WO2022095781A1

WO2022095781A1 - Molecular marker for predicting and monitoring disease course of novel coronavirus pneumonia and use thereof

Info

Publication number: WO2022095781A1
Application number: PCT/CN2021/127006
Authority: WO
Inventors: 于文强; 徐鹏; 杨帅; 茹道平; 徐建青
Original assignee: 上海市公共卫生临床中心
Priority date: 2020-11-03
Filing date: 2021-10-28
Publication date: 2022-05-12
Also published as: CN112345750A

Abstract

A molecular marker for predicting and monitoring the disease course of novel coronavirus pneumonia, the molecular marker being hyaluronic acid or a combination containing hyaluronic acid. Other markers in the combination comprise interleukin, D-dimer and/or C-reactive protein. The present invention also relates to the use of the molecular marker as a molecular marker for predicting and monitoring the disease course of novel coronavirus pneumonia, and the use of a detection reagent of the molecular marker in the preparation of a product for predicting and monitoring the disease course of novel coronavirus pneumonia. The level of hyaluronic acid in plasma of patients with severe novel coronavirus pneumonia is significantly increased, and the level of hyaluronic acid is closely related to other molecular changes in patients with novel coronavirus pneumonia, so that the content of hyaluronic acid in the plasma can be used as a molecular marker for distinguishing patients with mild and severe cases of novel coronavirus pneumonia, thus facilitating the treatment of the patients with novel coronavirus pneumonia.

Description

A molecular marker for predicting and monitoring the course of novel coronavirus pneumonia and its application

technical field

The invention belongs to the technical field of molecular biology, and in particular relates to a molecular marker for predicting and monitoring the course of novel coronavirus pneumonia and its application.

Background technique

Since January 2020, the new crown pneumonia (COVID-19) epidemic caused by the new coronavirus (SARS-CoV-2) has spread globally. As of October 25, 2020, more than 40 million people worldwide have been infected and over A million people died. Based on the clinical symptoms of the patients, patients with COVID-19 can be divided into mild, common, severe, and critical. Mild patients present with low fever, mild fatigue, and disturbance of smell and taste. The most common symptoms in hospitalized patients are ground-glass lesions in the lungs, while severe patients often develop dyspnea and/or hypoxemia one week after the onset, and severe cases can rapidly progress to acute respiratory distress syndrome (ARDS). , septic shock, difficult to correct metabolic acidosis and coagulation dysfunction and multiple organ failure, and eventually even death.

For patients of different severity, the "New Coronary Virus Pneumonia Diagnosis and Treatment Program (Trial Version 8)" issued by the General Office of the National Health Commission and the State Administration of Traditional Chinese Medicine recommends different treatment strategies. Therefore, to predict and monitor the new coronary pneumonia The development of new crown patients is particularly critical for the treatment of new crown patients.

The progression and outcome of COVID-19 infection are related to a variety of factors, such as age, gender, and the presence or absence of underlying diseases. At the molecular level, the plasma of patients with COVID-19 has decreased lymphocytes, especially CD4+ and CD8+ T cells, increased levels of interleukins IL-6 and IL-8, increased D-dimer levels, C-reactive protein (CRP) ) level rises. Among them, D-dimer is the most important laboratory index reflecting thrombosis and thrombolytic activity; CRP is an acute phase protein synthesized by hepatocytes when the body is subjected to inflammatory stimuli such as microbial invasion or tissue damage. Sensitive inflammatory markers. Although these molecular-level changes are closely related to the clinical manifestations and progression of COVID-19, there is still a lack of molecular markers that can effectively predict and monitor the course of COVID-19.

Hyaluronic acid (HA) is a glycosaminoglycan with disaccharides (D-glucuronic acid and N-acetylglucosamine) as the basic unit, also known as uronic acid and hyaluronic acid. Hyaluronic acid is synthesized by the hyaluronic acid synthase HAS (Hyaluronic Acid Synthase). The vertebrate HAS includes HAS1, HAS2 and HAS3. The inventors previously found that the HIS (Human Identical Sequence) sequence from the nucleic acid sequence of the new coronavirus that is consistent with the human genome can activate HAS2 and further promote the synthesis of hyaluronic acid, and found that hyaluronic acid was significantly increased in the peripheral plasma of patients with the new coronavirus. However, it is not known whether there is a close relationship between hyaluronic acid and the clinical symptoms and progression of patients with new coronary pneumonia.

SUMMARY OF THE INVENTION

In order to overcome the defects in the prior art, the present invention provides a molecular marker for predicting and monitoring the course of novel coronavirus pneumonia and its application, which can effectively predict and monitor the course of the novel coronavirus.

To achieve the above object, the present invention adopts the following technical solutions:

A first aspect of the present invention is to provide a marker for predicting and monitoring the course of COVID-19; wherein, the marker is hyaluronic acid or a combination containing hyaluronic acid, and other markers in the combination Including interleukins, D-dimer (D-D) and/or C-reactive protein (CRP).

In order to further optimize the above-mentioned technical scheme, the technical measures taken by the present invention also include:

Further, in the above markers, the hyaluronic acid is plasma hyaluronic acid.

Further, in the above markers, the hyaluronic acid is macromolecular hyaluronic acid and/or small molecular hyaluronic acid. Further, the molecular weight of the macromolecular hyaluronic acid is higher than 2000KDa, and the molecular weight of the small molecular hyaluronic acid is 10KDa-2000KDa.

Further, in the above markers, the interleukins include interleukin IL-6 and interleukin IL-8.

The second aspect of the present invention is to provide the use of any one of the above markers as a molecular marker for predicting and monitoring the course of COVID-19.

Further, in the above application, when the plasma hyaluronic acid is less than 10ng/mL, the patients with new coronary pneumonia are predicted to be mild; when the plasma hyaluronic acid is greater than or equal to 10 ng/mL, the patients with new coronary pneumonia are predicted to be common, severe or critical. Further, when 10ng/mL≤plasma hyaluronic acid<48.43ng/mL, the patients with new coronary pneumonia were predicted to be common type; when plasma hyaluronic acid≥48.43ng/mL, the patients with new coronary pneumonia were predicted to be severe or critical.

A third aspect of the present invention is to provide an application of any of the above-mentioned marker detection reagents in the preparation of a product for predicting and monitoring the course of COVID-19.

Further, in the above application, the product predicts that the patient belongs to mild or severe disease by detecting the level of hyaluronic acid in the plasma of patients with new coronary pneumonia. Based on the "New Coronary Virus Pneumonia Diagnosis and Treatment Plan (Trial Version 7)", patients with new crowns can be divided into light, common, severe and critical.

Further, in the above application, when the plasma hyaluronic acid is less than 10ng/mL, the patients with new coronary pneumonia are predicted to be mild; when the plasma hyaluronic acid is greater than or equal to 10 ng/mL, the patients with new coronary pneumonia are predicted to be common, severe or critical.

Further, in the above application, the threshold value of plasma hyaluronic acid determined to be severe is 48.43 ng/mL. That is, when 10ng/mL≤plasma hyaluronic acid<48.43ng/mL, the patients with new coronary pneumonia are predicted to be common type; when plasma hyaluronic acid≥48.43ng/mL, the patients with new coronary pneumonia are predicted to be severe or critical.

Further, in the above application, the product can also detect the levels of interleukin, D-dimer and/or C-reactive protein in the patient's plasma, so as to help predict whether the patient is mild or severe.

It can be understood that the above-mentioned other markers can be any suitable markers in the prior art used to assist in predicting or monitoring the course of new coronary pneumonia, and are not limited to the above examples; the above-mentioned reagents for detecting hyaluronic acid are any suitable ones. The substance that can be used to detect the level of hyaluronic acid in plasma; the above-mentioned reagent for detecting the level of interleukin, D-dimer and/or C-reactive protein can be any suitable substance that can be used to detect the level of the above-mentioned component in plasma .

Compared with the prior art, the present invention adopts the above-mentioned technical scheme, and has the following technical effects:

Based on the inventor's earlier discovery that 5 segments of the SARS-CoV-2 genome are identical to the human genome (named "human identical sequences (HIS)"), it was confirmed by molecular biology and cell biology experiments that HIS can activate The expression of hyaluronic acid synthase 2 (HAS2), thereby up-regulating the level of hyaluronic acid, the present invention found through further research that the level of hyaluronic acid in the plasma of patients with severe new coronary pneumonia was significantly increased, and the level of hyaluronic acid was closely related to other molecules in patients with new coronary pneumonia. (eg interleukin, D-dimer and, C-reactive protein) changes are closely related.

The present invention discovers and validates for the first time that plasma hyaluronic acid can be used as a molecular marker for distinguishing mild and severe cases of COVID-19, thereby making up for the lack of effective markers for predicting and monitoring the course of COVID-19 in the prior art, which is conducive to the development of Treatment of patients with new coronary pneumonia.

Description of drawings

Fig. 1 is the HIS (human identical sequences) sequence that exists in SARS-CoV-2 in one embodiment of the present invention and is completely identical to the human genome; wherein, SARS-CoV-2 is abbreviated as SARS2, and the five sequences are named respectively as HIS-SARS2-1, HIS-SARS2-2, HIS-SARS2-3, HIS-SARS2-4, HIS-SARS2-5; the numbers in brackets indicate the sequence in SARS-CoV-2 and human genomes, respectively Location, chr refers to chromosomes.

2 is a schematic diagram of the results of verifying that overexpressing HIS fragments in HEK293T and MRC5 can up-regulate the expression of hyaluronan synthase HAS2 in an embodiment of the present invention; the ordinate in the figure is the relative expression level of HAS2 RNA detected by RT-qPCR, the abscissa For the experimental group transfected with 5 kinds of HIS respectively; **, p<0.01; ***, p<0.001.

3 is a schematic diagram of the results of verifying that overexpression of HIS fragments in HEK293T and MRC5 can up-regulate the concentration of hyaluronic acid in the cell culture medium in an embodiment of the present invention; wherein, the ordinate is the concentration of hyaluronic acid in the cell culture medium (ng /mL), the abscissa is the transfection solvent control group mock, the transfection HIS-SARS2-3 and HIS-SARS2-4 groups; **, p<0.01; ns, not significance.

Figure 4 is a schematic diagram of the comparison of hyaluronic acid levels in the plasma of patients with mild symptoms of the new coronavirus and severe patients in an embodiment of the present invention; wherein, based on the "New Coronary Virus Pneumonia Diagnosis and Treatment Plan (Trial Version 7)", patients with new crowns can be classified as mild , common type, severe type and critical type, where mild disease refers to light type, and severe type includes common type, severe type and critical type; ****, p<0.0001.

Figure 5 is a schematic diagram of the results of dynamic changes of hyaluronic acid in plasma during the course of the new coronavirus in an embodiment of the present invention; wherein, the ordinate is the concentration of hyaluronic acid (ng/mL), and the abscissa is the course of the patient's disease, measured after blood collection according to time The number of times, 1, 2, and 3 are the 1st, 2nd, and 3rd times, respectively.

Figure 6 is a schematic diagram of the results of the lymphocyte levels in the plasma of new crown patients with different hyaluronic acid levels using the hyaluronic acid level as the classification standard in an embodiment of the present invention; ****, p<0.0001.

Figure 7 is a schematic diagram of the results of the D-dimer levels in the plasma of new crown patients with different hyaluronic acid levels using the hyaluronic acid level as the classification standard in an embodiment of the present invention; ****, p<0.0001.

Figure 8 is a schematic diagram of the results of the CRP levels in the plasma of new crown patients with different hyaluronic acid levels using the hyaluronic acid level as the classification standard in an embodiment of the present invention; ****, p<0.0001.

FIG. 9 is a schematic diagram of a result of bioinformatics analysis in an embodiment of the present invention.

Detailed ways

The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and embodiments. The following examples are only used to more clearly illustrate the technical solutions of the present invention, and cannot be used to limit the protection scope of the present invention. In the following examples, the experimental methods without specific conditions are selected according to the conventional methods and conditions in the art, or according to the product description.

Example 1 - Construction of vectors for HIS overexpression

The present embodiment is the construction of the carrier of HIS overexpression, and its steps include:

1. Sequence acquisition-identification of human identical sequence (HIS) in SARS-CoV-2

The new coronavirus SARS-CoV-2 gene sequence (NC_045512) was found from the Nucleotide database Genbank of NCBI (https://www.ncbi.nlm.nih.gov/), and then the whole genome nucleotide sequence of the virus and the human whole Blast alignment of the genome sequence (GRCh38.p13), and finally screened out more than 20 nucleotides and identical nucleotide sequences, and screened out 5 sequences that are completely complementary to the human genome in the new coronavirus SARS-CoV-2 HIS-SARS2-1, HIS-SARS2-2, HIS-SARS2-3, HIS-SARS2-4, HIS-SARS2-5 (as shown in Figure 1).

2. Amplify the target fragment

According to the use of Primer5 software to determine the upstream and downstream primers respectively, then add a protective base and EcoR I restriction site sequence (GAATTC) in front of the 5' end of the upstream primer, and add a protective base and BamH in front of the 5' end of the downstream primer. I restriction site sequence (GGATCC). The primer sequences are shown in Table 1 below, and the primers were entrusted to Shanghai Paisenuo Biotechnology Co., Ltd. for synthesis.

Table 1 - SARS-CoV-2 related primers

Use homologous recombination to artificially synthesize viral target fragments, after annealing the F and R1 primers designed according to the sequence, use F and R2 and F and R3 to carry out two rounds of nested PCR, Q5 enzyme to amplify the target gene fragment, the amplification system and The procedure is as follows:

PCR program: 98℃ for 30s;

98℃ for 10s, 55～72℃ for 30s, 72℃ for 30s/kb, 35 cycles;

72℃ for 2min.

3. Recovery, digestion and purification of PCR products

The PCR product was detected by 1% agarose gel electrophoresis, recovered by gel tapping, and the target fragment was recovered using a common agarose gel DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.); the enzyme digestion process was based on the enzyme digestion system of the NEB website at 37°C. Cut overnight, and use the PCR product recovery kit (Tiangen Biochemical Technology Co., Ltd.) to purify and recover.

4. Connection

The digested PCR product and the digested pCDH vector were connected by T4 ligase according to the following ligation system, overnight at 16°C. The connection system is as follows:

5. Transform and pick monoclonal ligation

10 μl of the ligation product was added to 50 μl of DH5α competent cells, and incubated on ice for 30 min. After heat shock competent cells at 42°C for 90s, they were immediately placed on ice for 5min. Add 300 μl of antibiotic-free LB liquid medium to the ultra-clean workbench, and shake the bacteria in a constant temperature shaker at 37°C for 30 min. The bacterial solution was centrifuged at 1000 g for 5 min, and the supernatant was discarded, leaving 50 μl of bacterial solution, which was evenly spread on an ampicillin-resistant LB solid plate, and cultured overnight in a 37°C constant temperature incubator. Pick an appropriate amount of monoclonal colonies from the plates cultured overnight, put them into EP tubes with 200 μl of ampicillin-resistant LB liquid medium, and shake the bacteria in a constant temperature shaker at 37°C for 2 hours before sending for sequencing identification. Finally, the target band can be obtained by vector PCR.

Example 2 - Effect of cells overexpressing HIS-SARS2 on gene expression levels

The present embodiment detects the expression level of HAS2 gene by overexpressing HIS-SARS2 sequence in HEK293T and MRC5 cells, and the steps are briefly described as follows:

1. Lentivirus packaged by liposome method: According to molecular cloning, SARS-CoV-2 overexpression plasmid, virus packaging plasmid psPAX2 and capsid plasmid pMD2.G-VSVG were transferred into HEK293T cells, and collected once after 48hr and 72hr. The supernatant was filtered with a 0.45 μm filter to obtain a lentiviral stock solution.

2. Infected cells: spread 200,000 infected cells (lentivirus stock solution) in a 6cm petri dish in advance, wait for the cells to adhere to the wall on the second day and carry out the first infection, and repeat the infection on the third day; the fourth day The cells were allowed to recover for one day without any stimulation; on the fifth day, drug screening was performed on the corresponding drug-killing markers carried by the plasmids.

3. Real-time fluorescent quantitative PCR

(1) Total RNA extraction

Prepare 10 ⁶ -10 ⁷ cells, resuspend in PBS, remove the supernatant by centrifugation, add 1 ml of TRIzol for lysis at room temperature for 5 min, then add 0.2 ml of chloroform, shake with a vortex shaker for 15 s, and stand at room temperature for 2 min. Centrifuge at 4°C for 15 min, 13,300 rpm. Transfer the upper colorless aqueous phase to another EP tube. Add an equal volume of isopropanol, mix thoroughly with a vortex shaker, and centrifuge at 4°C for 10 min at 13,300 rpm. Pour off the supernatant, add 1 ml of 75% ethanol prepared with DEPC water, invert up and down until the precipitate is suspended, and centrifuge in a 4°C centrifuge for 5 min at 13,300 rpm. Use a pipette to suck up the supernatant, dry at room temperature for 5-20 min, and observe the form of the precipitate during the period. When it just becomes transparent, dissolve it in 40-100 μl of DEPC water according to the amount of the precipitate. Take 1 μl to measure its concentration and OD260/OD280 on Nanodrop. The extracted RNA was stored in a -80°C refrigerator.

(2) Synthesis of cDNA by reverse transcription

Using the reverse transcription PCR kit of Takara (D2680A), the PCR reaction system and procedures are as follows:

Reverse transcription PCR program: 42°C for 10 min, 95°C for 2 min.

(3) RT-qPCR

Takara real-time fluorescence quantitative PCR kit was used to detect the expression of the target gene at the transcription level, and the reaction system was as follows:

The expressions of HIS-SAR2-1, HIS-SAR2-2, HIS-SAR2-3, HIS-SAR2-4, HIS-SAR2-5 and HAS2 were detected. The primers are shown in Table 2, and the detection results are shown in Figure 2.

Table 2 - Primer sequences for RT-qPCR

It can be seen from the detection results in Figure 2 that in HEK293T cells, the overexpression of HIS-SARS2-3 and HIS-SARS2-4 can up-regulate the expression of HAS2; while in MRC5, HIS-SARS2-1, HIS-SARS2-3 and HIS - SARS2-4 overexpression upregulates HAS2 expression. It can be seen that after overexpression of the HIS sequence, the hyaluronic acid synthase family HAS2 related to the new coronavirus can be significantly activated.

Example 3 - Identification of hyaluronic acid levels in cell culture media after HIS overexpression

The lentivirus and infected cells were prepared by the method of Example 2; the cell supernatant was collected after 24 hours, and the hyaluronic acid ELISA kit (R&D, DY3614-05) was used for detection. The steps are briefly described as follows:

1. ELISA plate coating: 100μl/well Capture Reagent was coated overnight at room temperature.

2. Closing: The clapper removes the Capture Reagent. Wash the plate 3 times with 400 μl/well Wash buffer and pat dry. Block with 100 μl/well Dilute Reagent for 1 h.

3. Wash the plate and incubate the sample: wash the plate 3 times with 400μl/well Wash buffer, add 100μl/well standard substance and the plasma to be tested (100μl of plasma from patients with mild and severe new coronary pneumonia are diluted with Dilute Reagent in the 200μl kit to a total volume of 300μl, Do 3 duplicate wells) and incubate at room temperature for 2h.

4. Wash the plate and incubate Detect Reagent. Wash the plate 3 times with 400 μl/well Wash buffer, add 100 μl/well Detect Reagent and incubate at room temperature for 2 h.

5. Wash the plate and incubate with HRP. Wash the plate 3 times with 400 μl/well Wash buffer, add 100 μl/well HRP and incubate at room temperature for 20 min.

6. Wash the plate and incubate the substrate. Wash the plate 3 times with 400 μl/well Wash buffer, add 100 μl/well A and B substrate mixture, and incubate at room temperature for 20 min.

7. Terminate color development. Add 50μl/well stop solution. Read absorbance at 450 nm within 15 minutes.

The detection result of the hyaluronic acid content in the above-mentioned cell culture medium is shown in Figure 3. In HEK293T, overexpressing HIS-SARS2-3 and HIS-SARS2-4 can significantly increase the hyaluronic acid concentration in the culture medium. Overexpression of HIS-SARS2-4 significantly increased the hyaluronic acid concentration in the medium. It can be seen that the level of hyaluronic acid can be significantly up-regulated after HIS overexpression.

Example 4-ELISA method to measure the concentration of hyaluronic acid in plasma

In this example, mild and severe cases were defined based on clinical CT results, and the hyaluronic acid content in the plasma of patients with mild and severe new coronary pneumonia was determined by ELISA, which was used according to the hyaluronic acid ELISA kit (R&D, DY3614-05) of R&D Company. Experiments are described, and the specific steps refer to Example 3.

The detection results of hyaluronic acid content in the above plasma are shown in Figure 4. Compared with mild new coronary pneumonia patients, the plasma hyaluronic acid content of severe new coronary pneumonia patients was significantly increased.

In this example, the plasma hyaluronic acid content of two new crown patients was also dynamically detected. The results are shown in Figure 5. The plasma hyaluronic acid level of the new crown patients showed dynamic changes during the course of the disease.

Embodiment 5-blood routine index detection

This example redefines the mild and severe cases of COVID-19 based on the plasma hyaluronic acid content, where hyaluronic acid <10ng/mL is defined as mild, and hyaluronic acid ≥10 ng/mL is defined as common and above (severe, critical) ELISA method was used to determine the absolute number of lymphocytes, D-dimer and CRP content in the plasma of patients with mild and severe new coronary pneumonia. Blood routine indexes were provided by Shanghai Public Health Clinical Center.

Based on the definition of hyaluronic acid level, it was found that the number of lymphocytes in patients with severe new coronary pneumonia was significantly lower than that in patients with mild new coronary pneumonia, which was consistent with the data reported in the previous literature, and suggested that the immune cells of patients decreased with the aggravation of the disease (as shown in Figure 6). ); and D-dimer is a fibrin degradation product, and the increased level of D-dimer indicates that there is a hypercoagulable state and secondary hyperfibrinolysis in the body, so the mass concentration of D-dimer is important for thrombotic diseases. It is of diagnostic significance, and the plasma D-dimer content of severe new coronary pneumonia patients is significantly higher than that of mild patients (as shown in Figure 7), which is consistent with the data reported in the previous literature, suggesting that the coagulation risk of patients increases with the progression of new coronary pneumonia. The aggravation and increase also indicate the feasibility of follow-up anticoagulation therapy; the CRP level in severe patients is higher than that in mild patients (as shown in Figure 8), which is consistent with the data reported in the previous literature, suggesting that new coronavirus infection can be triggered Inflammatory response of the host.

From the above results, it can be seen that compared with patients with mild new coronary disease with plasma hyaluronic acid <10ng/mL, the level of lymphocytes in the plasma of patients with severe new coronary disease with plasma hyaluronic acid level ≥10ng/mL was significantly lower, and the level of D-dimer was significantly lower. Significantly higher, and CRP levels were significantly higher, all suggesting that plasma hyaluronic acid can be used as an effective classification indicator for the course of disease in patients with new coronary pneumonia.

Example 6 - Bioinformatics analysis of cutoff value (threshold value) of hyaluronic acid

In this example, the plasma hyaluronic acid detection values of 137 patients with COVID-19 and the plasma hyaluronic acid detection values of 20 normal persons were used, and the hyaluronic acid value corresponding to the area under the curve with the largest sensitivity and specificity scores was screened. Figure 9 is the receiver operating characteristic curve (Receiver Operating Characteristic, ROC) curve, wherein the area under the curve (Area Under Curve, AUC) is 0.729, the threshold for calculating hyaluronic acid is 48.43ng/mL, which shows that the plasma hyaluronic acid The level of 48.43ng/mL can further distinguish common type, severe and critical type in the new crown, of which 10ng/mL≤plasma hyaluronic acid<48.43ng/mL is normal type, plasma hyaluronic acid≥48.43ng/mL is severe or critical type type, further indicating that the plasma hyaluronic acid level can be used as an indicator to distinguish the severity of the new crown.

The specific embodiments of the present invention have been described above in detail, but they are only used as examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention.

Claims

A marker, characterized in that, the marker is used to predict and monitor the course of new coronary pneumonia; wherein, the marker is hyaluronic acid or a combination containing hyaluronic acid, and other markers in the combination include leukocytes Interkines, D-dimers and/or C-reactive protein.
The marker according to claim 1, wherein the hyaluronic acid is plasma hyaluronic acid.
The marker according to claim 1, wherein the hyaluronic acid is macromolecular hyaluronic acid and/or small molecular hyaluronic acid.
The marker according to claim 3, wherein the molecular weight of the macromolecular hyaluronic acid is higher than 2000KDa, and the molecular weight of the small molecular hyaluronic acid is 10KDa-2000KDa.
The marker according to claim 1, wherein the interleukin comprises interleukin IL-6 and interleukin IL-8.
An application of the marker according to any one of claims 1 to 5 as a molecular marker for predicting and monitoring the course of novel coronavirus pneumonia.
The application according to claim 6, wherein when plasma hyaluronic acid is less than 10ng/mL, it is predicted that patients with new coronary pneumonia are mild; when plasma hyaluronic acid is greater than or equal to 10 ng/mL, patients with new coronary pneumonia are predicted to be common, severe or Critical.
An application of the marker detection reagent according to any one of claims 1 to 5 in the preparation of a product for predicting and monitoring the course of novel coronavirus pneumonia.
The application according to claim 8, wherein the product predicts whether the patient belongs to mild or severe disease by detecting the level of hyaluronic acid in the plasma of patients with new coronary pneumonia.
The application according to claim 8 or 9, characterized in that, when plasma hyaluronic acid<10ng/mL, it is predicted that the patient with new coronary pneumonia is mild; when 10ng/mL≤plasma hyaluronic acid<48.43ng/mL, it is predicted that new coronary pneumonia The patient is common type; when the plasma hyaluronic acid is ≥48.43ng/mL, the patient with new coronary pneumonia is predicted to be severe or critical.