Use of a pre-treatment reagent composition for the preparation of a mass spectrometry test kit for the detection of respiratory pathogens

Abstract

The application provides a use of a pretreatment reagent composition for preparing a mass spectrometry detection kit for detecting respiratory pathogens, the pretreatment reagent composition comprising all reagents for mass spectrometry detection, such as a nasal swab preservative, a mass spectrometry matrix, a mass spectrometry calibrant, a quality control product, etc. The detection object of the application is a nasal swab sample, sampling is convenient, interference is small, and the pretreatment reagent composition can quickly inactivate and keep the sample for a long time, so that a variety of respiratory pathogens can be quickly, accurately and high-throughput mass spectrometry detected by cooperating the reagent composition with an existing detection reagent or kit.

Description

Technical Field

[0001] This invention belongs to the field of protein proteometry detection. It relates to a product that utilizes time-of-flight mass spectrometry (TOF-MS) technology for the rapid detection of novel coronavirus and other respiratory pathogen infections. Background Technology

[0002] In recent years, mass spectrometry techniques have emerged for detecting characteristic peptides in patients infected with the novel coronavirus. This means that after a patient is infected with the novel coronavirus, specific characteristic peptides will appear in their body; detecting these peptides can provide a relatively accurate identification of the infection. For example, the study "Detection of SARS-CoV-2 in nasal swabs using MALDI-MS" reported a study on the direct detection of nasal swab samples using MALDI-MS. This study used residual samples from novel coronavirus nucleic acid testing for analysis and established a mass spectrometry detection model based on machine learning, achieving a maximum accuracy of 93.9%. However, this study did not improve the sample preservation reagents, and therefore, some components in the preservation solution severely affected the quality of the mass spectra.

[0003] A novel application of automated machine learning with MALDI-TOF-MS for rapid high-throughput screening of COVID-19: a proof of concept. This study presents a novel coronavirus entry detection solution jointly developed using machine learning and MALDI-TOF technology. The solution boasts performance comparable to existing commercial tests with shorter processing times, achieving a daily testing capacity of 1104 tests per instrument. In this study, nasal swab samples were preserved in saline solution, and virus inactivation was achieved by irradiating the mass spectrometry target plate with ultraviolet light. While the simple preservation solution composition significantly reduced interference with the mass spectrometry signal, the degradation of sample peptides during preservation remained a concern. Furthermore, simple ultraviolet disinfection and delayed disinfection steps still could not guarantee the safety of the detection process.

[0004] Chinese patent application 202010251209.0 discloses a novel coronavirus N protein extraction and preservation solution, its preparation method, and its application. This solution comprises casein, antibiotics, bovine serum albumin, ethylphenyl polyethylene glycol, and anti-erythrocyte monoclonal antibodies. The novel coronavirus extraction and preservation solution provided by this invention exhibits good compatibility with various samples, including nasal swabs, pharyngeal swabs, oral swabs, bronchoalveolar lavage fluid, sputum, and saliva, providing a suitable and stable buffer environment for viral samples, which is beneficial for sample detection and preservation. However, this invention is only intended for immunoassay purposes, and the complex composition of the preservation solution makes it unsuitable for large-scale and rapid detection of explosive novel coronavirus samples.

[0005] Chinese patent application 202010587255.8, "A Preservative Solution for Improving Nucleic Acid Stability and Its Application," discloses a preservative solution for improving nucleic acid stability and its application. The preservative solution described in this invention includes one or more of tris(hydroxymethyl)aminomethane (Tris), a nonionic surfactant, EDTA, an inorganic salt, and sorbitol. However, this preservative solution is used to preserve nucleic acids in samples and is not suitable for preserving peptides containing characteristic polypeptides that appear after viral infection.

[0006] As the closest prior art, Chinese patent application 201610150642.9, "A Sample Pretreatment Solution for Detecting and Separating Respiratory Viruses in Samples," discloses a sample pretreatment solution for detecting and separating respiratory viruses in samples, comprising: (i) a virus protectant selected from the group consisting of calcium chloride, anhydrous magnesium sulfate, potassium chloride, sodium chloride, anhydrous sodium dihydrogen phosphate, anhydrous glucose, or combinations thereof; (ii) a chromogenic agent; and (iii) a sterilizing agent. The chromogenic agent is selected from the group consisting of phenol red, neutral red, or combinations thereof. Although this invention introduces a chromogenic agent to indicate the sample preservation status, it still focuses on the preservation of the virus, rather than the preservation of characteristic polypeptides produced after viral infection. Therefore, the above-mentioned pretreatment solution is still unsuitable for preserving characteristic polypeptides that appear after viral infection. Furthermore, the pretreatment solution contains salt compounds such as calcium chloride, magnesium sulfate, and potassium chloride, which generate impurities during laser mass spectrometry ionization, affecting the mass spectrometry signal. Therefore, it is unsuitable as a component of the pretreatment solution and matrix for protein spectrometry.

[0007] In summary, existing mass spectrometry detection kits for detecting respiratory pathogens, including the novel coronavirus, all have limitations in sample pretreatment, such as the inability to rapidly inactivate the virus, or the inactivated virus being easily degraded or interfering with the mass spectrometry signal. These limitations lead to the detection of characteristic peptides or immune peptides present in the patient's body.

[0008] Therefore, there is a current need for a mass spectrometry detection kit for pretreatment of respiratory pathogens such as the novel coronavirus. Summary of the Invention

[0009] The first principle of this invention lies in the fact that, in the prior art, although it is known that ethanol can inactivate and lyse viruses, it also denatures and inactivates proteins or peptides. Therefore, ethanol is usually only used as a virus inactivation and lysis agent, and not as a preservation solution or pretreatment solution for peptides or proteins. However, the inventors unexpectedly discovered that in characteristic peptide mass spectrometry, even denatured peptide fragments, after being excited and lysed by an electric field to produce specific peptide fragments, do not change in molecular weight, thus not affecting the acquisition of indicative characteristic spectra.

[0010] The second principle of this invention is that the inventors first proposed that after being infected by a virus, the human body produces characteristic polypeptides (which can be antibody fragments or non-antibody characteristic polypeptides produced after an immune response, i.e., immune response polypeptide products or immune polypeptides). Through the pretreatment agent of this invention, these characteristic polypeptides (immune response polypeptide products) can be detected, thereby completing the final identification.

[0011] The third principle of this invention is that it has been verified that the added phenol red not only does not affect the mass spectrometry response and peptide stability, but can also act as an indicator to show the stability of the pretreatment solution.

[0012] The fourth principle of this invention is that the added formic acid in the pretreatment solution can assist the ionization of peptides in the sample in the mass spectrometer, thereby increasing the intensity of the mass spectrometer signal.

[0013] Therefore, the first objective of this invention is to provide a pretreatment reagent composition for laser mass spectrometry detection of respiratory pathogens, comprising a nasal swab preservation solution and a nasal swab, wherein the nasal swab preservation solution contains 70%–80% ethanol (V / V) and phenol red (10–200 mg / L), wherein the respiratory pathogens include novel coronavirus, influenza A virus, influenza B virus, SARS virus, MERS virus, etc.

[0014] In one embodiment, the ethanol has the effect of inactivating respiratory pathogens such as viruses. When the nasal swab sample is collected, it is immediately placed in the nasal swab preservation solution and mixed evenly. The preservation solution can inactivate the virus while preserving the virus, thus shortening the detection time.

[0015] In another implementation, phenol red is present as an indicator, which can visually indicate the state of the nasal swab sample in the sample preservation solution. When the color of phenol red changes significantly, it indicates that the pH of the solution system has changed, which in turn indicates that the protein and polypeptide components in the nasal swab have undergone an acid-base conversion chemical reaction, indicating that the sample components are no longer in their initial state and can no longer be used for mass spectrometry detection.

[0016] In other embodiments, the nasal swab preservation solution contains ethanol, phenol red, and formic acid (0.2%–5.0%, V / V), wherein formic acid can assist in the ionization of peptides in the sample in mass spectrometry, thereby increasing the intensity of the mass spectrometry signal.

[0017] In any of the above embodiments, the volume of the nasal swab preservation solution is 0.2 to 4.0 ml / tube, preferably 1.0 to 3.0 ml / tube.

[0018] In any of the above embodiments, the laser mass spectrometer is selected from MALDI TOF MS or Clin-ToF.

[0019] A second objective of this invention is to provide a mass spectrometry detection kit comprising the above-described reagent composition for detecting respiratory pathogens, which further includes matrix powder, matrix solvent, mass spectrometry calibrators, positive control and negative control, wherein the respiratory pathogens include novel coronavirus, influenza A virus, influenza B virus, SARS virus, MERS virus, etc.

[0020] In one embodiment, the kit includes a calibration tube to ensure the accuracy of molecular weight measurements by the mass spectrometer. In a specific embodiment, the calibrator, when detected by mass spectrometry, yields 3 to 10 characteristic peptide peaks, with the molecular weights of each peak relatively uniformly distributed within the target molecular weight range. The sample in the calibration tube is used for calibrating the mass axis of the mass spectrometer, and parallel mass spectrometry tests with the target sample are performed to determine the accuracy and reliability of the target sample's molecular weight information.

[0021] In a preferred embodiment, the calibrators include adrenocorticotropic hormone, cytochrome C, and myoglobin, with characteristic peptide peaks at m / z values ​​of 2465 m / z, 4121 m / z, 6181 m / z, 8477 m / z, 12362 m / z, and 16952 m / z, respectively.

[0022] In another embodiment, the kit also includes quality control samples to determine the reliability of the test results. These quality control samples are formulated from a mixture of protein peptides; the positive control has a characteristic profile similar to that of individuals infected with respiratory pathogens, while the negative control has a characteristic profile similar to that of healthy individuals. Parallel mass spectrometry tests are performed between the quality control samples and the test samples to determine the accuracy and reliability of the test results.

[0023] In any of the above embodiments, the laser mass spectrometer is selected from MALDI TOF MS or Clin-ToF.

[0024] A third objective of this invention is to provide the use of the reagent composition in the preparation of a mass spectrometry detection kit for detecting respiratory pathogens, including novel coronavirus, influenza A virus, influenza B virus, SARS virus, MERS virus, etc.

[0025] In one embodiment, the kit includes a nasal swab preservation solution and a nasal swab, wherein the nasal swab preservation solution contains 70%–80% ethanol (V / V) and phenol red (10–200 mg / L).

[0026] In other embodiments, the nasal swab preservation solution contains ethanol, phenol red, and formic acid (0.2%–5.0%, V / V), wherein formic acid can assist in the ionization of peptides in the sample in mass spectrometry, thereby increasing the intensity of the mass spectrometry signal.

[0027] In any of the above embodiments, the volume of the nasal swab preservation solution is 0.2 to 4.0 ml / tube, preferably 1.0 to 3.0 ml / tube.

[0028] In any of the above embodiments, the kit further includes matrix powder, matrix solvent, mass spectrometry calibrators, positive control, and negative control.

[0029] In any of the above embodiments, the kit includes a calibration tube to ensure the accuracy of the molecular weight measured by the mass spectrometer. In one specific embodiment, the calibrator, when detected by mass spectrometry, yields 3 to 10 characteristic peptide peaks, with the molecular weights of each peptide peak relatively uniformly distributed within the range of the molecular weight to be measured. The sample in the calibration tube is used for calibrating the mass axis of the mass spectrometer, and the accuracy and reliability of the molecular weight information of the sample to be measured are determined by performing parallel mass spectrometry tests with the sample to be measured.

[0030] In any of the above embodiments, the calibrators include adrenocorticotropic hormone, cytochrome C, and myoglobin, with characteristic peptide peaks at m / z values ​​of 2465 m / z, 4121 m / z, 6181 m / z, 8477 m / z, 12362 m / z, and 16952 m / z, respectively.

[0031] In any of the above embodiments, the kit further includes a quality control to determine the reliability of the test results. This quality control is formulated from a mixture of protein peptides; the positive control has a characteristic profile similar to that of individuals infected with respiratory pathogens, and the negative control has a characteristic profile similar to that of healthy individuals. Parallel mass spectrometry tests are performed between the quality control and the test sample to determine the accuracy and reliability of the test results.

[0032] In any of the above embodiments, the reagent composition can be combined with existing mass spectrometry detection reagents to prepare the mass spectrometry detection kit.

[0033] In any of the above embodiments, the laser mass spectrometer is selected from MALDI TOF MS or Clin-ToF.

[0034] Technical effect

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] 1. The reagent composition or test kit of the present invention can effectively inactivate viruses that may exist in nasal swab samples in 10 seconds, and at the same time convert the peptides in the sample into mass spectrometry peak signals. By detecting changes in a set of characteristic peptide mass spectrometry patterns, the diagnosis of diseases such as novel coronavirus and influenza can be performed.

[0037] 2. This invention features simple sample pretreatment, low requirements for the testing environment, low testing cost, and allows for one-person-one-testing without the need for pooling. Compared to existing methods, the mass spectrometry-based detection method for novel coronavirus immune response nasal swab peptide characteristics is more suitable for rapid screening of novel coronavirus infections in large populations, as well as rapid detection of other respiratory pathogens such as influenza. The current practice of pooled testing in nucleic acid testing stems from its complexity, long testing time, and low throughput. One-person-one-testing is inefficient for large-scale screening. Pooled testing is a necessary compromise that sacrifices sensitivity for increased efficiency. Mass spectrometry's main advantages are speed, simplicity, and high throughput. One-person-one-testing offers high accuracy and is more conducive to tracing the source and tracking the patient's disease progression. Therefore, while this invention also supports pooled testing, it is generally unnecessary to use it for patient testing in most cases.

[0038] 3. Unlike traditional methods that only target the virus itself, this invention detects multiple characteristic protein combinations that differ between infected individuals and healthy individuals with different respiratory pathogens, and simultaneously monitors changes in multiple immune characteristic peptides.

[0039] 4. Compared with nucleic acid testing, the method of this invention has advantages such as no need for additional virus inactivation steps, simple operation, low testing cost, and high throughput, which can shorten the overall testing time to less than 1 minute per sample. This is not only beneficial for rapid screening of large populations, but also has high application prospects in scenarios such as emergency rooms where rapid test results are required.

[0040] 5. This kit achieves 100% accuracy, 100% sensitivity, and 100% specificity in detecting novel coronavirus infection. This result demonstrates that the present invention can meet practical testing needs.

[0041] 6. This invention can be combined with existing mass spectrometry detection reagents (such as detection matrices, viral characteristic peptide markers, etc.) for detecting respiratory pathogens such as the novel coronavirus to prepare related mass spectrometry detection kits. These existing mass spectrometry detection reagents include, but are not limited to, the prior applications of this invention, 202110155492.1, 2021101552589, and 2021101589526, etc., as well as products and uses for nasal swab detection of the novel coronavirus based on the characteristic peptide spectrum of immune responses (Invention Application No.: 202211292701.8). Attached Figure Description

[0042] Figure 1 : Color change of the preservation solution. The solution in the left preservation tube (tube A) is yellow, indicating that the sample can be used normally for mass spectrometry detection; the solution in the right preservation tube (tube B) is colorless, indicating that the sample has degraded and is no longer suitable for mass spectrometry detection.

[0043] Figure 2 Mass spectra obtained from nasal swab samples of the same subject treated with different preservation solutions, where:

[0044] Preservation Solution A: The nasal swab preservation solution of the present invention is used;

[0045] Preservation solution B: derived from the preservation solution (Cary-Blair transport medium) used in the study of Detection of SARS-CoV-2 in nasal swabs using MALDI-MS.

[0046] Preservative solution C: The preservative solution (i.e., physiological saline) used in "Novel application of automated machine learning with MALDI-TOF-MS for rapid high-throughput screening of COVID-19: a proof of concept".

[0047] Figure 3: Effect of nasal swab preservation solution volume (0.3 ml, 0.6 ml, 1.0 ml, 2.0 ml, and 3.0 ml) on the chromatogram. Wherein:

[0048] Figure 3a This is a full mass spectrum of the sample after being treated with different volumes of nasal swab preservation solution, with a molecular weight range of 2000–20000 m / z;

[0049] Figures 3b-3d They are respectively Figure 3a The magnified view shows that some spectral peaks (peaks within the box) are unstable in the 0.3 ml and 0.6 ml groups.

[0050] Figure 4: Mass spectra of the three preservation solutions at different preservation times. (Note:)

[0051] Figure 4a Use the nasal swab preservation solution of the present invention;

[0052] Figure 4b The preservation solution (Cary-Blair transport medium) used in the Detection of SARS-CoV-2 in nasal swabs using MALDI-MS was employed.

[0053] Figure 4c The preservation solution (physiological saline) used in the concept of using a novel application of automated machine learning with MALDI-TOF-MS for rapid high-throughput screening of COVID-19: a proof of concept.

[0054] Figure 5 Sample testing process.

[0055] Figure 6: Validation set confusion matrix. Wherein, Figure 6-1 The confusion matrix for the validation set is based on 20 characteristic peaks;

[0056] Figure 6-2 This is a confusion matrix based on 5 characteristic peaks.

[0057] Figure 7 Mass spectra of different samples obtained by this kit. From bottom to top: Mass spectra of individuals infected with the novel coronavirus; Mass spectra of individuals infected with influenza; Mass spectra of healthy individuals. Detailed Implementation

[0058] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0059] Example 1. Determination of the composition of nasal swab preservation solution

[0060] This invention is the first nasal swab preservation solution specifically designed for MALDI-TOF mass spectrometry. The main components of the preservation solution are ethanol and phenol red, or ethanol, formic acid, and phenol red. The concentration of ethanol is 70-80% (V / V), which has the effect of inactivating viruses. Nasal swab samples are immediately placed in the nasal swab preservation solution after collection and mixed thoroughly. The preservation solution can inactivate the virus while preserving it, shortening the detection time. The nasal swab preservation solution contains formic acid. Formic acid can assist in the ionization of peptides in the sample during mass spectrometry, improving the mass spectrometry signal intensity. Phenol red acts as an indicator, visually indicating whether the sample is still suitable for mass spectrometry detection. When the sample degrades and is no longer suitable for detection, the preservation solution changes from yellow to colorless. The preservation solution before and after color change is as follows: Figure 1 As shown.

[0061] Table 1 shows a comparison between the nasal swab preservation solutions of this invention and those reported in the literature for MALDI mass spectrometry. Preservative solution A is the preservation solution of this invention; preservation solution B is the preservation solution used in the article "Detection of SARS-CoV-2 in nasal swabs using MALDI-MS"; and preservation solution C is the preservation solution used in the article "Novel application of automated machine learning with MALDI-TOF-MS for rapid high-throughput screening of COVID-19: a proof of concept". Mass spectra obtained from nasal swab samples of the same subject treated with different preservation solutions are shown below. Figure 2 As shown, the sample exhibits low mass spectrometry noise, a large number of peaks, and stable peaks in the preservation solution of this invention.

[0062] Table 1. Comparison of Nasal Swab Preservation Solutions

[0063]

[0064] As shown in Table 1, the nasal swab preservation solution of the present invention has advantages such as rapid inactivation of viruses, facilitating the conversion of peptides into mass spectrometry signals, and stable preservation, which is of positive significance.

[0065] Example 2. Determination of Nasal Swab Preservation Solution Volume

[0066] Dispense the nasal swab preservation solution into sampling tubes according to the volumes listed in the table below, and add nasal swab samples to each tube. Mix well and then perform mass spectrometry analysis. Compare the effects of different volumes of preservation solution on the spectra.

[0067] Table 2. List of reagent quantities for nasal swab preservation solution

[0068] experimental group Reagent volume (ml) 1 0.3 2 0.6 3 1.0 4 2.0 5 3.0

[0069] Mass spectra of each group are as follows Figure 3a As shown, all spectra meet the requirements of high peak count, low noise, and high resolution. It can be considered that a qualified spectrum can be detected with a nasal swab preservation solution volume of 0.3–3.0 ml.

[0070] A magnified view of a portion of the spectrum ( Figures 3b-3d After careful comparison, it can be seen that... Figure 3b In the 3400–3600 m / z range, the peak shapes and intensities of the characteristic peaks from 0.6 ml to 3 ml are relatively consistent, while the intensity of the 0.3 ml peak is weaker. In the 3600–3800 m / z range, the peak shapes and intensities of the characteristic peaks from 1 ml to 3 ml are relatively consistent, while a characteristic peak is missing from 0.3 ml to 0.6 ml. In the 4000–4200 m / z range, the peak shapes and intensities of the characteristic peaks from 1 ml to 3 ml are relatively consistent, while a characteristic peak is missing from 0.3 ml to 0.6 ml. Figure 3c In the range of 5400 m / z, the peak shape and intensity of the characteristic peaks in the range of 1 ml to 3 ml are relatively consistent, while the peak shape in the range of 0.3 ml to 0.6 ml fluctuates more. In the range of 7600 m / z, the peak shape and intensity of the characteristic peaks in the range of 1 ml to 3 ml are relatively consistent, while the peak intensity in the range of 0.3 ml to 0.6 ml is higher. Figure 3d In the 8500–9000 m / z range, the peak shape and intensity of the characteristic peaks are relatively consistent between 1 ml and 3 ml, while a characteristic peak is missing between 0.3 ml and 0.6 ml. In the 11500–12000 m / z range, the peak shape and intensity of the characteristic peaks are relatively consistent between 1 ml and 3 ml, while the peak intensity is weaker between 0.3 ml and 0.6 ml. The variation in relative peak intensity is related to the sample concentration in the preservation solution. To ensure the stability of the characteristic peak positions and relative intensities as much as possible in practical applications, we prioritize the range with higher tolerance to changes in sample concentration. The spectra are basically consistent when the volume of the nasal swab preservation solution is 1.0–3.0 ml. This means that the spectrum is stable within this sample concentration range. Therefore, we prioritize a volume of 1.0–3.0 ml of preservation solution per nasal swab tube.

[0071] Example 3. Sample Collection and Virus Inactivation

[0072] The kit of this invention requires testing nasal swab samples. First, blow your nose with a tissue. Open the outer packaging of the nasal swab, tilt your head slightly back, hold the swab near the nasal septum, and gently push the swab into your nose, 1-1.5 cm behind the bottom of the inferior nasal meatus. Rotate the swab at least 4 times and hold it for at least 15 seconds. Remove the swab from the nostril, immerse the swab tip in the nasal swab preservation solution, and break the swab. Replace the cap of the nasal swab preservation solution tube and gently shake to dissolve the sample in the solution. The ethanol in the nasal swab preservation solution automatically inactivates the virus.

[0073] Samples should be tested immediately after collection. If testing cannot be completed within 72 hours, the samples should be frozen at -80°C to avoid repeated freeze-thaw cycles.

[0074] Example 4. Detection of virus inactivation effect of nasal swab preservation solution

[0075] The virus inactivation effect was tested according to the "Disinfection Technical Specifications" (2002 edition). The experimental steps are as follows:

[0076] a) Remove the frozen host cells (MDCK cells) from liquid nitrogen, thaw them rapidly in 37°C water, wash them twice with cell maintenance medium, and then transfer them to culture flasks containing 10 ml of complete culture medium. Observe cell growth daily, and use the cells in experiments when they have formed a complete monolayer.

[0077] b) Remove the cryopreserved strain (Influenza A virus A / PR / 8 / 34 / H1N1), thaw it in a 37°C water bath, dilute it 10-fold with cell maintenance medium, and then inoculate it into cell culture flasks containing a confluent monolayer of cells. Place the flasks at 37°C to allow the virus to adhere to and grow with the cells. Observe the lesions daily, and harvest the virus when 3 / 4 of the cells show lesions.

[0078] c) Take an appropriate amount of the nasal swab preservation solution to be tested, dilute it with sterile hard water to the required concentration of 1.25 times, and keep it in a water bath at 20℃±1℃ for later use;

[0079] d) Mix 100 μl of organic interfering substance with 100 μl of virus stock solution, incubate in a water bath at 20℃±1℃ for 5 min, add 0.8 ml of the test solution, mix immediately and record the time. After 10 s of incubation, immediately remove 0.1 ml and treat with a qualified removal method.

[0080] e) Perform 10-fold serial dilutions of the sample to be tested using cell maintenance culture medium, and titrate the residual virus amount in each dilution sample on a 96-well culture plate. Perform 4 wells for each dilution, incubate at 37°C for 1-2 hours, remove the culture plate, and replace the cell maintenance culture medium.

[0081] f) Continue to incubate in a carbon dioxide incubator (37℃, 5% CO2), observe cell pathogenesis under a microscope daily for 3 consecutive days, and observe and record cell pathogenesis in each well.

[0082] g) In the positive control group test, sterile deionized water was used instead of the test sample mixture;

[0083] h) Virus titers were determined in each group using the endpoint dilution method;

[0084] i) The experiment was repeated 3 times.

[0085] The test results are shown in Table 3. The virus inactivation rate after 10 seconds of contact with the nasal swab preservation solution was greater than 99.99%. This indicates that the nasal swab preservation solution in this kit has excellent virus inactivation capabilities.

[0086] Table 3. Virus inactivation test results

[0087]

[0088] Example 5. Sample Preservation Stability Test

[0089] Large-scale screening often requires several hours of sample collection, followed by the centralized delivery of a batch of samples to a testing laboratory. This necessitates that the samples remain stable in the preservation solution. This invention collects nasal swab samples from individuals infected with the novel coronavirus, those infected with influenza, and healthy individuals, and places them in a nasal swab preservation solution. Mass spectrometry analysis is performed after preservation at room temperature for 0 hours, 48 ​​hours, and 72 hours. The mass spectra of the three preservation solutions at different preservation times are shown in Figure 4. Figure 4a Use the nasal swab preservation solution of the present invention; Figure 4b The preservation medium (Cary-Blair transport medium) used in the article "Detection of SARS-CoV-2 in nasal swabs using MALDI-MS" was employed. Figure 4c The preservation solution (physiological saline) used in the article "Novel application of automated machine learning with MALDI-TOF-MS for rapid high-throughput screening of COVID-19: a proof of concept" is employed. Figure 4a

[0090] The medium spectrum exhibits a large number of peaks, high characteristic peak resolution, and low baseline noise. Furthermore, the spectrum remains essentially unchanged after storage at room temperature for 48 hours and 72 hours. This means that the preservation solution of this invention is highly suitable for mass spectrometry detection. Figure 4b The mid-spectral peaks have good shape and number, but the noise level is high. Noisy peaks may interfere with the identification of meaningful characteristic peaks, affecting the stability of the method. Figure 4c The 0-hour chromatogram showed significant changes compared to the spectra after 48 hours and 72 hours of storage. This indicates that the sample is not stable in this preservation solution. In large-scale screening, it is often necessary to collect a batch of samples and transport them to the testing laboratory for analysis. The inability to stably preserve samples can cause operational problems. Furthermore, it cannot be guaranteed that the sample has not undergone uncontrollable changes by the time of testing, increasing the probability of testing errors.

[0091] Example 5 demonstrates that the present invention has a good effect on stabilizing peptides in samples, and can meet the requirements of practical applications.

[0092] Example 6. Detection of Novel Coronavirus Infection

[0093] The novel coronavirus infection testing process is as follows: Figure 5 As shown, the specific steps are as follows:

[0094] 1. Preparation of matrix solution: Completely transfer one vial of matrix solvent into the matrix powder tube. Vortex at 4000 rpm for 5 minutes to completely dissolve the solid, thus obtaining the matrix solution. If some solid remains undissolved, the vortexing time can be increased appropriately. The prepared matrix solution can be stored at 4°C for one month.

[0095] 2. Mass spectrometry calibrator testing:

[0096] a) Take one mass spectrometry calibration sample. Centrifuge briefly using a handheld centrifuge to remove the powder to the bottom of the tube.

[0097] b) Add 10 μl of deionized water to the calibration tube. Vortex at 1200 rpm for 30 s to obtain the mass spectrometry calibration solution.

[0098] c) Apply 1 μl of mass spectrometry calibrator solution to the corresponding target point on the target plate. Allow to air dry.

[0099] d) Pipette 1 μl of matrix solution and cover the calibrator spot. Allow to air dry naturally; the calibrator spotting is now complete.

[0100] e) Mass spectrometry calibrators should be tested at least once a day.

[0101] 2. Positive control sample testing:

[0102] a) Take one positive control sample. Centrifuge briefly using a handheld centrifuge to collect the powder until it reaches the bottom of the tube.

[0103] b) Pipette 10 μl of the nasal swab preservation solution into a positive control tube. Vortex at 1200 rpm for 30 s to obtain the positive control solution.

[0104] c) Apply 1 μl of the positive control solution to the corresponding target point on the target plate. Allow to air dry.

[0105] d) Take 1 μl of matrix solution and cover it on the quality control spot. Let it air dry naturally to complete the positive control spotting.

[0106] e) Positive control samples should be tested at least once a day.

[0107] 3. Negative quality control testing:

[0108] a) Take one negative control sample. Centrifuge briefly using a handheld centrifuge until the powder reaches the bottom of the tube.

[0109] b) Pipette 10 μl of the nasal swab preservation solution into a negative control tube. Vortex mix at 1200 rpm for 30 s. Obtain the negative control solution.

[0110] c) Apply 1 μl of the negative control solution to the corresponding target point on the target plate. Allow to air dry.

[0111] d) Take 1 μl of matrix solution and cover it on the quality control spot. Let it air dry naturally to complete the spotting of the negative quality control.

[0112] e) Negative control samples should be tested at least once a day.

[0113] 4. Sample testing:

[0114] a) Place the broken nasal swab sample into the nasal swab preservation solution and mix thoroughly.

[0115] b) Remove the swab and apply it to the corresponding target point on the target plate. Allow it to air dry.

[0116] c) Take 1 μl of matrix solution and cover it on the sample spot. Allow it to air dry before performing mass spectrometry detection.

[0117] 5. Mass spectrometry data acquisition:

[0118] All calibrators, control samples, and samples were analyzed using MALDI-TOF MS. Mass spectrometer model: Clin-ToF (Beijing Yixin Bochuang Biotechnology Co., Ltd.). A suitable laser energy was used to acquire data at a single point of the sample crystallization. Fifty laser bombardment positions were selected for each sample point, with 10 bombardments at each position, resulting in 500 laser bombardments for each sample crystallization point. The spectrum was collected. Laser frequency: 30 Hz. Data collection range: m / z 2000–20000. External standard calibration was performed using standards before each sample crystallization point acquisition, with an average molecular weight deviation of less than 500 ppm.

[0119] 6. Result Interpretation:

[0120] The mass spectrometry data was imported into the COVID-19 immune response mass spectrometry flash detection software (Beijing Biotechnology Co., Ltd.), and the software automatically scored each mass spectrometry spectrum and obtained qualitative detection results.

[0121] Table 4. Result Interpretation Criteria

[0122] Judgment results Score Positive 60～100 gray area 40～60 Negative 0～40

[0123] Example 7. Blind selection test of clinical samples infected with novel coronavirus

[0124] After preparing a kit using the reagent composition of this invention and existing mass spectrometry detection reagents, a blind selection test for novel coronavirus infection was performed on 31 clinical samples. All samples were nasal swab samples, which were immediately placed in nasal swab preservation solution after collection and mass spectrometry was performed within 48 hours. The accuracy of the detection results of this method (mass spectrometry) was calculated using the control nucleic acid detection results as the standard. The results of the blind selection test using mass spectrometry are shown in Tables 5-6.

[0125] The results are shown in Tables 5 and 6, and the confusion matrix, respectively. Figure 6-1 , Figure 6-2 As shown. The control nucleic acid test (qPCR method) is currently the gold standard for detecting novel coronavirus infection. In the confusion matrix, the vertical axis represents the actual grouping of samples, with the top row representing the number of negative samples and the bottom row representing the number of positive samples; the horizontal axis represents the model prediction results, with the left column representing the number of samples judged as negative by the model and the right column representing the number of samples judged as positive by the model.

[0126] Table 5. Comparison of blind selection tests for mass spectrometry using 20 characteristic peptide variables.

[0127]

[0128]

[0129] The comparison results show that: from Table 5 and Figure 6-1 The results for the test group samples show that: among the 9 cases of novel coronavirus infection, 9 were correctly identified, with a sensitivity of 100%; among the 22 normal individuals, 22 were correctly identified, with a specificity of 100%.

[0130] Table 6. Comparison of blind selection tests for mass spectrometry using five characteristic peptide variables.

[0131]

[0132] From Table 6 and Figure 6-2 The results for the test group samples show that: among 9 patients infected with the novel coronavirus, 8 were correctly identified, with a sensitivity of 88.9%; among 22 healthy individuals, 21 were correctly identified, with a specificity of 95.5%. This indicates that the model composed of the input variables of 5 characteristic peptides only produced a small number of misclassifications. This model already meets the needs of rapid clinical screening.

[0133] Furthermore, the results above show that the accuracy of the prediction results for the novel coronavirus infected group and the normal group using complete variables of 20 characteristic peptides in this invention reached 100%. This indicates that the diagnostic results are reliable and trustworthy, minimizing missed diagnoses and / or misdiagnoses. Therefore, it has positive significance.

[0134] For details, please refer to the inventor's prior invention application, "Product and Use of Nasal Swab for Detecting SARS-CoV-2 Based on Immune Response Characteristic Peptide Profile (Invention Application No.: 202211292701.8)".

[0135] Example 8. Detection of other respiratory pathogen infections

[0136] 1. Preparation of matrix solution: Completely transfer one vial of matrix solvent into the matrix powder tube. Vortex at 4000 rpm for 5 minutes to completely dissolve the solid, thus obtaining the matrix solution. If some solid remains undissolved, the vortexing time can be increased appropriately. The prepared matrix solution can be stored at 4℃ for one month.

[0137] 2. Mass spectrometry calibrator testing:

[0138] a) Take one mass spectrometry calibration sample. Centrifuge briefly using a handheld centrifuge to remove the powder to the bottom of the tube.

[0139] b) Add 10 μl of deionized water to the calibration tube. Vortex at 1200 rpm for 30 s to obtain the mass spectrometry calibration solution.

[0140] c) Apply 1 μl of mass spectrometry calibrator solution to the corresponding target point on the target plate. Allow to air dry.

[0141] d) Pipette 1 μl of matrix solution and cover the calibrator spot. Allow to air dry naturally; the calibrator spotting is now complete.

[0142] e) Mass spectrometry calibrators should be tested at least once a day.

[0143] 3. Positive control sample testing:

[0144] a) Take one positive control sample. Centrifuge briefly using a handheld centrifuge to collect the powder until it reaches the bottom of the tube.

[0145] b) Pipette 10 μl of the nasal swab preservation solution into a positive control tube. Vortex at 1200 rpm for 30 s to obtain the positive control solution.

[0146] c) Apply 1 μl of the positive control solution to the corresponding target point on the target plate. Allow to air dry.

[0147] d) Take 1 μl of matrix solution and cover it on the quality control spot. Let it air dry naturally to complete the positive control spotting.

[0148] e) Positive control samples should be tested at least once a day.

[0149] 4. Negative quality control testing:

[0150] a) Take one negative control sample. Centrifuge briefly using a handheld centrifuge until the powder reaches the bottom of the tube.

[0151] b) Pipette 10 μl of the nasal swab preservation solution into a negative control tube. Vortex mix at 1200 rpm for 30 s. Obtain the negative control solution.

[0152] c) Apply 1 μl of the negative control solution to the corresponding target point on the target plate. Allow to air dry.

[0153] d) Take 1 μl of matrix solution and cover it on the quality control spot. Let it air dry naturally to complete the spotting of the negative quality control.

[0154] e) Negative control samples should be tested at least once a day.

[0155] 5. Sample testing:

[0156] a) Place the broken nasal swab sample into the nasal swab preservation solution and mix thoroughly.

[0157] b) Remove the swab and apply it to the corresponding target point on the target plate. Allow it to air dry.

[0158] c) Take 1 μl of matrix solution and cover it on the sample spot. Allow it to air dry before performing mass spectrometry detection.

[0159] 6. Mass spectrometry data acquisition:

[0160] All calibrators, control samples, and samples were analyzed using MALDI-TOF MS. Mass spectrometer model: Clin-ToF (Beijing Yixin Bochuang Biotechnology Co., Ltd.). A suitable laser energy was used to acquire data at a single point of the sample crystallization. Fifty laser bombardment positions were selected for each sample point, with 10 bombardments at each position, resulting in 500 laser bombardments for each sample crystallization point. The spectrum was collected. Laser frequency: 30 Hz. Data collection range: m / z 2000–20000. External standard calibration was performed using standards before each sample crystallization point acquisition, with an average molecular weight deviation of less than 500 ppm.

[0161] 7. Mass spectrometry spectrum

[0162] Influenza and other respiratory pathogens cause symptoms similar to those of the novel coronavirus, potentially interfering with COVID-19 testing. The mass spectrometry spectra of novel coronavirus infected individuals, influenza infected individuals, and healthy subjects are shown in Figure 6. Compared to novel coronavirus infected individuals, the characteristic peaks at 2614 m / z, 3354 m / z, 3405 m / z, 2688 m / z, 5444 m / z, 4839 m / z, 4710 m / z, 2234 m / z, 2189 m / z, 3440 m / z, 2208 m / z, 2277 m / z, 4980 m / z, 2924 m / z, and 2488 m / z in influenza infected individuals showed statistically different results from those in novel coronavirus infection. The characteristic peaks of influenza samples at 2189 m / z, 2208 m / z, 2234 m / z, 2277 m / z, 2614 m / z, 2688 m / z, 4710 m / z, 4839 m / z, and 5444 m / z showed an upward trend, while the characteristic peaks at 2488 m / z, 2924 m / z, 3354 m / z, 3405 m / z, 3440 m / z, and 4980 m / z showed a downward trend. ROC curve validation showed that the AUC of the ROC curve for novel coronavirus infection and influenza infection was 1. The model's accuracy in distinguishing between novel coronavirus and influenza was 100%.

[0163] 8. Result Interpretation:

[0164] The mass spectrometry data is imported into the respiratory disease immune response mass spectrometry flash detection software (Beijing Biotechnology Co., Ltd.), and the software automatically reports the qualitative detection results of the sample.

[0165] Table 6. Test Results

[0166] Project Name Test results novel coronavirus Positive / Negative influenza Positive / Negative

[0167] 9. Results of blinded testing of influenza patients

[0168] The results of the blinded selection test of clinical samples from influenza patients are shown in Table 7. Thirty influenza samples were used to validate the model; after interpretation by the detection software, all 30 samples were detected as influenza samples. The detection accuracy was 100%.

[0169] Table 7. Results of blind selection test of influenza patient samples

[0170]

[0171] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a pretreatment reagent composition in the preparation of a mass spectrometry detection kit for detecting respiratory pathogen infections, characterized in that, The reagent composition includes a nasal swab preservation solution and a nasal swab, wherein the nasal swab preservation solution comprises a 70%–80% (V / V) ethanol solution and a 10–200 mg / L phenol red solution, wherein the respiratory pathogen is a novel coronavirus, the mass spectrometry is selected from MALDI TOF MS, and the nasal swab sample to be tested contains characteristic polypeptides produced by respiratory pathogen infection.

2. The use according to claim 1, wherein the nasal swab preservation solution further contains 0.2% to 5.0% (V / V) formic acid solution, wherein formic acid can assist the ionization of peptides in the sample in mass spectrometry and improve the mass spectrometry signal intensity.

3. The use according to claim 2, wherein the volume of the nasal swab preservation solution is 0.2 to 4.0 ml / tube.

4. The use according to claim 3, wherein the kit further comprises matrix powder, matrix solvent, mass spectrometry calibrator, positive control and negative control.

5. The use according to claim 3, wherein the kit includes a calibrator tube to ensure the accuracy of molecular weight measurements by the mass spectrometer, the calibrator including adrenocorticotropic hormone, cytochrome C, and myoglobin.

6. The use according to claim 5, wherein the kit further comprises a quality control for determining whether the test results are reliable, the quality control being prepared from a mixture of protein peptides, wherein the positive quality control has a characteristic spectrum similar to that of a person infected with a respiratory pathogen; and the negative quality control has a characteristic spectrum similar to that of a healthy person, and the quality control and the test sample are subjected to parallel mass spectrometry testing to determine whether the test results are accurate and reliable.

7. The use according to any one of claims 1-6, wherein the reagent composition is combined with existing mass spectrometry detection reagents to prepare the mass spectrometry detection kit.

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