette or a 1.0 mL pipette, draw 0.5–1.0 mL from a test tube containing 5 mL of liquid to dissolve Mycoplasma pneumoniae particles.

[0075] (b) Under aseptic conditions, aspirate the dissolved Mycoplasma pneumoniae culture back into the test tube and mix well.

[0076] (c) A series of gradient dilution steps. Dilution is important not only for titration but also for maintaining the culture at different growth stages. Many strains will die rapidly when certain pH conditions are reached. It is recommended to dilute the bacterial culture several times from the original tube, as cryoprotectants often inhibit growth during freeze-drying.

[0077] (d) At the same time, prepare a test tube without bacterial culture as a control tube.

[0078] (e) Plate inoculation allows for observation of colony morphology. Inoculate each dilution of the bacterial culture onto plates (four or more plates per dilution) to determine the colony-forming unit.

[0079] (f) Incubate all plates and tubes under the recommended conditions and at the appropriate temperature, pick single colonies to prepare pure culture medium, and obtain Mycoplasma pneumoniae culture.

[0080] III. Culture of Legionella pneumophila (ATCC VR-33152)

[0081] (a) Take 0.5 mL of CYE charcoal yeast extract broth and add it to the bottle to dissolve the Legionella pneumophila lyophilized powder. Transfer all contents to a test tube containing 5-6 mL of broth. This process must be performed under aseptic conditions. Prepare another test tube containing 5-6 mL of broth and take 0.5 mL of the broth from the first tube.

[0082] (b) Pick the first tube of broth medium and inoculate it onto a CYE charcoal yeast extract plate; incubate at 37°C and 5% CO2 for 48-72 hours, pick a single colony to prepare a pure culture medium, and obtain Legionella pneumophila culture.

[0083] IV. Culture of Streptococcus pneumoniae (ATCC VR-49619)

[0084] (a) Take 0.5 mL of brain heart perfusion broth and add it to the bottle to dissolve the lyophilized powder. Transfer all contents to a test tube containing 5-6 mL of broth. This process must be performed under aseptic conditions. Prepare another test tube containing 5-6 mL of broth. Take 0.5 mL of the broth from the first test tube.

[0085] (b) Pick the first tube of broth culture medium and inoculate it onto a trypsin-soybean agar plate.

[0086] (c) Incubate at 37°C and 5% CO2 for 24 hours, pick a single colony to prepare a pure culture medium, and obtain a Streptococcus pneumoniae culture.

[0087] Example 3: Inactivation treatment of pathogen cultures

[0088] The pathogen cultures described above were then inactivated.

[0089] 1. Physical inactivation treatment: First, place the pathogen culture in a 56℃ constant temperature water bath and soak for 30 minutes to carry out physical inactivation treatment;

[0090] 2. Chemical inactivation treatment: Then, nucleic acid dilution solution is added to the above pathogen culture for chemical inactivation treatment. Guanidine isothiocyanate and SDS can denature proteins and thus inactivate them.

[0091] 3. Preparation of nucleic acid dilution buffer:

[0092] (a) Measure 40 mL of sterile pure water using a graduated cylinder and add it to a sterile beaker;

[0093] (b) Add 5 mL of DMSO to a beaker and stir well;

[0094] (c) Weigh 0.19g of EGTA into a beaker and stir to dissolve;

[0095] (d) Weigh 0.1g of SDS into a beaker and stir to dissolve;

[0096] (e) Weigh 1g of BSA into a beaker and stir to dissolve;

[0097] (f) Weigh 1g of guanidine isothiocyanate into a beaker and stir to dissolve;

[0098] (g) Weigh 1.21g of Tris-HCl and add it to a beaker, then stir to dissolve.

[0099] (h) Add 10 mg LEDTA (5 mol / L) to a beaker and stir well;

[0100] (i) Add 5 mL of glycerol to a beaker and stir well; adjust the pH to 7.2-7.6; transfer the pH-adjusted solution to a 100 mL volumetric flask, bring the volume to 100 mL with sterile pure water, seal and autoclave, and store at room temperature.

[0101] Example 4: Extraction of nucleic acids from pathogen cultures

[0102] This invention uses a magnetic bead-based viral DNA / RNA extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP438) to extract nucleic acids from the pathogen culture. The specific operation is as follows:

[0103] (1) Add 20 μL of proteinase K to a clean 1.5 mL centrifuge tube.

[0104] (2) Add 200 μL of virus solution (the virus solution is the pathogen culture medium prepared above) to the centrifuge tube.

[0105] (3) Add 400 μL of Carrier RNA working solution (a mixture of buffer GB and Carrier RNA solution, prepared by adding 6 μL of Carrier RNA to 400 μL of buffer GB) to the sample. Cap the tube and vortex for 10 seconds to mix.

[0106] (4) Incubate at 56°C or room temperature for 10 minutes, then briefly centrifuge to collect the liquid adhering to the tube wall and tube cap.

[0107] (5) Add 400 μL of anhydrous ethanol. At this time, flocculent precipitate may appear. Cover the tube and vortex for 10 seconds to mix thoroughly.

[0108] (6) Add 15 μL of magnetic bead suspension G, cap the tube and vortex for 10 seconds to mix thoroughly. Let stand at room temperature (15-25℃) for 5 minutes.

[0109] (7) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. When the magnetic beads are completely attracted, carefully remove the liquid.

[0110] (8) Remove the centrifuge tube from the magnetic rack, add 500 μL of washing solution GD, and vortex to mix.

[0111] (9) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. When the magnetic beads are completely attracted, carefully remove the liquid.

[0112] (10) Remove the centrifuge tube from the magnetic rack, add 500 μL of rinsing solution RW, and vortex to mix.

[0113] (11) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. When the magnetic beads are fully attracted, carefully remove the liquid.

[0114] (12) Repeat steps 10 and 11 to remove as much liquid as possible.

[0115] (13) Place the centrifuge tubes on a magnetic rack and let them air dry at room temperature for 5-10 minutes.

[0116] (14) Remove the centrifuge tube from the magnetic rack, add 50-100 μL of Nase-free ddH2O, vortex to mix, and incubate at room temperature for 10 minutes.

[0117] (15) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. When the magnetic beads are completely adsorbed, carefully transfer the nucleic acid solution to a new centrifuge tube and store it under appropriate conditions.

[0118] Example 5: Method for determining the value of quality control materials according to the present invention

[0119] This embodiment uses the preparation of H1N1 RNA quality control material as an example to illustrate the method for determining the quality control material of the present invention.

[0120] I. Preparation of H1N1 RNA quality control samples

[0121] 1. Design primers and probes for H1N1

[0122] Based on the H1N1 gene sequence data from NCBI, two pairs of PCR primers and probes were designed using Primer Premier 5.0 biological software. The specific sequence listing is shown in Table 2:

[0123] Table 2: Primers and probes for PCR amplification of H1N1 matrix protein gene (M)

[0124]

[0125] 2. Preparation of H1N1 RNA quality control materials and Q-PCR method

[0126] Using H1N1 influenza virus nucleic acid as a template, primers were used for amplification. The amplified product was ligated into a plasmid vector and then transformed into Trans T1 competent cells. Bacteria containing positive recombinant plasmids were screened and sequenced for identification. Then, using purified SPE I linearized product as a template, the concentration was determined using a NanoDrop2000 UV spectrophotometer, the copy number was calculated, and 10-fold serial dilutions were performed. The DNA copy number per microliter volume was calculated using the following formula: y(copies / μL)=[x(g / μL)DNA / (transcript length in nucleotides×340)]×6.02×10 23 .

[0127] A Q-PCR method was established using well-designed primers and probes. The Q-PCR reaction system consisted of 2* The probeqPCR mixture consisted of 10 μL dUTP Master Mix, 1 μL 10 μM primers, 0.5 μL 10 μM probe, 7.5 μL water, and 2 μL template. Amplification conditions were: 50℃ for 15 min; 95℃ for 15 min; 94℃ for 15 s → 55℃ for 45 s; 98℃ for 10 min; 55℃ for 45 s, for 40 cycles. The constructed quality control samples were serially diluted 10-fold to plot the Q-PCR standard curve. The copy number of the amplification curve was 5.37 × 10⁻⁶. 6 ~5.37×10 1 copies / μL.

[0128] 3. Methods and optimization of droplet digital PCR for H1N1 influenza virus

[0129] Droplet digital PCR (ddPCR) involves four steps: reaction system preparation, droplet generation, PCR amplification, and droplet reading. Optimized results are determined by comparing the number of droplets generated, the droplet distribution, and the intensity of the droplet fluorescence signal in ddPCR.

[0130] (1) Extraction of H1N1 viral nucleic acid: The viral DNA / RNA was extracted using a magnetic bead method viral DNA / RNA extraction kit. For details, please refer to Example 4.

[0131] (2) Formulation system

[0132] The four primers and two probes selected from Table 2 were randomly combined to form eight primer-probe combinations, as shown in Table 3:

[0133] Table 3: Random combinations of four primers and two probes

[0134]

[0135]

[0136] One-step RT-ddPCR probe premix (20 μl): 10 μL of 2xProbe Q-PCR dUTP Master mix, 1 μL of 10 μM upstream and downstream primers, 0.5 μL of 20 μM specific probe, and 7.5 μL of DNase-free distilled water.

[0137] The 20 μL RT-ddPCR reaction solution is formulated as follows: 20 μL of one-step RT-ddPCR probe premix and 2 μL of RNA template from the sample to be tested.

[0138] PCR reaction program: 50℃ for 15 min; 95℃ for 15 min; 94℃ for 15 s → 55℃ for 45 s, 40 cycles; 98℃ for 10 min; 55℃ for 45 s.

[0139] (3) Droplet formation

[0140] a. Place a new DG8 cartridge into the holder.

[0141] b. Add 20L of the reaction mixture into the eight holes in the middle row of the DG8 cartridge.

[0142] c. Add 70 μL of microdroplet-generated oil (DG Oil) to each of the eight holes at the bottom of the DG8 cartridge.

[0143] d. Cover with a gasket.

[0144] e. Gently and steadily place the holder into the droplet generator to begin generating droplets.

[0145] f. Pipette 40 μL of the microdrop from the top well into a single row of 96-well plates.

[0146] g. Immediately after transferring the droplets, seal the PCR plate with aluminum foil to prevent evaporation. Set the sealing temperature to 180°C for 5 seconds.

[0147] (4) Detection results: The detection value of combination 1 was 1770 copies / μL, the detection value of combination 2 was 2000 copies / μL, the detection value of combination 3 was 1894 copies / μL, the detection value of combination 4 was 1822 copies / μL, the detection value of combination 5 was 2006 copies / μL, the detection value of combination 6 was 2818 copies / μL, the detection value of combination 7 was 2454 copies / μL, and the detection value of combination 8 was 2158 copies / μL.

[0148] The results showed that the optimal primer-probe combination 6 in the reaction system was FORWARD-1, REVERSE-1, and Probe-1.

[0149] (5) The final droplet digital PCR reaction system and reaction conditions were determined as follows:

[0150] Table 4. Droplet-based digital PCR reaction system

[0151]

[0152]

[0153] Table 5. Reaction conditions for droplet digital PCR

[0154]

[0155] Example 6: Test on the determination of the quality control material and the evaluation of its uncertainty.

[0156] This embodiment uses the determination test of the quality control material for the novel coronavirus as an example to illustrate the determination of the nucleic acid values ​​of various pathogens and the uncertainty evaluation test of the quality control material of the present invention.

[0157] (1) Five tubes of novel coronavirus quality control material were extracted and nucleic acid extraction and standard digital PCR were performed on the original samples by four different testing personnel at different times. Each person tested five tubes and tested each tube once, and finally obtained 20 sets of experimental data.

[0158] Table 6: Experimental data from four different testing personnel

[0159]

[0160] Within-group suspicious value test

[0161] For each group of independent measurement results, use appropriate statistical methods, such as Grubbs' method, Dixon's method, t-test, etc., combined with technical judgment, to eliminate suspicious values.

[0162] Table 7: Results of within-group suspicious value test

[0163]

[0164] (3) Accuracy verification of intergroup data, etc.

[0165] The standard deviation of each group of data was tested for equal precision using the Cochrane test or the F-test. Data groups with significant differences were reviewed technically before a decision was made on whether to remove them.

[0166] When all groups of data have the same precision, use a t-test to check whether there is a significant difference in the average values ​​of the groups. If there is no significant difference in the average values, first merge the data and then use an appropriate method to test the normality of the data distribution. If the data conforms to a normal distribution, the average of the multiple average values ​​can be calculated again to obtain the overall average value, which is the standard value.

[0167] Table 8: Results of Homogeneity of Variance Test

[0168]

[0169] Table 9: Results of Multiple Comparisons (LSD)

[0170] Dependent variable: Detected concentration

[0171]

[0172]

[0173] Table 10

[0174]

[0175] a. Lilliefers significance level correction; *. This is the lower bound of the true significance level.

[0176] The results of the Levene test for homogeneity of variance show that the Levene statistic is 2.972 and the significance P-value is 0.063 > 0.05. Therefore, the four groups of data are homogeneous in variance.

[0177] The results of multiple comparisons show that the significance of the LSD method is greater than 0.05, indicating that there is no statistically significant difference between the data in each group.

[0178] Both the Shapiro-Wilk and Kolmogorov-Smirnov tests can be used to perform normality tests. The results show that the Sig values ​​are all >0.05, indicating that the data distribution conforms to a normal distribution.

[0179] (4) Calculation of standard values

[0180]

[0181] (5) Determination of final uncertainty

[0182] To summarize the components that constitute the uncertainty U of the quality control material:

[0183] Table 11

[0184]

[0185] Final value and uncertainty:

[0186] Table 12

[0187]

[0188] Based on the above-described method of value determination and uncertainty assessment test, the standard values ​​of the quality control materials of this invention are obtained, as shown in Table 13:

[0189] Table 13: Standard values ​​of the quality control materials of this invention

[0190]

[0191]

[0192] The standard values ​​described in this invention are the concentrations of nucleic acids of each pathogen. In the table, Severe Acute Respiratory Syndrome Coronavirus is SARS-CoV, and Middle East Respiratory Syndrome Coronavirus is MERS-CoV.

[0193] Example 7: Uniformity detection of the respiratory pathogen assay control material of the present invention

[0194] (1) Take 1 mL of the positive sample of respiratory pathogens with a fixed value and add it to 499 mL of diluent. Mix thoroughly to obtain the quality control material for respiratory pathogens with a fixed value.

[0195] (2) Detection of uniformity

[0196] Thirty tubes of the above quality control samples were randomly selected, and each tube was tested three times. The minimum sample size was 200 μL. The nucleic acid extraction method, reaction conditions, and reaction system of Example 2 were used for testing. Analysis of variance was used to analyze the homogeneity of the quality control samples. A randomized sequential repeated measurement method was employed. The samples were first sequentially coded, and a random number table was used to determine the sample number for each sample. Three repeated measurements were performed. The measurement protocol is as follows:

[0197] Repeated determination 1:

[0198] 141-80-12-77-48-07-189-159-148-114-135-169-174-63-03-195-150-74-21-15-14-100-128-137-124-56-24-63-83-192

[0199] Repeated determination 2:

[0200] 83-141-15-195-74-14-63-114-159-07-48-03-128-24-148-174-124-21-12-80-192-77-63-56-137-100-150-169-135-189

[0201] Repeated measurements 3:

[0202] 56-192-148-195-114-63-24-15-77-189-141-159-124-80-137-12-63-14-48-174-07-83-128-100-21-74-150-03-169-135

[0203] The test results are shown in Table 14:

[0204] Table 14 Analysis results of uniformity test

[0205] serial number name Between-group variance Within-group variance F value 1 Human coronavirus HCoV-NL63 3.55E+06 1.15E+07 0.683 2 Human coronavirus HCoV-229E 3.60E+06 1.43E+07 0.561 3 Human coronavirus HCoV-HKU1 3.33E+06 8.83E+06 0.591 4 Human coronavirus HCoV-OC43 2.74E+07 5.05E+07 1.21 5 Severe Acute Respiratory Syndrome Coronavirus 1.76E+07 1.24E+08 0.315 6 Middle East Respiratory Syndrome Coronavirus 7.56E+06 2.22E+07 0.756 7 novel coronavirus 1.49E+07 3.52E+07 0.941 8 H1N1 influenza A virus 2.45E+07 5.57E+07 0.978 9 H5N1 influenza A virus 1.35E+07 2.22E+07 1.35 10 H7N9 influenza A virus 8.58E+06 1.19E+07 1.59 11 H9N2 influenza A virus 5.00E+06 1.31E+07 0.856 12 Influenza B virus 3.74E+07 5.23E+07 1.58 13 Respiratory syncytial virus 4.55E+07 6.43E+07 1.57 14 Parainfluenza virus 5.67E+06 1.95E+07 0.646 15 adenovirus 1.20E+07 1.67E+07 1.58 16 Mycoplasma pneumoniae 1.17E+08 1.44E+08 1.81 17 Streptococcus pneumoniae 3.84E+06 9.14E+06 0.933 18 rhinovirus 1.78E+08 7.78E+08 0.507 19 Legionella pneumophila 3.15E+08 1.05E+09 0.665

[0206] The critical value of F0.05(10,30) obtained from the F distribution critical value table is 2.39. Since the F values ​​of the respiratory pathogen control samples obtained in the homogeneity experiment are all less than F0.05(10,30), it can be considered that there are no significant differences within or between groups. Therefore, the sample of this quality control is homogeneous and meets the requirements.

[0207] Example 8: Stability detection of the respiratory pathogen assay control material of the present invention

[0208] Long-term stability studies focus on the stability of respiratory pathogen control materials under specific storage conditions. In this study, the storage conditions were -20±5℃, and the number of samples was sufficient for testing up to the 14th month or longer. Testing was conducted at 1, 2, 5, 8, 12, 14... months. Five tubes of control material were sampled each time, and each sample was tested twice. The mean values ​​were calculated, and a linear regression method was used for stability monitoring.

[0209] Starting from the third stability test, statistical analysis was performed after each stability experiment. X represents time, and Y represents the characteristic value of the standard substance (logarithmic mean of concentration). The result was fitted to a straight line, and its slope b1 and intercept b0 were calculated.

[0210] The t-factor values ​​with n-2 degrees of freedom and a significance level of p = 0.95 (95% confidence level) are as follows:

[0211] Table 15: t-factor values

[0212]

[0213] Table 16: Results of 12-month stability test (at -20±5℃)

[0214]

[0215]

[0216] Results and Analysis: The stability of the respiratory pathogen control materials under storage conditions of -20±5℃ was analyzed, and the results showed that the values ​​were all less than the corresponding t values. 0.05 The quantile of the n-2 factor indicates that the slope is not significantly different, and no instability was observed, thus meeting the needs of practical measurement. Considering the influence of factors such as transportation conditions on the stability of the quality control material, it is determined that the quality control material is stable for 12 months when stored at -20±5℃.

[0217] Example 9: Specificity test of the quality control material of the present invention

[0218] The following combinations of the present invention were used: Combination 1: Human coronavirus HCoV-NL63, Human coronavirus HCoV-229E, Human coronavirus HCoV-HKU1, Human coronavirus HCoV-OC43, SARS-CoV, MERS-CoV, and novel coronavirus; Combination 2: Influenza A virus H1N1, Influenza A virus H5N1, Influenza A virus H7N9, Influenza A virus H9N2, Influenza B virus (IVB), Respiratory syncytial virus (RSV), and Parainfluenza virus (PIV); Combination 3: Adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Rhinovirus, and Legionella pneumophila. Nucleic acid was extracted from each combination, and then digital PCR was performed to verify its specificity. The test results are as follows:

[0219] Table 17: Results of Combined Detection

[0220]

[0221]

[0222] Table 18: Results of Combination II Detection

[0223]

[0224]

[0225] Table 19: Results of Combined Three Detection

[0226] serial number Virus Name abbreviation Test results 1 Human coronavirus HCoV-NL63 HCoV-NL63 Negative 2 Human coronavirus HCoV-229E HCoV-229E Negative 3 Human coronavirus HCoV-HKU1 HCoV-HKU1 Negative 4 Human coronavirus HCoV-OC43 HCoV-OC43 Negative 5 Severe Acute Respiratory Syndrome Coronavirus SARS-CoV Negative 6 Middle East Respiratory Syndrome Coronavirus MERS-CoV Negative 7 novel coronavirus 2019-nCoV Negative 8 H1N1 influenza A virus H1N1 Negative 9 H5N1 influenza A virus H5N1 Negative 10 H7N9 influenza A virus H7N9 Negative 11 H9N2 influenza A virus H9N2 Negative 12 Influenza B virus IVB Negative 13 Respiratory syncytial virus RSV Negative 14 Parainfluenza virus PIV Negative 15 adenovirus ADV Positive 16 Mycoplasma pneumoniae MP Positive 17 Streptococcus pneumoniae / Positive 18 rhinovirus RhV Positive 19 Legionella pneumophila / Positive

[0227] Example 10: Comparison Test of the Quality Control Material of the Present Invention with Domestic Similar Novel Coronavirus Pseudovirus Products

[0228] The novel coronavirus 2019-nCoV nucleic acid detection kits (fluorescent PCR method) from Sun Yat-sen University Da An Gene Co., Ltd., Sansure Biotech Co., Ltd., Shanghai ZJ Biotech Co., Ltd., BGI Genomics (Wuhan) Co., Ltd., Guangdong Kaipu Biotechnology Co., Ltd., Shanghai Berger Medical Technology Co., Ltd., and Beijing Zhuocheng Huisheng Biotechnology Co., Ltd. were used to detect the virus. China Metrology The novel coronavirus nucleic acid standard materials GBW(E)091090 and GBW(E)091089 [ORF1ab gene fragment, genomic coordinates: 14911-15910; full length of N gene; full length of E gene] from the Chinese Academy of Sciences, the novel coronavirus in vitro transcribed RNA standard materials GBW(E)091111 and GBW(E)091112 [ORF1ab gene fragment, genomic coordinates: 13321-15540; full length of N gene; full length of E gene] from the Shanghai Institute of Metrology, and this invention were used for testing, strictly following the instructions of the kit.

[0229] Table 20: Detection results of the N gene

[0230]

[0231] Table 21: Detection results of the ORF1ab gene

[0232]

[0233] Table 22: Detection results of the E gene

[0234]

[0235] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A quality control material for detecting nucleic acids of respiratory pathogens, characterized in that, The respiratory pathogens of the quality control materials are: coronavirus, influenza virus, respiratory syncytial virus, adenovirus, rhinovirus, mycoplasma pneumoniae, streptococcus pneumoniae, and Legionella pneumophila. The nucleic acid of the coronavirus consists of six 4000-5500bp nucleic acid fragments; The coronaviruses include: human coronavirus HCoV-NL63, human coronavirus HCoV-229E, human coronavirus HCoV-HKU1, human coronavirus HCoV-OC43, SRSA-CoV, MERS-CoV, and novel coronavirus; the influenza viruses include: influenza A virus H1N1, influenza A virus H5N1, influenza A virus H7N9, influenza A virus H9N2, influenza B virus IVB, and parainfluenza virus PIV; The standard value for the human coronavirus HCoV-NL63 is 1.2 × 10⁻⁶. 4 The standard value for human coronavirus HCoV-229E is 1.9 × 10 copies / μL. 4 The standard value for human coronavirus HCoV-HKU1 is 1.3 × 10 copies / μL. 4 The standard value for human coronavirus HCoV-OC43 is 2.0 × 10 copies / μL. 4 The standard value for SRSA-CoV is 2.0 × 10 copies / μL. 4 The standard value for MERS-CoV is 1.8 × 10⁻⁶ copies / μL. 4 The standard value for the novel coronavirus is 1.6 × 10⁶ copies / μL. 4 The standard value for influenza A virus H1N1 is 1.1 × 10 copies / μL. 4 The standard value for influenza A virus H5N1 is 1.9 × 10 copies / μL. 4 The standard value for influenza A virus H7N9 is 1.5 × 10 copies / μL. 4 The standard value for influenza A virus H9N2 is 1.3 × 10 copies / μL. 4 The standard value for influenza B virus is 2.3 × 10 copies / μL. 4 The standard value for respiratory syncytial virus (RSV) is 2.2 × 10⁻⁶ copies / μL. 4 The standard value for parainfluenza virus is 1.5 × 10 copies / μL. 4 The standard value for adenovirus is 1.3 × 10⁻⁶ copies / μL. 4 The standard value for Mycoplasma pneumoniae is 1.9 × 10⁻⁶ copies / μL. 4 The standard value for Streptococcus pneumoniae is 1.3 × 10⁻⁶ copies / μL. 4 The standard value for rhinovirus is 2.3 × 10⁻⁶ copies / μL. 4 The standard value for Legionella pneumophila is 2.1 × 10⁻⁶ copies / μL. 4 copies / μL.

2. The quality control material as described in claim 1, characterized in that, It also includes a diluent comprising the following components: dimethyl sulfoxide, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, sodium dodecyl sulfate, bovine serum albumin, guanidine isothiocyanate, Tris-HCl, ethylenediaminetetraacetic acid, and glycerol; the pH of the diluent is 7.2 to 7.

6.

3. A method for preparing the quality control material as described in claim 2, characterized in that, Includes the following steps: S1. Prepare respiratory pathogen cultures and extract pathogen nucleic acids; the respiratory pathogens include coronaviruses, influenza viruses, respiratory syncytial viruses, adenoviruses, Mycoplasma pneumoniae, Streptococcus pneumoniae, rhinoviruses, and Legionella pneumophila; the coronaviruses include: human coronavirus HCoV-NL63, human coronavirus HCoV-229E, human coronavirus HCoV-HKU1, human coronavirus HCoV-OC43, SRSA-CoV, MERS-CoV, and novel coronavirus; the influenza viruses include: influenza A virus H1N1, influenza A virus H5N1, influenza A virus H7N9, influenza A virus H9N2, influenza B virus IVB, and parainfluenza virus PIV; S2 The nucleic acid content obtained in step S1 was detected by real-time PCR to obtain the original concentration value of nucleic acid for each pathogen; S3 uses the diluent described in claim 2 to dilute the respiratory pathogen from step S2 to the desired concentration value, thereby obtaining a quality control material containing the standard value of the respiratory pathogen described in claim 1.

4. The preparation method according to claim 3, characterized in that, Step S1 is specifically implemented using the following method: (A) Extraction of the nucleic acid of the coronavirus: First, the full-length genome sequence of the coronavirus is divided into 6 fragments, which are then ligated to lentiviral expression vectors using a seamless cloning method to obtain 6 expression plasmids; then, the expression plasmids that have been identified as having qualified sequences are co-transfected with packaging vectors into packaging cells to obtain coronavirus liquid. At this time, the extracted coronavirus nucleic acid consists of 6 nucleic acid fragments containing partial genome sequences. (B) Extraction of nucleic acids from the influenza virus, respiratory syncytial virus, adenovirus and rhinovirus: After mixing and culturing the influenza virus solution, respiratory syncytial virus solution, adenovirus solution and rhinovirus solution with infected host cells respectively, the viruses are harvested and their nucleic acids are extracted. (C) Extraction of nucleic acids from Mycoplasma pneumoniae, Legionella pneumophila and Streptococcus pneumoniae: Mycoplasma pneumoniae, Legionella pneumophila and Streptococcus pneumoniae were inoculated into the culture medium, and their culture solutions were obtained and their nucleic acids were extracted.

5. The preparation method according to claim 4, characterized in that, The nucleic acid was extracted using a magnetic bead-based nucleic acid extraction kit.

6. The preparation method according to claim 3, characterized in that, The method further includes the following steps: (1) The nucleic acid of S3 was aliquoted and its performance was evaluated and analyzed; (2) The quality control material in step S3 was absolutely quantitatively detected by droplet digital PCR to determine the quantification value.

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