CN116298303A

CN116298303A - Use of pretreatment reagent composition for preparing mass spectrum detection kit for detecting respiratory tract pathogens

Info

Publication number: CN116298303A
Application number: CN202211416245.3A
Authority: CN
Inventors: 马庆伟; 吕倩; 许彬
Original assignee: Beijing Clin Bochuang Biotechnology Co Ltd
Current assignee: Beijing Clin Bochuang Biotechnology Co Ltd
Priority date: 2022-11-12
Filing date: 2022-11-12
Publication date: 2023-06-23

Abstract

The invention provides application of a pretreatment reagent composition to preparation of a mass spectrometry detection kit for detecting respiratory pathogens, wherein the pretreatment reagent composition comprises all reagents for mass spectrometry detection, such as a nasal swab preservation solution, a mass spectrometry matrix, a mass spectrometry calibrator, a quality control product and the like. The detection object of the invention is a nose swab sample, the sampling is convenient, the interference is small, meanwhile, the pretreatment reagent composition which can quickly inactivate and keep the sample for a long time can be used for detecting various respiratory pathogens by matching the reagent composition with the existing detection reagent or kit, and the detection of various respiratory pathogens can be quickly, accurately and high-throughput mass spectrometry.

Description

Use of pretreatment reagent composition for preparing mass spectrum detection kit for detecting respiratory tract pathogens

Technical Field

The invention belongs to the field of protein mass spectrum detection. Relates to a product for rapidly detecting novel coronaviruses and other respiratory pathogen infections by utilizing a time-of-flight mass spectrometry technology.

Background

In recent years, mass spectrometry technology has been developed for detecting characteristic polypeptides of patients infected with novel coronaviruses, that is, specific characteristic polypeptides appear in vivo after patients are infected with novel coronaviruses, and by detecting these characteristic polypeptides, the infection results can be identified more accurately. For example, detection of SARS-CoV-2in nasal swabs using MALDI-MS reports studies based on MALDI-MS direct Detection of nasal swab samples. The novel coronavirus nucleic acid detection residual sample is used for analysis, a mass spectrum detection model is established based on a machine learning method, and the highest accuracy is 93.9%. However, the sample preservation reagent is not improved in the study, so that part of components in the preservation solution seriously affect the mass spectrogram quality.

Novel application of automated machine learning with MALDI-TOF-MS for rapid high-throughput screening of COVID-19:a proof of concept propose a novel coronavirus entry detection scheme developed in combination with machine learning and MALDI-TOF techniques. The detection performance of the scheme is equivalent to that of the existing commercial test, and the detection time is shorter. The daily measurement of 1104 times per instrument can be completed. The nasal swab samples in this study were stored in physiological saline and virus inactivation was achieved by uv irradiation of the mass spectrometry target plate. Although simple preservation solution components greatly reduce interference on mass spectrum signals, the problem that sample polypeptides are easy to degrade in preservation still exists. And the safety of the detection process still cannot be ensured by simple ultraviolet disinfection and delayed disinfection steps.

Chinese patent application 202010251209.0, a novel coronavirus N protein extraction preservation solution, and a preparation method and application thereof, disclose a novel coronavirus N protein extraction preservation solution, comprising: casein, antibiotics, bovine serum albumin, ethylphenyl polyethylene glycol, anti-erythrocyte monoclonal antibodies, and the like. The novel coronavirus extraction preservation solution provided by the invention has good compatibility to different samples such as nasal swab, pharyngeal swab, oral swab, alveolar lavage fluid, sputum, saliva and the like, provides a proper and stable buffer environment for a virus sample, and is beneficial to sample detection and preservation. However, this invention is only used for immunodetection purposes, and the preservation solution is complex in composition and is not suitable for sample processing for mass and rapid detection of explosive novel coronaviruses.

Chinese patent application 202010587255.8, a preservation solution for improving nucleic acid stability and application thereof disclose a preservation solution for improving nucleic acid stability and application thereof, wherein the preservation solution comprises one or more of Tris (hydroxymethyl) aminomethane), nonionic surfactant, EDTA (ethylene diamine tetraacetic acid), inorganic salt and sorbitol. However, this preservation solution is used for preserving sample nucleic acids and is not suitable for preserving nucleic acids containing characteristic polypeptides that occur after infection by viruses.

As the closest prior art, chinese patent application 201610150642.9, a sample pretreatment liquid for detecting and separating respiratory viruses of a sample, discloses a sample pretreatment liquid for detecting and separating respiratory viruses of a sample, comprising: (i) A viral protective agent selected from the group consisting of: calcium chloride, anhydrous magnesium sulfate, potassium chloride, sodium chloride, anhydrous sodium dihydrogen phosphate, anhydrous dextrose, or a combination thereof; (ii) a color developer; (iii) a sterilant. Wherein the color-developer is selected from the group consisting of: phenol red, neutral red, or a combination thereof. Although the invention introduces a color reagent to indicate the preservation status of the sample, the invention is still concerned with preservation of viruses rather than preserving characteristic polypeptides produced after infection by viruses, and thus the pretreatment liquid is still unsuitable for preserving polypeptides containing characteristics that occur after infection by viruses. In addition, since the pretreatment liquid contains salt compounds such as calcium chloride, magnesium sulfate, and potassium chloride, impurities are generated during ionization by laser mass spectrometry, which affect mass spectrum signals, the pretreatment liquid is not suitable as a pretreatment solution component and a matrix component for protein mass spectrometry.

In summary, existing mass spectrometry kits for detecting respiratory pathogens including novel coronaviruses suffer from the disadvantage that it is difficult to pre-treat the sample, such as the inability to rapidly inactivate viruses, or the susceptibility of inactivated viruses to degradation or the occurrence of interfering mass spectrometry signals, which all result in the detection of characteristic or immune polypeptides present in the patient.

Thus, there is a need for a mass spectrometry detection kit for pretreatment of novel respiratory pathogens such as coronaviruses.

Disclosure of Invention

The first principle of the invention is that: in the prior art, although it is known that ethanol inactivates and cleaves viruses, it also denatures and inactivates proteins or polypeptides, so that ethanol is generally used only as a virus inactivating and cleaving agent and not as a preserving or pretreatment solution for polypeptides or proteins. However, the inventors have unexpectedly found that in characteristic polypeptide mass spectra, even denatured polypeptide fragments, the molecular weight of the specific polypeptide fragments produced by electric field-induced cleavage does not change, and thus do not affect the acquisition of indicative characteristic spectra.

The second principle of the invention is that: the inventors propose for the first time that after infection by virus, characteristic polypeptides (which may be antibody fragments or non-antibody characteristic polypeptides generated after immune reaction, i.e. immune reaction polypeptide products or immune polypeptides) produced by human body can be detected by the pretreatment agent of the present invention, thereby completing the final identification.

The third principle of the invention is that: the added phenol red is verified, so that the stability of the mass spectrum corresponding and polypeptide is not affected, and the stability of the pretreatment liquid can be displayed as an indicator.

The fourth principle of the invention is that: in the pretreatment liquid, the added formic acid can assist the ionization of the polypeptide in the sample in the mass spectrum, so that the signal intensity of the mass spectrum is improved.

It is therefore a first object of the present invention to provide a pretreatment reagent composition for laser mass spectrometry detection of respiratory pathogens, comprising a nasal swab preservation solution, a nasal swab, wherein the nasal swab preservation solution comprises 70% -80% ethanol (V/V) and phenol red (10-200 mg/L), wherein the respiratory pathogens comprise novel coronaviruses, influenza a viruses, influenza b viruses, SARS viruses, MERS viruses, etc.

In one embodiment, the ethanol has the effect of inactivating respiratory pathogens such as viruses, and when the nasal swab sample is collected, the nasal swab sample is immediately placed into a nasal swab preservation solution and uniformly mixed, and the preservation solution can inactivate the viruses while preserving the viruses, so that the detection time is shortened.

In another embodiment, the phenol red is used as an indicator to visually indicate the state of the nasal swab sample in the sample preservation liquid, and when the color of the phenol red is obviously changed, the pH value of a solution system is changed, so that the chemical reaction of acid-base conversion of the protein polypeptide component in the nasal swab is indicated, and the sample component is not in an initial state and can not be used for mass spectrum detection.

In other embodiments, the nasal swab preservation solution contains ethanol, phenol red and formic acid (0.2% -5.0%, V/V) at the same time, wherein formic acid can assist in ionization of polypeptides in the sample in mass spectrometry, and improve mass spectrometry signal intensity.

In any of the above embodiments, the nasal swab holding solution is contained in an amount of 0.2 to 4.0 ml/tube, preferably 1.0 to 3.0 ml/tube.

In any of the above embodiments, the laser mass spectrometry is selected from MALDI TOF MS or Clin-TOF.

The second object of the present invention is to provide a mass spectrometry detection kit comprising the above reagent composition and used for detecting respiratory pathogens, which further comprises matrix powder, matrix solvent, mass spectrometry calibrator, positive quality control and negative quality control, wherein the respiratory pathogens comprise novel coronavirus, influenza a virus, influenza b virus, SARS virus, MERS virus, etc.

In one embodiment, the kit includes a calibrator tube that ensures that the molecular weight measured by the mass spectrometer is accurate. In a specific embodiment, 3 to 10 characteristic polypeptide peaks can be obtained by mass spectrometry detection of the calibrator, and the molecular weight of each polypeptide peak is relatively uniformly distributed in the molecular weight range to be detected. The sample in the calibrator tube is used for calibrating a mass axis of the mass spectrometer, and whether the molecular weight information of the sample to be measured is accurate and reliable is judged by carrying out parallel mass spectrometry test with the sample to be measured.

In a preferred embodiment, the calibrator comprises corticotropin, cytochrome C and myoglobin, and the characteristic polypeptide peak has an m/z of 2465m/z, 4121m/z, 6181m/z, 8477m/z, 12362m/z, 16952m/z, respectively.

In another embodiment, the kit further comprises a quality control for determining whether the test result is reliable. The quality control product is prepared from mixed protein polypeptide, and the positive quality control product has a characteristic spectrum similar to that of a respiratory pathogen infected person; the negative quality control has a similar profile to healthy people. And carrying out parallel mass spectrometry test when the quality control product and the sample to be tested are subjected to mass spectrometry so as to judge whether the result to be tested is accurate and reliable.

It is a third object of the present invention to provide the use of the reagent composition in the preparation of a mass spectrometry detection kit for detecting respiratory pathogens, wherein the respiratory pathogens comprise novel coronaviruses, influenza a viruses, influenza b viruses, SARS viruses, MERS viruses, etc.

In one embodiment, the kit comprises a nasal swab preservation solution, a nasal swab, wherein the nasal swab preservation solution comprises 70% -80% ethanol (V/V) and phenol red (10-200 mg/L).

In any of the above embodiments, the kit further comprises a matrix powder, a matrix solvent, a mass spectrometry calibrator, a positive quality control, and a negative quality control,

in any of the above embodiments, the kit comprises a calibrator tube that ensures that the molecular weight measured by the mass spectrometer is accurate. In a specific embodiment, 3 to 10 characteristic polypeptide peaks can be obtained by mass spectrometry detection of the calibrator, and the molecular weight of each polypeptide peak is relatively uniformly distributed in the molecular weight range to be detected. The sample in the calibrator tube is used for calibrating a mass axis of the mass spectrometer, and whether the molecular weight information of the sample to be measured is accurate and reliable is judged by carrying out parallel mass spectrometry test with the sample to be measured.

In any of the above embodiments, the calibrator comprises corticotropin, cytochrome C and myoglobin, and the characteristic polypeptide peaks have m/z of 2465m/z, 4121m/z, 6181m/z, 8477m/z, 12362m/z, 16952m/z, respectively.

In any of the above embodiments, the kit further comprises a quality control for determining whether the detection result is reliable. The quality control product is prepared from mixed protein polypeptide, and the positive quality control product has a characteristic spectrum similar to that of a respiratory pathogen infected person; the negative quality control has a similar profile to healthy people. And carrying out parallel mass spectrometry test when the quality control product and the sample to be tested are subjected to mass spectrometry so as to judge whether the result to be tested is accurate and reliable.

In any of the above embodiments, the reagent composition is combined with existing mass spectrometry detection reagents, and the mass spectrometry detection kit can be prepared.

Technical effects

Compared with the prior art, the invention has the following advantages:

1. the reagent composition or the detection kit can effectively inactivate viruses possibly existing in the nasal swab sample within 10 seconds, simultaneously convert the polypeptides in the sample into mass spectrum peak signals, and diagnose diseases such as novel coronaviruses, influenza and the like by detecting the change of a group of characteristic polypeptide mass spectrum.

2. The invention has simple sample pretreatment, low requirement on detection environment and low detection cost, and can realize one-person one-test without mixing samples. Compared with the existing method, the novel coronavirus immunoreaction nasal swab polypeptide characteristic spectrum detection method based on mass spectrum is more suitable for rapid screening of novel coronavirus infected persons in large population and rapid detection of infection of other respiratory pathogens such as influenza. The reason why the existing nucleic acid detection adopts a mixed detection mode is that the detection method is complex, the detection time is long and the detection flux is low. If 1 person 1 checks, the large-scale screening requirement cannot be met in efficiency. Mixed detection is a non-trivial choice in exchange for detection efficiency at the expense of sensitivity. The main advantages of mass spectrometry are fast speed, simple detection and high throughput. The 1 person 1 detection accuracy is high, and is more beneficial to tracing the source and tracking the course change of the patient. Thus, while the present invention also supports mixed testing, there are more cases where mixed testing is not required to test patients.

3. Unlike traditional methods which are directed only against the virus itself, the present invention detects multiple combinations of characteristic proteins that differ from normal human and are directed against different respiratory pathogen infectors, while simultaneously focusing on the changes in multiple immune characteristic polypeptides.

4. Compared with nucleic acid detection, the method provided by the invention has the advantages of no need of extra virus inactivation step, simplicity in operation, low detection cost, high flux and the like, and the whole detection time can be shortened to be within 1 min/sample. Not only is beneficial to the rapid screening of large-scale crowd, but also has a very high application prospect in the scenes such as emergency treatment and the like which need to rapidly obtain the detection result.

5. The accuracy of the kit for detecting the novel coronavirus infected person reaches 100%, the sensitivity is 100%, and the specificity is 100%. The result shows that the invention can meet the actual detection requirement.

6. The invention can be combined with mass spectrum detection reagents (such as detection matrix, virus characteristic polypeptide markers and the like) for detecting respiratory pathogens such as novel coronaviruses and the like in the market to prepare a related mass spectrum detection kit. Such existing mass spectrometric detection reagents include, but are not limited to, 202110155492.1, 2021101552589, 2021101589526, etc. of the prior application of the present invention. And, products and uses of nasal swabs for detection of novel coronaviruses based on immune response signature polypeptide profiles (invention application number 202211292701.8).

Drawings

Fig. 1: the preservation solution changes color. The solution in the left preservation tube (tube A) is yellow, which represents that the sample can be normally used for mass spectrometry detection; the solution in the right holding tube (tube B) was colorless, indicating that the sample had degraded and was no longer suitable for mass spectrometry.

Fig. 2: a mass spectrum obtained from nasal swab samples of the same subject treated with different preservation solutions, wherein:

preservation solution A: the nasal swab preservation solution of the invention is used;

preservation solution B: preservation fluid (Cary-Blair's transport Medium) from Detection of SARS-CoV-2in nasal swabs using MALDI-MS

Preservation solution C: from the preservation 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.

Fig. 3: effects of nasal swab preservative fluid loading (0.3 ml, 0.6ml, 1.0ml, 2.0ml and 3.0 ml) on spectra. Wherein:

FIG. 3a is a full view of a mass spectrum of a sample after being treated by nasal swab preservation solutions with different volumes, wherein the molecular weight ranges from 2000m/z to 20000m/z;

FIGS. 3 b-3 d are partial enlarged views of FIG. 3a, with partial spectral peaks (in-box spectral peaks) at the 0.3ml group and 0.6ml group spectral instability, respectively.

Fig. 4: mass spectrograms of 3 kinds of preservation solutions with different preservation times. Wherein:

FIG. 4a shows a nasal swab preservation solution using the present invention;

FIG. 4b uses the preservation fluid used in Detection of SARS-CoV-2in nasal swabs using MALDI-MS (Cary-Blair's transport medium);

FIG. 4c shows the use of a preservation solution (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.

Fig. 5: sample detection flow.

Fig. 6: the validation set confusion matrix. Wherein FIG. 6-1 is a validation set confusion matrix based on 20 characteristic peaks;

fig. 6-2 is a confusion matrix based on 5 characteristic peaks.

Fig. 7: mass spectrograms of different samples obtained by detection of the kit. The method comprises the following steps of: mass spectrogram of new coronavirus infected person; mass spectrogram of influenza infected person; mass spectrogram of healthy people.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

EXAMPLE 1 nasal swab preservative fluid composition determination

The invention relates to a nasal swab preservation solution specially 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 the ethanol is 70-80% (V/V), and the ethanol has the effect of inactivating viruses. The nasal swab sample is immediately placed into the nasal swab preservation solution after collection and is uniformly mixed. The preservation solution can complete the inactivation of the virus while preserving the virus, and shortens the detection time. The nasal swab preservation solution contains formic acid. The formic acid can assist the ionization of the polypeptide in the sample in the mass spectrum, and improves the signal intensity of the mass spectrum. Phenol red as an indicator can visually indicate whether the sample is suitable for continued use in 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 discoloration is shown in FIG. 1.

The comparison of the present invention with the nasal swab preservation solution protocol reported in the literature for use in MALDI mass spectrometry is shown in table 1. Wherein the preserving fluid A is the preserving fluid of the invention, and the preserving fluid B is the preserving fluid used in the article Detection of SARS-CoV-2in nasal swabs using MALDI-MS; preservation solution C was the preservation solution used in article Novel application of automated machine learning with MALDI-TOF-MS for rapid high-throughput screening of COVID-19:a proof of concept. A mass spectrum obtained by treating nasal swab samples of the same subject with different preservation solutions is shown in fig. 2. The sample in the preservation solution has low mass spectrum noise, a large number of spectrum peaks and stable spectrum peaks.

TABLE 1 comparison of nasal swab preservative fluid

As shown in Table 1, the nasal swab preservation solution has the advantages of being capable of rapidly inactivating viruses, facilitating transformation of polypeptides into mass spectrum signals, being stable in preservation and the like, and has positive significance.

EXAMPLE 2 determination of nasal swab preservation fluid

The nasal swab preservation solution was dispensed into sampling tubes and nasal swab samples were added separately according to the following table volumes. And (5) mixing uniformly and detecting by mass spectrum. The effect of different volumes of preservation solution on the spectra were compared.

TABLE 2 nasal swab preservative fluid reagent dose list

Test group	Amount of reagent (ml)
		1	0.3
2	0.6
		3	1.0
4	2.0
		5	3.0

The mass spectrograms of each group are shown in figure 3a, and all spectrograms can meet the requirements of large number of peaks, low noise and high resolution. The volume of the nasal swab preservation solution is 0.3-3.0 ml, and a qualified spectrogram can be detected.

As can be seen from detailed comparison of the partial enlarged images (fig. 3 b-3 d) of the spectrograms, in fig. 3b, 3400-3600 m/z parts, 0.6 ml-3 ml characteristic peak shapes and peak intensities are relatively consistent, and 0.3ml peak intensities are weaker; the peak shape and the peak intensity of the characteristic peaks are relatively consistent between 1ml and 3ml in 3600m/z part and the characteristic peak is missing between 0.3ml and 0.6 ml; the 4000-4200 m/z part, the peak shape and the peak intensity of the characteristic peak are relatively consistent between 1ml and 3ml, and a characteristic peak is missing between 0.3ml and 0.6 ml. In FIG. 3c, around 5400m/z, the peak shape and peak intensity are relatively uniform between 1ml and 3ml, while the peak shape fluctuation is larger between 0.3ml and 0.6 ml; about 7600m/z, the characteristic peak-to-peak shape and peak intensity are relatively uniform between 1ml and 3ml, and the peak intensity is higher between 0.3ml and 0.6 ml. In FIG. 3d, in the 8500-9000 m/z section, the peak shape and peak intensity of the characteristic peaks are relatively uniform from 1ml to 3ml, and a characteristic peak is absent from 0.3ml to 0.6 ml; 11500-12000 m/z, 1-3 ml characteristic peak shape and peak intensity are relatively consistent, and 0.3-0.6 ml peak intensity is weaker. The change in relative peak intensity is related to the concentration of the sample in the preservation solution. In order to ensure the stability of the characteristic peak position and the relative intensity in practical application, a part with stronger tolerance to the change of the sample concentration is preferably selected. The spectrogram is basically consistent when the volume of the nasal swab preservation solution is 1.0-3.0 ml. This means that the spectrum of the sample concentration is stable in this interval. Therefore, 1.0-3.0 ml of liquid is preferably contained in each nasal swab storage tube.

Example 3 sample collection and Virus inactivation

The kit provided by the invention is used for detecting a nose swab sample. The nasal discharge is blown by toilet paper, the outer package of the nasal swab is opened, the head is turned slightly, the swab is kept near the nasal septum, meanwhile, the swab is gently pushed into the nose and is rotated for at least 4 circles and the residence time is kept for more than 15 seconds along the bottom of the lower nasal passage backwards by 1-1.5 cm. The swab is removed from the nostril, the swab head is immersed in the nasal swab preservation solution and the nasal swab is broken. The nasal swab is capped with a preserving fluid tube cap and gently shaken to dissolve the sample in the preserving fluid. The ethanol in the nasal swab preservation solution automatically completes virus inactivation.

The sample should be detected immediately after collection. If the detection can not be completed within 72 hours, the sample needs to be frozen in a refrigerator at the temperature of minus 80 ℃ so as to avoid repeated freezing and thawing.

EXAMPLE 4 detection of the Virus inactivation Effect of nasal swab preservative fluid

The virus inactivation effect was tested according to the disinfection technical Specification (2002 edition). The experimental procedure was as follows:

a) Frozen test host cells (MDCK cells) were removed from liquid nitrogen, thawed rapidly in warm water at 37℃and, after washing twice with cell maintenance liquid, transferred to a flask containing 10ml of complete medium. Observing the growth condition of cells day by day, and testing when the cells are full of a monolayer;

b) Taking out the frozen strain (influenza A virus A/PR/8/34/H1N 1), melting in water bath at 37 ℃, diluting 10 times with cell maintenance solution, inoculating into a cell bottle containing cells which are already full of monolayer, and placing into a 37 ℃ incubator to adsorb and grow the cells. Observing pathological changes day by day, and harvesting viruses when 3/4 cells have pathological changes;

c) Taking a proper amount of nasal swab preservation solution to be tested, diluting the nasal swab preservation solution to 1.25 times of the required concentration by using sterilized hard water, and placing the nasal swab preservation solution in a water bath at 20+/-1 ℃ for standby;

d) Mixing 100 μl of organic interfering substance with 100 μl of virus stock solution, allowing to act in water bath at 20deg.C+ -1deg.C for 5min, adding 0.8ml of the mixture to be detected, immediately mixing, and recording. Taking out 0.1ml immediately after the action for 10s, and treating by a qualified chemical removal method;

e) Serial dilution of the sample to be tested is carried out by 10 times by using a cell maintenance culture solution, the residual virus amount in each dilution sample is titrated on a 96-hole culture plate, each dilution is 4 holes, the culture plate is taken out after being placed at 37 ℃ for 1-2 hours, and the cell maintenance culture solution is replaced;

f) Continuously placing the cells in a carbon dioxide incubator (37 ℃ and 5% CO 2) for culturing, observing cytopathy under a microscope day by day, continuously observing for 3 days, observing hole by hole and recording cytopathy conditions;

g) In the positive control group test, sterile deionized water is used for replacing the test sample mixed solution;

h) Each group of virus titer is measured by adopting an end point dilution method;

i) The test was repeated 3 times.

The test results are shown in Table 3, and the virus inactivation rate of 10s of the virus-contact nasal swab preservation solution is more than 99.99%. The nasal swab preservation solution of the kit has excellent virus inactivating capability.

TABLE 3 Virus inactivation test results

Example 5 sample preservation stability test

In large-scale screening, it is often necessary to collect samples for several hours and then to send a collection of samples collectively to a testing laboratory for testing. This requires that the sample remain stable in the sample-holding fluid. The invention respectively collects nose swab samples of novel coronavirus infected persons, influenza infected persons and healthy persons, and puts the nose swab samples into a nose swab preservation solution. And (5) performing mass spectrometry detection after preserving for 0h, 48h and 72h at room temperature. The mass spectrum of 3 kinds of preservation solutions with different preservation times is shown in fig. 4. Wherein FIG. 4a uses the nasal swab preservation solution of the present invention; FIG. 4b uses the preservative fluid (Cary-Blair's shipping medium) used in article Detection of SARS-CoV-2in nasal swabs using MALDI-MS; FIG. 4c uses the preservation solution (physiological saline) used in article Novel application of automated machine learning with MALDI-TOF-MS for rapid high-throughput screening of COVID-19:a proof of concept. FIG. 4a

The middle spectrogram has more peaks, high characteristic peak resolution and low baseline noise, and the spectrogram is basically unchanged after being stored for 48 hours and 72 hours at room temperature. This means that the preservation solution of the present invention is very suitable for mass spectrometry detection. The spectrum in fig. 4b has good peak shape and number of peaks, but high noise. Noise peaks may interfere with the identification of meaningful feature peaks, affecting the stability of the process. The spectrum at 0h in FIG. 4c is greatly altered from the spectrum after 48h and 72h of storage. This means that the sample does not exist stably in this preservation solution. In large-scale screening, it is often necessary to collect a batch of samples and then to transport the samples to a testing laboratory for testing. The failure to stably preserve the sample can cause trouble to the actual operation. And moreover, whether the sample has uncontrollable changes during detection cannot be guaranteed, so that the risk probability of detection errors is increased.

Example 5 demonstrates that the invention has good effect of stabilizing the polypeptide in the sample, and can meet the practical application requirements.

EXAMPLE 6 novel coronavirus infection detection

The novel coronavirus infection detection flow is shown in fig. 5, and the specific steps are as follows:

1. preparing a matrix solution: 1 branch of matrix solvent was completely transferred into the matrix powder tube. Vortex mixing at 4000rpm for 5min to dissolve the solid completely to obtain matrix solution. If some of the solids are undissolved, the swirling time can be increased appropriately. The prepared matrix solution can be stored at 4deg.C for 1 month.

2. Mass spectrometry calibrator detection:

a) Taking 1 mass spectrum calibrator. The palm centrifuge is used for instantaneous centrifugation to centrifuge the powder to the bottom of the tube.

b) Mu.l of deionized water was pipetted into the calibrator tube. Vortex mixing at 1200rpm for 30s. Obtaining the mass spectrum calibrator solution.

c) 1 μl of mass spectrometry calibrator solution was spotted on the corresponding target spot of the target plate. Naturally airing.

d) Mu.l of the matrix solution was pipetted onto the calibrator spots. Naturally airing to finish the sample application of the calibrator.

e) Mass spectrometry calibrants were tested at least 1 time per day.

2. Detecting positive quality control:

a) Taking 1 positive quality control product. The palm centrifuge is used for instantaneous centrifugation to centrifuge the powder to the bottom of the tube.

b) 10 μl of preserving solution of the nasal swab is sucked up and added to the positive quality control tube. Vortex mixing at 1200rpm for 30s. To obtain the cationic quality control solution.

c) 1 μl of positive quality control solution is pipetted onto the corresponding target of the target plate. Naturally airing.

d) Mu.l of the matrix solution was aspirated and covered on the quality control spots. Naturally airing to finish the sample application of the cationic quality control product.

e) The positive quality control is tested at least 1 time per day.

3. And (3) negative quality control detection:

a) Taking 1 negative quality control product. The palm centrifuge is used for instantaneous centrifugation to centrifuge the powder to the bottom of the tube.

b) 10 μl of preserving solution of the nasal swab is sucked up and added to the negative quality control tube. Vortex mixing at 1200rpm for 30s. To obtain the negative quality control product solution.

c) 1 μl of the negative quality control solution was pipetted onto the corresponding target of the target plate. Naturally airing.

d) Mu.l of the matrix solution was aspirated and covered on the quality control spots. Naturally airing to finish the sample application of the negative quality control product.

e) The negative quality control is tested at least 1 time per day.

4. Sample detection:

a) And placing the broken nose swab sample into the nose swab preservation solution, and fully and uniformly mixing.

b) And taking out the swab and smearing the swab on a corresponding target point of the target plate. Naturally airing.

c) Mu.l of the matrix solution was pipetted off and covered on the sample spots. Naturally airing, and then carrying out mass spectrum detection.

5. And (3) mass spectrum data acquisition:

all of the above calibrators, quality controls and samples were detected by MALDI-TOF MS. Mass spectrometer model: clin-ToF (Beijing-perseveration New Bo-Chuan biotechnology Co., ltd.). Invoking proper laser energy to collect one point of the crystallization point of the sample. Each sample point selects 50 laser bombardment positions, each position is bombarded for 10 times, namely, each sample crystallization point is subjected to 500 laser bombardment, and a spectrogram is collected. Laser frequency: 30Hz. Data collection range: m/z is 2000-20000. External standard calibration was performed with standard before each sample crystallization point was collected, with an average molecular weight deviation of less than 500ppm.

6. Interpretation of the results:

the mass spectrum data are imported into new crown immune reaction mass spectrum flash test software (Beijing biosciences Co., ltd.) and each mass spectrum is automatically scored by the software to obtain a qualitative detection result.

TABLE 4 results interpretation criteria

Discrimination result	Score of
		Positive and negative	60～100
Ash zone	40～60
		Negative of	0～40

EXAMPLE 7 novel clinical sample blind selection test for coronavirus infection

After the reagent composition and the existing mass spectrum detection reagent are used for preparing the kit, 31 clinical samples are subjected to blind selection test of novel coronavirus infection. All samples were nasal swab samples, which were placed into nasal swab stock immediately after collection and mass spectrometric detection was performed within 48 hours. And (3) calculating the accuracy of the detection result of the method (mass spectrometry) by taking the detection result of the reference nucleic acid as a standard. The results of the mass spectrometry blind selection test are shown in tables 5-6.

The results are shown in tables 5 and 6, and the confusion matrices are shown in FIGS. 6-1 and 6-2, respectively. Control nucleic acid detection (qPCR method) is now the gold standard for detecting novel coronavirus infection. The vertical axis in the confusion matrix represents the real grouping situation of the samples, the upper row represents the number of negative samples, and the lower row represents the number of positive samples; the horizontal axis represents the model prediction result, the left column represents the number of samples judged negative by the model, and the right column represents the number of samples judged positive by the model.

TABLE 5 comparison of blind selection tests on Mass Spectrometry by 20 characteristic polypeptide variables

The comparison result shows that: from Table 5 and FIG. 6-1, it can be seen that the results for the test group samples are: of 9 cases of novel coronavirus infected patients, 9 cases were judged correctly, and the sensitivity was 100%;22 out of 22 normal people were judged to be correct, and the specificity was 100%.

TABLE 6 Blind test comparison of Mass Spectrometry with 5 characteristic polypeptide variables

From Table 6 and FIG. 6-2, it can be seen that the results for the test group samples are: of 9 cases of novel coronavirus infected patients, 8 cases were judged correctly, and the sensitivity was 88.9%; 21 out of 22 normal people were judged to be correct, and the specificity was 95.5%. This illustrates that a model consisting of 5 characteristic polypeptides with input variables only a few erroneous decisions occur. The model meets the requirement of rapid screening clinically.

In addition, it can be seen from the above results that: the accuracy of the prediction results of the novel coronavirus infection group and the normal human group by using the complete variable of 20 characteristic polypeptides reaches 100%. The method is true and reliable for the diagnosis result, and missing diagnosis and/or misdiagnosis are avoided to the greatest extent. Therefore, has positive significance.

In a specific process, see the prior invention application of the inventor (product for detecting the novel coronavirus based on the nasal swab of immune response characteristic polypeptide spectrum) and application thereof (invention application number: 202211292701.8) "

Example 8 other respiratory pathogen infection detection

2. Mass spectrometry calibrator detection:

e) Mass spectrometry calibrants were tested at least 1 time per day.

3. Detecting positive quality control:

e) The positive quality control is tested at least 1 time per day.

4. And (3) negative quality control detection:

e) The negative quality control is tested at least 1 time per day.

5. Sample detection:

6. And (3) mass spectrum data acquisition:

7. Mass spectrogram

Other respiratory pathogen infections such as influenza are similar to the symptoms of the novel coronaviruses and can become interference items for the detection of the novel coronaviruses. The mass spectrum of the novel coronavirus infected person, influenza infected person and healthy subjects is shown in figure 6. The characteristic peaks that appear in influenza infected persons at 2614m/z, 3354m/z, 3405m/z, 2688m/z, 5444m/z, 4839m/z, 4710m/z, 2234m/z, 2189m/z, 3440m/z, 2208m/z, 2277m/z, 4980m/z, 2924m/z, 2488m/z compared to the novel coronavirus infected persons are statistically different from the novel coronavirus infected persons. Wherein the characteristic peak of the influenza sample at 2189m/z, 2208m/z, 2234m/z, 2277m/z, 2614m/z, 2688m/z, 4710m/z, 4839m/z, 5444m/z is an up-regulation trend; while characteristic peaks at 2488m/z, 2924m/z, 3354m/z, 3405m/z, 3440m/z, 4980m/z are in a downward trend. The ROC curve verifies that the AUC of ROC of the novel coronavirus infected person and the influenza infected person is 1. The accuracy of the model in distinguishing the novel coronavirus from the influenza is 100%.

8. Interpretation of the results:

the mass spectrum data is imported into the respiratory tract disease immune reaction mass spectrum flash test software (Beijing biosciences Co., ltd.) and the qualitative detection result of the sample is automatically reported by the software.

TABLE 6 detection results

Project name	Detection result
		Novel coronavirus	Positive/negative
Influenza virus	Positive/negative

9. Blind test results for influenza patients

The results of the blind test on clinical specimens of influenza patients are shown in table 7. The 30 influenza samples were used to validate the model, interpreted by the detection software, and all 30 samples were detected as influenza samples. The detection accuracy was 100%.

TABLE 7 blind test results for influenza patient samples

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the scope of the present invention.

Claims

1. Use of a pretreatment reagent composition for preparing a mass spectrometry detection kit for detecting respiratory pathogens, wherein the reagent composition comprises a nasal swab preservation solution and a nasal swab, wherein the nasal swab preservation solution comprises 70% -80% ethanol (V/V) and phenol red (10-200 mg/L), wherein the respiratory pathogens comprise novel coronaviruses, influenza a viruses, influenza b viruses, SARS viruses, MERS viruses and the like.

2. The use of claim 1, wherein the kit comprises a nasal swab preservation solution, a nasal swab, wherein the nasal swab preservation solution comprises 70% -80% ethanol (V/V) and phenol red (10-200 mg/L).

3. The use of claim 1 or 2, wherein the nasal swab preservation solution contains ethanol, phenol red and formic acid (0.2% -5.0%, V/V) at the same time, wherein formic acid can assist the ionization of polypeptides in a sample in mass spectrometry, and improves the signal intensity of the mass spectrometry.

4. The use according to claim 3, wherein the nasal swab holder is provided in an amount of 0.2 to 4.0 ml/tube, preferably 1.0 to 3.0 ml/tube.

5. The use of claim 4, wherein the kit further comprises a matrix powder, a matrix solvent, a mass spectrometry calibrator, a positive quality control, and a negative quality control.

6. The use of claim 4, wherein the kit comprises a calibrator tube for ensuring accurate molecular weight measurements by a mass spectrometer, the calibrator comprising corticotropin, cytochrome C and myoglobin, the characteristic polypeptide peaks having m/z of 2465m/z, 4121m/z, 6181m/z, 8477m/z, 12362m/z, 16952m/z, respectively.

7. The use of claim 5 or 6, wherein the kit further comprises a quality control product for judging whether the detection result is reliable, the quality control product is prepared from the mixed protein polypeptide, and the positive quality control product has a characteristic spectrum similar to that of a respiratory pathogen infected person; the negative quality control has a similar profile to healthy people. And carrying out parallel mass spectrometry test when the quality control product and the sample to be tested are subjected to mass spectrometry so as to judge whether the result to be tested is accurate and reliable.

8. The use of claims 1-7, wherein the mass spectrometry detection kit can be prepared by combining the reagent composition with an existing mass spectrometry detection reagent.

9. The use of claim 8, wherein the laser mass spectrometry is selected from MALDI TOF MS or Clin-TOF.