Application of MXA in preparation of detection reagent for screening virus infection

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to application of MXA in preparation of a detection reagent for screening virus infection. The level of mucus virus resistance protein A (MXA) is detected by using a nasopharynx secretion sample, and a non-invasive diagnosis method for early stage (morbidity for 1-2 days) of common cold is carried out. The method can also be used for screening the risk of potential major viral infectious diseases (such as influenza or novel coronavirus infection and the like) with initial symptoms of common cold.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and particularly relates to the use of detecting myxovirus resistance protein A (MXA) in the preparation of a detection reagent for differentiating virus infection. Background Art

[0002] Acute upper respiratory tract infection, abbreviated as upper respiratory tract infection, is a general term for acute inflammation mainly invading the nose, pharynx or larynx caused by various viruses and / or bacteria. Viruses are more common, accounting for about 70% - 80%. It includes common cold, viral pharyngitis, laryngitis, herpangina, pharyngoconjunctival fever, bacterial pharyngo-tonsillitis, etc.

[0003] Among them, the common cold is the most common in the population, commonly known as "cold", which is a viral infection of the upper respiratory tract, with catarrhal symptoms in the nasopharynx as the main clinical manifestation. It is mostly caused by rhinovirus, followed by coronavirus, parainfluenza virus, respiratory syncytial virus, echovirus, coxsackievirus, etc. In the Guidelines for Primary Care of Acute Upper Respiratory Tract Infection in China (2018), the common cold is described as having a relatively acute onset, mainly manifested as nasal symptoms such as sneezing, nasal congestion, and runny nose with clear water, and can also be manifested as cough, dry throat, itchy throat or burning sensation, and even a sense of postnasal drip. Symptoms such as sneezing, nasal congestion, and runny nose with clear water may occur simultaneously or several hours after the onset. After 2 - 3 days, the nasal discharge becomes thick, often accompanied by sore throat, tearing, loss of taste, shortness of breath, hoarseness, etc. Generally, there are no fever and systemic symptoms, or only low fever, discomfort, mild chills, and headache. Physical examination shows nasal mucosa congestion, edema, and secretions, and mild pharyngeal congestion. Generally, the course of the disease is 5 - 7 days and it can recover.

[0004] The present invention defines 'early common cold' as the stage within 1 - 2 days of onset, where the patient only shows nasopharyngeal symptoms (such as sneezing, nasal congestion, and runny nose with clear water), without systemic symptoms (such as fever, myalgia, chills), which conforms to the initial characteristics of the common cold described in the Guidelines for Primary Care of Acute Upper Respiratory Tract Infection in China (2018), but the guidelines do not clarify the diagnostic method for this stage.

[0005] In addition, the initial stages of influenza virus, novel coronavirus, and other major viral infectious diseases can all show common cold-like symptoms. Therefore, diagnosing early common cold is of great significance. The 'early stage' (1 - 2 days) is the best diagnostic window period, but existing methods are limited due to low sensitivity or strong invasiveness. In the early stage of the disease, the viral load may be low, the sensitivity of virus nucleic acid detection is limited, and the sampling and detection processes are relatively complex; the sensitivity of virus antigen detection (such as rapid influenza antigen detection) is also generally low at this stage (usually less than 20%).

[0006] During the process of viral infection, a cellular response occurs that secretes type I interferons (IFNs), and the antiviral activity of type I IFNs can be mediated by the production of myxovirus resistance protein A (MXA). Our research team discovered for the first time that in early-stage common cold patients (1-2 days after onset, with only nasopharyngeal symptoms), the MXA protein can be stably detected in nasopharyngeal samples with a significantly increased concentration, while the MXA concentration in the whole blood and oropharyngeal samples of the same patients is at the background level (usually below the diagnostic threshold). This specific distribution pattern reveals a strong type I interferon response characteristic in the local area (nasopharynx) at the initial stage of viral infection, exceeding the current understanding of the prior art. This discovery not only identifies the nasopharynx as the best and only effective biological sample source for detecting MXA protein related to early viral infection, but also lays the foundation for developing a highly sensitive, highly specific, and non-invasive early common cold diagnosis method based on nasopharyngeal MXA detection. Using this method to accurately identify viral common colds at the very early stage (1-2 days) of symptom onset has important clinical significance and application value for timely providing symptomatic supportive treatment, reducing unnecessary antibiotic use, assessing potential severe disease risks (such as influenza, novel coronavirus, etc.), and implementing early public health intervention measures, filling the gap in the prior art for accurate diagnosis at the very early stage of the disease. Summary of the Invention

[0007] The present invention provides a method for non-invasively diagnosing early-stage common cold (1-2 days after onset) by detecting the level of myxovirus resistance protein A (MXA) in nasopharyngeal secretion samples. This method can also be used to screen the risk of potential major viral infectious diseases (such as influenza or novel coronavirus infection, etc.) with initial symptoms similar to those of common cold.

[0008] The first aspect of the present invention is to provide the use of MXA or a preparation specifically binding to MXA in the preparation of a detection reagent for differentiating common cold. If the MXA content is higher than a predetermined threshold, it is diagnosed as common cold, and the cut-off value of the detection reagent in the detection is 20-40 ng / mL.

[0009] Furthermore, the course of the common cold is 1-7 days.

[0010] Furthermore, the differentiation refers to differentiating common cold from allergic rhinitis and / or bacterial sinusitis.

[0011] Furthermore, the preparation specifically binding to MXA is an antibody or antibody fragment specifically binding to MXA.

[0012] Furthermore, the cut-off value of the detection reagent is 30 ng / mL.

[0013] Furthermore, the detection reagent is used to detect the MXA content in nasopharyngeal secretion samples of the subject to be tested.

[0014] Further, the nasopharyngeal secretion sample is a nasopharyngeal swab sample.

[0015] Further, the diagnosis is based on the content of MXA in the nasopharyngeal swab sample being higher than the predetermined threshold, while the content of MXA in the whole blood sample and oropharyngeal swab sample of the same patient is lower than the predetermined threshold.

[0016] Further, the diagnostic reagent further comprises a reagent that specifically binds to influenza virus, for detecting influenza virus antigen while detecting the content of MXA in the nasopharyngeal secretion sample, so as to screen whether the patient has a risk of influenza virus infection while diagnosing early common cold.

[0017] Further, the common cold is an early common cold, and the early common cold refers to a disease course within 1 - 2 days.

[0018] Further, the diagnosis of the early common cold is used to indicate whether the patient is suffering from a major viral infectious disease.

[0019] Further, in the uses, diagnostic methods or diagnostic products provided by the present invention above: the diagnostic reagent mentioned can detect the content of MXA by any method through its structure and / or component composition, including but not limited to electrophoresis, immunofluorescence, direct competition method, indirect competition method, ELISA method, RIA method, flow cytometry method or immunochromatography method, etc.

[0020] Further, the common cold is caused by viruses such as rhinovirus, and the symptoms include runny nose and cough.

[0021] The second aspect of the present invention is to provide an MXA detection kit, the detection kit comprising a conjugate that specifically binds to MXA, a standard product, a buffer solution, and a warning line mark, the warning line mark including a detection result pattern corresponding to the cut-off value of the MXA content in the sample; the warning line mark further comprises a prominent and eye-catching mark corresponding to the detection result when the MXA content in the sample is greater than 20 - 40 ng / mL.

[0022] Further, the cut-off value is 30 ng / mL.

[0023] The beneficial effects of the present invention include: In the early stage of the onset of the common cold (1-2 days, with only nasopharyngeal symptoms), nasopharyngeal swab MXA detection (threshold 30 ng / mL) has extremely high sensitivity (100%) and specificity (98.2%) for the diagnosis of viral common cold, and is significantly superior to oropharyngeal or whole blood MXA detection and rapid influenza antigen detection during the same period. At the same time, this method also shows excellent early warning ability in the early stage of influenza virus infection (sensitivity 100%, specificity 100%) Description of the Drawings

[0024] Figure 1 Sensitivity and specificity of early common cold screening after nasopharyngeal MXA detection. Detailed Implementation Manner

[0025] The following further elaborates on the concept and technical effects of the present invention in combination with specific embodiments to fully understand the purpose, features, and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. It should be noted that, without conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0026] Example 1 The MXA detection kit was used as the research and verification test object The MXA detection kit can be prepared with reference to the MXA immunoassay method (for example, the specific antibodies used, detection principles such as immunochromatography) described in the applicant's prior Chinese invention patent CN113866428B (invention name: A diagnostic reagent for differentiating children's viral influenza prepared with MXA and SAA). By testing 264 samples of nasopharyngeal secretions from healthy people, the normal value reference range of MXA in nasopharyngeal secretions of healthy people was obtained, and the MXA diagnostic threshold was determined to be 30 ng / ml accordingly.

[0027] Example 2 A test was conducted to determine whether a patient has early common cold using the MXA level Thirty-two patients with early common cold (1-2 days of onset, no systemic symptoms, only nasopharyngeal symptoms such as sneezing and nasal congestion) from the Department of Infectious Diseases of the Second Affiliated Hospital of Chongqing Medical University were selected as the test participants, and virus infection was confirmed by virus nucleic acid detection. And thirty patients with allergic rhinitis and twenty-six patients with bacterial sinusitis, who also showed early common cold symptoms, were selected, and virus infection was excluded by imaging examination and virus nucleic acid detection.

[0028] 1. Nasopharyngeal and oropharyngeal samples were collected from 88 patients using 10-µL quantitative swabs with quantitative absorption capacity. At the same time, 10-µL venous whole blood samples were collected from 88 patients. The 10-µL whole blood samples collected and the 10-µL quantitative swabs were further eluted with 100 times the volume of lysis buffer containing 0.1% CHAPS and then detected using the MXA detection kit. The detection results of MXA in whole blood, nasopharyngeal secretions, and oropharyngeal secretions were recorded in Tables 1 and 2 below. 2. Combining the clinical diagnosis results of the patients in Tables 1 and 2, statistical analysis was performed on the detected concentration results of MXA in whole blood, nasopharyngeal secretions, and oropharyngeal secretions shown in Tables 1 and 2.

[0029] 3. According to the aforementioned test methods, the test verification results are shown in Tables 1 and 2.

[0030] Table 1 Test Results of 88 Patients with Early Common Cold Symptoms

[0031] Table 2 Test Results of 88 Patients with Early Common Cold Symptoms at 3 - 7 Days of the Disease Course

[0032] 4. Analysis of the Results in Tables 1 and 2 Statistical analysis of the data in Table 1: Among the 32 clinically diagnosed patients with early common cold, 30 patients with allergic rhinitis, and 26 patients with bacterial sinusitis, after nasopharyngeal MXA detection, 32 cases of early common cold were correctly diagnosed. Among the 56 patients with allergic rhinitis and bacterial sinusitis, 55 cases were correctly diagnosed after nasopharyngeal MXA detection. Using SPSS software for statistical analysis of the data, 30 ng / ml can be used as the diagnostic threshold. The sensitivity and specificity of nasopharyngeal MXA detection for differentiating early common cold were 100% and 98.2% respectively, as Figure 1 shown.

[0033] At the same time, MXA detection in oropharynx and whole blood could not distinguish between early common cold and non-early common cold, and there was no significant difference between the two groups of data statistically.

[0034] It should be noted that among 56 non-early-stage patients with common cold (30 cases of allergic rhinitis + 26 cases of bacterial sinusitis), 1 false positive occurred (a patient with bacterial sinusitis, case 88, with a nasopharyngeal MXA level of 178.5 ng / mL). Moreover, the nasopharyngeal MXA level in this case continued to increase, which is in line with the general law of viral infection. After reviewing the clinical data, it was found that due to the limitations of the types or categories of viruses detected by the current nucleic acid test, there is a high possibility of undetected co-infection with other viruses. This positive case is included in the above specific calculation results (98.2%).

[0035] Similarly, the data in Statistical Table 2 indicate that in the middle and late stages (3 - 7 days) of common cold, the MXA protein can still be stably detected in nasopharyngeal samples, and its concentration is significantly higher than that in the early stage. The concentration of MXA protein in nasopharyngeal samples does not show a downward trend as the condition of common cold improves. At the same time, MXA detection in oropharyngeal and whole blood samples still cannot distinguish between common cold and non-common cold, and there is no significant difference between the two groups of data statistically.

[0036] Example 3: Using MXA level to pre-warn of influenza virus infection test A prospective study was conducted on patients in the Department of Infectious Diseases of the Second Affiliated Hospital of Chongqing Medical University who had a history of close contact with influenza. According to the final influenza nucleic acid diagnosis results, 22 patients were diagnosed with influenza. 10 patients with allergic rhinitis and bacterial sinusitis presenting with common cold symptoms were excluded from viral infection through imaging examinations and viral nucleic acid tests and served as non-influenza controls. All 32 patients above were tested for MXA in whole blood, nasopharyngeal secretions, and oropharyngeal secretions during the early stage of common cold symptoms (within 1 - 2 days of onset, without systemic symptoms, only nasopharyngeal symptoms such as sneezing and nasal congestion), with a cut-off value of 30 ng / ml. A value greater than or equal to 30 ng / ml was marked as positive, and a value less than 30 ng / ml was marked as negative. In addition, 32 patients in the early stage of common cold symptoms were simultaneously tested using a commercially available influenza antigen detection test strip.

[0037] Table 3: Experimental results of 32 patients in the early stage of common cold symptoms with influenza

[0038] Data in Statistical Table 3: Among 22 clinically diagnosed influenza patients and 10 non-influenza patient controls, after nasopharyngeal MXA testing during the early stage of common cold symptoms, 22 influenza patients were correctly diagnosed. Among the 10 non-influenza patient controls, 10 were correctly diagnosed after nasopharyngeal MXA testing. Using 30 ng / ml as the diagnostic threshold, the sensitivity and specificity of nasopharyngeal MXA testing in pre-warn of influenza virus infection during the early stage of common cold symptoms were 100%.

[0039] The commercially available influenza antigen test strips were used to test 22 influenza patients with early common cold symptoms, and 4 influenza patients were correctly diagnosed. A total of 10 non-influenza patients were compared, and 10 were correctly diagnosed after testing. The sensitivity and specificity of distinguishing influenza from non-influenza were 18.2% and 100%, respectively.

[0040] At the same time, in the early stage of common cold symptoms of influenza, oropharyngeal and whole blood MXA tests could not distinguish between influenza and non-influenza, and there was no statistically significant difference between the two groups of data.

[0041] The above clinical validation results fully confirm that: in the early stage of the disease (1-2 days, only nasopharyngeal symptoms), the nasopharyngeal swab MXA test (threshold 30 ng / mL) has extremely high sensitivity (100%) and specificity (98.2%) for diagnosing viral common colds, and is significantly better than the oropharyngeal or whole blood MXA test and influenza antigen rapid test in the same period. At the same time, this method also shows excellent early warning ability in the early stage of influenza virus infection (sensitivity 100%, specificity 100%).

[0042] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. Use of MXA or a preparation specifically binding to MXA in the preparation of a detection reagent for differentiating common cold, characterized in that, If the MXA content is higher than a predetermined threshold, it is diagnosed as a common cold. The cut-off value of the detection reagent in the detection is 20-40 ng / mL, and the detection reagent is used to detect the MXA content in the nasopharyngeal secretion sample of the subject to be tested.

2. The use according to claim 1, characterized in that, The preparation that specifically binds to MXA is an antibody or antibody fragment that specifically binds to MXA.

3. The use according to claim 1 or 2, characterized in that, The cut-off value of the detection reagent is 30 ng / mL.

4. Use of MXA or a preparation specifically binding to MXA in the preparation of a detection reagent for differentiating early-stage common cold, characterized in that, If the MXA content is higher than a predetermined threshold, it is diagnosed as an early common cold. The cut-off value of the detection reagent in the detection is 20-40 ng / mL, where the early common cold refers to the course of the disease within 1-2 days.

5. The use according to claim 4, wherein The preparation that specifically binds to MXA is an antibody or antibody fragment that specifically binds to MXA.

6. The use according to claim 4 or 5, characterized in that, The cut-off value of the detection reagent is 30 ng / mL.

7. Use according to claim 4 or 5, characterized in that, The detection reagent is used to detect the MXA content in the nasopharyngeal secretion sample of the subject to be tested.

8. Use of MXA or a preparation specifically binding to MXA in the preparation of a detection reagent for influenza early warning, characterized in that, If the MXA content is higher than a predetermined threshold, it is diagnosed as potential influenza. The cut-off value of the detection reagent in the detection is 20-40 ng / mL, and the detection reagent is used to detect the MXA content in the nasopharyngeal secretion sample of the subject to be tested.

9. The use according to claim 8, wherein, The diagnosis is based on the MXA content in the nasopharyngeal swab sample being higher than the predetermined threshold, while the MXA content in the whole blood sample and oropharyngeal swab sample of the same subject is lower than the predetermined threshold.

10. A MXA detection kit, characterized in that, The detection kit contains a conjugate that specifically binds to MXA, a standard product, and a buffer, as well as a warning line marker. The warning line marker includes a detection result pattern corresponding to the cut-off value of the sample MXA content; the warning line marker also includes a prominent and eye-catching marker corresponding to the detection result when the sample MXA content is higher than the cut-off value of 20-40 ng / mL.

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